ZEITSCHRIFT FÜR NATURFORSCHUNG · 2012. 5. 22. · Carotenoid Composition of...

ZEITSCHRIFT FÜR N A T U R F O R S C H U N G

SECTION C

B I O S C I E N C E S

Council

E . BÖNNING, Tübingen A. BUTENANDT, München M. E I G E N , Göttingen

Editorial Board

A. HÄGER, Tübingen W. HASSELBACH, Heidelberg P. KARLSON, Marburg F. KAÜDEWITZ, München E. W E C K E R , Würz bürg

Advisory Editorial Board P. BÖGER, Konstanz D . BÜCKMANN, Ulm K . G. GÖTZ, Tübingen G. GOTTSCHALK, Göttingen H . HOFFMANN-BERLING, Heidelberg R . JAGNICKE, Regensburg G. F. M E Y E R , TÜ Inngen M . R A J E W S K T , Essen

H . SCHIMASSEK, Heidelberg D . SCHULTE-FROHLINDE, Mülheim/R F . F . SEELIG, Tübingen J . SEELIG, Basel H . SIMON, München W. STBGLICH, Bonn A. TREBST, Bochum

EDITED IN COLLABORATION

WITH THE INSTITUTES OF THE MAX-PLANCK-GESELLSCHAFT

V O L U M E 37c N U M B E R 1/2 J A N U A R Y / F E B R U A R Y 1982

V E R L A G D E R Z E I T S C H R I F T F Ü R N A T U R F O R S C H U N G

T Ü B I N G E N

1 W

Anschrift des Verlages: Postfach 26 45, D-74 Tübingen Satz und Druck: Konrad Triltsch, Wurzburg

Nachdruck — auch auszugsweise — nur mit schriftlicher Genehmigung des Verlags

Bayerisch« j Staatsbibliothek I

München J

Section a Physics, Physical Chemistry, Cosmic Physics

Section b Inorganic and Organic Chemistry

Volume 37 c Zeitschrift für Naturforschimg 1982

Conten t s

Con ten t s of N u m b e r 1/2

O r i g i n a l C o m m u n i c a t i o n s

Antibiotics from Basidiomycetes. X V [1]. 1-Hydroxy-2-nonyn-4-one, an Antifungal and Cytotoxic Meta-bolite from I s c h n o d e r m a benzoinum (Wahl.) Karst. (In German) T. A N K E , B . - M . G I A N N E T T I , and W. S T E G L I C H 1

Vinyl-Olefines and Sesquiterpenes in the Root-Oil of Senecio isatideus W. B O L A N D , L . J A E N I C K E , and A . B R A U N E R 5

Carotenoid Composition of Chlorophyll-Carotenoid-Proteins from Radish Chloroplasts H . K . L I C H T E N T H A L E R , U . P R E N Z E L , and G. K U H N 10

Carotenoid Biogenesis in the Stick Insect, C a r a u s i u s morosus, during a Larval Instar H . K A Y S E R 13

Hydroxylation of the B-Ring of Flavonoids in the 3'- and 5'-Position with Enzyme Extracts from Flowers of Verbena h y b r i d a G. S T O T Z and G. F O R K M A N N 19

Dissociation of F A D from The N A D (P) H-Nitrate Reductase Complex from Ankistrodesmus b r a u n i i and Role of Flavin in Catalysis M . A . D E L A R O S A , A . J. M Ä R Q U E Z , and J. M . V E G A 24

Spectrophotometric Isolation of Kinetically Different Pools of P-700 and Their Correlation to the Re-duction of N A D P by Isolated Chloroplasts. I. The Effect of Light Quality and Intensity H . J . R U R A I N S K I , R. G E R H A R D T , and G. M A D E R 31

Chemical Aspects of the Mutagenic Activity of the Ascorbic Acid Autoxidation System W. H . K A L U S , W. G. F I L B Y , and R. M Ü N Z N E R 40

Mevinolin: A Highly Specific Inhibitor of Micro-somal 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase of Radish Plants T. J . B A C H and H . K . L I C H T E N T H A L E R 46

Free Activation Energies and Activation Volumes f or the Amide Rotation in Some Peptides Studied by High Pressure ^ - H i g h Resolution N M R H . H A U E R , H.-D. L Ü D E M A N N , and R. J A E N I C K E 51

Investigation of Large Intramolecular Movements within Metmyoglobin by Rayleigh Scattering of Mössbauer Radiation (RSMR) Y . F . K R U P Y A N S K I I , F . P A R A K , V . I. G O L D A N S K I I , R. L . M Ö S S B A U E R , E . E . G A U B M A N , H . E N G E L M A N N , and I. P. S U Z D A L E V 57

On the Free RadicaUnduced Aggregation of Ribo-nuclease — A Pulse Radiolysis Study Using the Light Scattering Detection Method H . S E K I and W. S C H N A B E L 63

Dark Oxidation of Unsaturated Lipids by the Pho-toxidized 8-Methoxypsoralen A . Ya . P O T A P E N K O , M . V . M O S H N I N , A . A . K R A S N O V S K Y J R . , and V . L . S U K H O R U K O V 70

Growth Specific Thylakoid Differentiation of the Endocyanelle C y a n o c y t a k o r s c h i k o f f i a n a in the Symbiotic Association with C y a n o p h o r a p a r a d o x a (In German) P. B R A N D T , S. M A R T E N , B . M Ü L L E R , and W. WlESSNER 75

Differential Fluorescence and Kinetic Studies on the Template-Binding of R N A Polymerase from Pars-ley and E s c h e r i c h i a c o l i K . G R O S S M A N N , H . B I S S W A N G E R , and H . U . SEITZ 81

Sensitivity of E s c h e r i c h i a c o l i to Viral Nucleic Acid. X V I . Temperature Conditions for C a 2 + Dependent D N A Uptake in E s c h e r i c h i a c o l i A . T A K E T O 87

Studies on 1 80 2-Uptake in the Light by Entire Plants of Different Tobacco Mutants R. ISHII and G. H . S C H M I D 93

Immobilization of Yeast Gells by Radiation-Induced Polymerization T. F U J I M U R A and I. K A E T S U 102

Contents IV

N a n o c h l o r u m e u c a r y o t u m : a Very Reduced Coccoid Species of Marine Chlorophyceae C. W I L H E L M , G. EISENBEIS, A . W I L D , and R. Z A H N 1 0 7

Growth and Photosynthesis of N a n o c h l o r u m eu-c a r y o t u m , a New and Extremely Small Eucaryotic Green Alga C. W I L H E L M and A . W I L D 1 1 5

Kinetics of Proton-Hydroxyl Transport across Leci-thin Vesicle Membranes as Measured with a Lipoid pH-Indicator W . G. P O H L 1 2 0

Notes 1 H-NMR- and ESR-Investigations on the Structures

of Dehydroascorbic Acid and the Semidehydro-ascorbate Radical (In German) H . S A P P E R , A . P L E Y E R - W E B E R , and W . L O H M A N N 1 2 9

Hydroxyrutacridone Epoxide, a New Acridone A l -kaloid from R u t a g r a v e o l e n s (In German) U . E I L E R T , B. W O L T E R S , A . N A H R S T E D T , and V . W R A Y 1 3 2

Flavonol 3-O-Methyltransferase in Plant Tissues V . D E L U C A , G. B R U N E T , H . K H O U R I , R. I B R A H I M , and G. H R A Z D I N A 1 3 4

Enzymatic Hydroxylation of Ruthenocen and Osmo-cene (In German) M . S C H N E I D E R , M . W E N Z E L , and G. S C H A C H S C H N E I D E R 1 3 6

Neutral Red Fluorescence of Chromatin: Specificity and Binding Mechanism R. H . E S P E L O S I N and J. C. S T O C K E R T 1 3 9

Proteinase-Inhibitors in Albumin Glands of A c h a t i n a f u l i c a

R. 0 . O K O T O R E and G. U H L E N B R U C H 1 4 2

Erratum 144

Con ten t s of N u m b e r 3/4


Structure Elucidation of Pterosupin from P t e r o -c a r p u s m a r s u p i u m , the First Naturally Occurring C-Glycosyl-jß-hydroxy-dihydrochalcone D . A D I N A R A Y A N A , K . V . S Y A M A S U N D A R , 0 . S E L I G M A N N , and H . W A G N E R 145

A New Catechin Glycoside from P o l y p o d i u m v u l g a r e (In German) C. K A R L , G. M Ü L L E R , and P . A . P E D E R S E N 148

On the Essential Oil Components from Blossoms of A r t e m i s i a v u l g a r i s L . (In German) K . M I C H A E L I S , 0 . V O S T R O W S K Y , H . P A U L I N I , R . Z I N T L , and K . K N O B L O C H 152

Phenazine and Phenoxazinone Biosynthesis in B r e v i b a c t e r i u m i o d i n u m R . B . H E R B E R T , J . M A N N , and A . R Ö M E R 159

Separation, Partial Purification and Characteriza-tion of a Fatty Acid Hydroperoxide Cleaving Enzyme from Apple and Tomato Fruits P. S C H R E I E R and G. L O R E N Z 165

The Site of Indole-3-acetic Acid Synthesis in Mesophyll Cells of S p i n a c i a o l e r a c e a B . H E T L M A N N , W . H Ä R T U N G , and H . G I M M L E R 174

Rubins and Rubinoid Addition Products from Phycocyanin W . K U F E R and H . S C H E E R 179

Isolation of a 6-Type Cytochrome Oxidase from Membranes of the Phototrophic Bacterium Ehodopseudomonas c a p s u l a t a H . H Ü D I G and G. D R E W S 193

Tetrapyrrol Derivatives Shown by Fluorescence Emission and Excitation Spectroscopy in Cells of Ehodopseudomonas c a p s u l a t a Adapting to Phototrophic Conditions J . B E C K and G. D R E W S 199

Formation of Hydrocarbons by Photobleaching Cyanobacterium, Anacystis n i d u l a n s G. S C H M E T T E R E R 205

Peptidoglycan and Protein, the Major Cell Wall Constituents of the Obligate Halophilic Bac-terium R h o d o s p i r i l l u m salexigens M . H . T A D R O S , G. D R E W S , and D . E V E R S 210

CO2 Fixation in A n a b a e n a c y l i n d r i c a G. D Ö H L E R 213

On the Occurrence of Monoacylglycerol Derivatives in L ip id Metabolism of Chloroplasts A . S A U E R and K . - P . H E I S E 218

Contents

Possible Models of Murein and Their Fourier Transforms H . F O R M A N E K 226

Binding of Antibodies onto the Thylakoid Mem-brane. V I I . Localization of Coupling Factor of Photophosphorylation in the Lamellar System of Chloroplasts from A n t i r r h i n u m m a j u s A . R A D U N Z and R . M E I E R 236

Photophosphorylation by Chloroplasts: Effects of Low Concentrations of Ammonia and Methyl-amine C. G I E R S C H 242

Digitonin Precipitable Sterols Inducing the Ag-gregation of Prolamellar Body-Like Structures from Completely Dissolved P L B Components A . Nis ius , M . M Ü L L E R , and H . G. R U P P E L 251

Isolation of PS II-Particels with i n v i v o Charac-teristics from E u g l e n a g r a c i l i s , Stamm Z (In German) F . S C H U L E R , P . B R A N D T and W . W I E S S N E R 256

Absorption and Picosecond Fluorescence Charac-teristics of Chlorophyll Vesicles as a Function of Temperature S. S. B R O D Y 260

Herbicides which Inhibit Electron Transport or Produce Chlorosis and Their Effect on Chloro-plast Development in Radish Seedlings. I. Chloro-phyll a Fluorescence Transients and Photosys-tem II Activity K . H . G R U M B A C H 268

Use of Isolated Leaf Cells of A b u t i l o n t h e o p h r a s t i to Localize the Action of Two Aminotriazinone Herbicidal Derivatives K . K . H A T Z I O S 276

Structure Investigations of Agonists of the Natural Neurotransmitter Acetylcholine I I [1]. X-ray Structure Analysis of Trimethyl(4-oxopentyl)-ammonium-chloride A . G I E R E N and M . K O K K I N I D I S 282

Bile Salt Delipidation, Residual Phospholipids and Reactivation of the Ca 2 + -ATPase from Sarco-plasmic Reticulum G. S W O B O D A and W . H A S S E L B A C H 289

V

The Influence of Detergents on the C a 2 + - and M g 2 + -Dependent Adenosine Triphosphatase of the Sarcoplasmic Reticulum H . L Ü D I , B . R A U C H , and W . H A S S E L B A C H 299

Inhibition of Acetyl CoA Carboxylase by a High Molecular Weight Protein in Rat Liver M . N . A B D E L - H A L I M and S. Y . K . Y O U S U F Z A I 308

Subcellular Fractionation of Bone Marrow-Derived Macrophages: Localization of Phospholiphase A i and A2 and AcyI-CoA:l-Acylglycero-3-phos-phorylcholine-O-acyltransferase E . E . K R Ö N E R , H . F I S C H E R , and E . F E R B E R 314

Periodicities of Dinucleotide Self-Information Val-ues in 0X174 D N A N . B . F U R L O N G and C. F . B E C K N E R 321

Growth Characteristics of i n v i t r o Cultured Ehrlich Ascites Tumor Cells under Anaerobic Conditions and affcer Rearation

R. M E R Z and F . S C H N E I D E R 326

Notes

: 3-O-Methyl, 8-C-methyl Quercetin a New Flavonoid from D a s y l i r i o n a c r o t r i c h u m and X a n t h o r r h o e a h a s t i l i s , Liliales (In German) C. L A R A C I N E , J . F A V R E - B O N V I N , and P . L E B R E T O N 335

New Flavonoids from the Farina of P i t y r o g r a m m a Species C. H I T Z , K . M A N N , and E . W O L L E N W E B E R 337

Actinioerythrin in M u l l u s b a r b a t u s L . (Pisces; Teleostei; Mullidae) (In German) F . -C. C Z Y G A N and A . K R Ü G E R 340

The Importance of the Position of the Adenosine Linkage in N A D + for Its Coenzyme Activity v i a Lactate Dehydrogenase (In German) E . S C H L I M M E , R . M A T T E R N , and E . SCHOTT 342

Characterization of the Nuclear Lamina in an Insect by Differential Staining R. M A R X 345

VI Contents

Trail Orientation of Wood Ants ( F o r m i c a p o l y c t e n a Förster) (In German) K . H O R S T M A N N 348

Erratum

Conten t s of N u m b e r 5/6

O r i g i n a l Communications

Structure of Echinacoside (In German) H . B E C K E R , W . C H . H S I E H , R . W Y L D E , C . L A F F I T E , and C . A N D A R Y

3 5 0

3 5 1

Synthesis of Naturally Occurring Prenylated Naph-thalene Derivates. Isolation of a New Prenyl-naphthoquinone from G a l i u m m o l l u g o (In German) L . H E I D E and E . L E I S T N E R 3 5 4

Biosynthesis of Isoflavonoid Phytoalexins: Incor-poration of Sodium [l,2- 1 3C2]Acetate into Phase-ollin and Kievitone P. M . D E W I C K , M . J . S T E E L E , R . A . D I X O N , and I. M . W H I T E H E A D 3 6 3

Diurnal Fluctuation of Quinolizidine Alkaloid Ac-cumulation in Legume Plants and Photomixotro-phic Cell Suspension Cultures M . W I N K and T. H A R T M A N N 3 6 9

Volatile Compounds from Cephalic Secretions of Females in two Cleptoparasite Bee Genera, Epeolus (Hym., Anthophoridae) and Coelioxys (Hym., Megachilidae) J . T E N G Ö , G. B E R G S T R Ö M , A . - K . B O R G - K A R L S O N , I . G R O T H , and W . F R A N C K E 3 7 6

Purification and Properties of Glucosaminephos-phate Isomerase of Proteus m i r a b i l i s B . C I F U E N T E S and C. V I C E N T E 381

Effects of Trypan Blue and Related Compounds on Production and Activi ty of Streptolysin S Y . T A K E T O and A . T A K E T O 3 8 5

Antiviral Activi ty of 2-(a-Hydroxybenzyl)-benz-imidazole and other 2-Substituted Benzimid-azoles against T M V in Tomato Leaf Discs A . C. C A S S E L L S and F . M . C O C K E R 3 9 0

Proton NMR-Relaxation Dispersion in Meconium Solutions and Healthy Amniotic F lu id : Possible Applications to Medical Diagnosis G. J . B E N E , B . B O R C A B D , V . G R A F , E . H I L T -B R A N D , P . M A G N I N , and F . N O A C K 4 9 3

Monospecific Antibody against 5-Methyl-Cytidine for the Structural Analysis of Nucleic Acids S. K . M A S T R O N I C O L I S , V . M . K A P O U L A S , and H . K R Ö G E R 3 9 9

On the Oxidative Main-Chain Scission of Nucleic Acids — Pulse Radiolysis Studies (In German) 0 . D E N K and W . S C H N A B E L 4 0 5

Binding Constants, Binding Enthalpies and E n -tropies of the Non-Competitive and the Com-petitive Binding of Acriflavine, Tetramethylacri-flavine and Acridine Orange to D N A (In German) H . W I L L E , J . P A U L U H N , and H . W . Z I M M E R -mANN 4 1 3

Possible Orientation of the Fatty Acid Chains in Lipopolysaccharide H . F O R M A N E K 4 2 8

Screening for Highly Active Plasmid Promoters via Fusion to ß-Galactosidase Gene A . R O S N E R , M . G O R E C K I , and H . A v i v 4 4 1

Anomalous Reduction of Cytochrome b in Highly Purified Complex I I I from Baker's Yeast F . F . D E L A R O S A and G. P A L M E R 4 4 5

Changes in the Fluorescence Emission Spectrum of C h l o r e l l a emersonii Induced by Cold Treatment; a Possible Regulative Feature of Energy Uptake G. H A R N I S C H F E G E R and H . J A R R Y 4 4 8

Studies of Photosynthesis Inhibition by Phytolu-minography R . B L A I C H , 0 . B A C H M A N N , and I. B A U M -B E R G E R 4 5 2

A Study of State Changes in Chlorella: The Effect of Un coupler and Energy Transfer Inhibitors P. V . S A N E , D . F U R T A D O , T. S. D E S A I , and V . G. T A T A K E 4 5 8

Adaptation of Chloroplast-Ultrastructure and of Chlorophyll-Protein Levels to High-Light and Low-Light Growth Conditions H . K . L I C H T E N T H A L E R , G. K U H N , U . P R E N Z E L , C. B U S C H M A N N , and D . M E I E R 4 6 4

Contents VII

Protochlorophyllide Photoeonversion Initiates the Transformation of Reaggregated Prolamellar Body Tubules i n v i t r o M . M Ü L L E R and A . N I S I U S 476

The Sarcoplasmic Reticulum of Smooth Muscle Fibers L . R A E Y M A E K E R S 481

Histochemical Demonstration of an ATP-Dependent Ca 2 + -Pump in Bullfrog Myocardial Cells R . M E Y E R , W . S T O C K E M , M . S C H M I T Z , and H . G. H A A S 489

Ca Entry and Contraction as Studied in Isolated Bovine Ventricular Myocytes G. I S E N B E R G 502

Fluorescence Energy Transfer between ATPase Monomers in Sarcoplasmic Reticulum Recon-stituted Vesicles, in the Presence of Low Con-centrations of a Nonionic Detergent P . C H A M P E I L , J . - L . R I G A U D , and M . P . G I N -G O L D 513

Functional Significance of Quaternary Organization of the Sarcoplasmic Reticulum Ca 2 + -ATPase J . V . M 0 L L E R , T. S. M A H R O U S , J . P . A N D E R S E N , and M . L E M A I R E 517

Equilibrium Constants for Some Steps of the Reac-tion Cycle of the Sarcoplasmic Reticulum Calcium Pump C. T A N F O R D and D . W . M A R T I N 522

Phosphorylation of Ca 2 + -ATPase by Inorganic Phosphate in Water-Organic Solvent Media: D i -electric Constant and Solvent Hydrophobicity Contribution A . D E SOTJZA O T E R O and L . D E M E I S 527

ATPases of the Cat Carotid Body and of the Neigh-bouring Ganglia

H . S T A R L I N G E R 532

Notes Synthesis of 6-Chlorogenistein

A . L E V A I and A . L . T Ö K E S 5 4 0

Effect of Penicillins on the Level of Inorganic Pyrophosphatase in E s c h e r i c h i a c o l i K 12 E . I. K U K K O and J . K . H E I N O N E N 542

Hyperglycemia in the Fresh Water Field Crab ( O z i o t e l p h u s a senex senex) Produced by a Pesti-cide (BHC) P . S R E E N I V A S U L A R E D D Y , S. B . R A M E S H B A B U , and R . R A M A M U R T H I 5 4 5

Coenzyme Binding at Different Ionization States of Cytoplasmic and Mitochondrial Malate Dehydro-genase K . S C H W E R D T F E G E R , C. W O E N C K H A U S , D . M . P A R K E R , and J . J . H O L B R O O K 5 4 7

A New Method to Prepare Membrane Fractions Containing Ionophore-Stimulated ATPase from Pumpkin Hypocotyls ( C u c u r b i t a m a x i m a , L . ) G. F . E . S C H E R E R 5 5 0



E S R Studies on Platinum Uracil Blue H . N E U B A C H E R , M . S E U L , and W . L O H M A N N 5 5 3

Roseanolone: A New Diterpene from Hypoestes rosea

J . I. O K O G U N , A . A . A D E S O M O J U , G. A . A D E S I D A , H . J . L I N D N E R , and G. H A B E R M E H L 5 5 8

6-C-a-L-Rhamnopyranosylapigenin 7-0-/?-D-gluco-pyranoside (Isofurcatain 7-0-/?-D-glucoside), a New Flavone Glycoside from M e t z g e r i a f u r c a t a K . R . M A R K H A M , R . T H E O D O R , R . M U E S , and H . D . Z I N S M E I S T E R 5 6 2

On the Essential Oil Components from K u m u l u s l u p u l u s L . var. neomexicanus Nels. & Cockerell. I. Contribution K . K N O B L O C H , H . P A U L I N I , C. E L E Y , J . H . E L E Y , E . Z I E G L E R , H . B R A N D A U E R , K . M I C H A E L I S , and O. V O S T R O W S K Y 5 6 5

Demethylation, Methylation and 3'-Hydroxylation of Isoflavones by F u s a r i u m Fungi K . - M . W E L T R I N G , K . M A C K E N B R O C K , and W . B A R Z 5 7 0

Accumulation of Acridone-Epoxides in Callus Cul-tures of R u t a graveolens Increased by Coculture with Not Host-Specific Fungi (In German) B . W O L T E R S and U . E L L E R T 5 7 5

VIII Contents

Precursors of Cyclic Polysulphides in Seeds of P a r -k i a speciosa Hassk. (In German) R. S U S I L O and R. G M E L I N 584

Formation of Flavonol 3-O-Diglycosides and Fla-vonol 3-O-Triglycosides by Enzyme Extracts from Anthers of T u l i p a cv. Apeldoorn G . K L E I N E H O L L E N H O R S T , H . B E H R E N S , G . P E G E L S , N . SRTTNK, and R . W I E R M A N N 587

Polynucleotide Phosphorylase from a Cyanobac-terium (Synechococcus s p . ) : Subunit Composition and Properties W . - T . N O L D E N and G . R I C H T E R 600

DEAE-Dextran Induced Increase of Membrane Permeability and Inhibition of Photosynthesis in D u n a l i e l l a p a r v a H . G I M M L E R and G . L O T T E R 609

High- and Low-Affinity Binding of Photosystem II Herbicides to Isolated Thylakoid Membranes and Intact Algal Cells H . L A A S C H , K . P F I S T E R , and W . U R B A C H 620

Herbicides which Inhibit Electron Transport or Produce Chlorosis and Their Effect on Chloro-plast Development in Radish Seedlings. II. Pig-ment Excitation, Chlorophyll Fluorescence and Pigment-Protein Complexes C. B Ü S C H M A N N and K . H . G R U M B A C H 632

Herbicides which Inhibit Electron Transport or Produce Chlorosis and Their Effect on Chloro-plast Development in Radish Seedlings. III . Plastid Pigment and Quinone Composition K . H . G R U M B A C H 642

Effects of Substituted 2-Phenylamino-l,4,5,6-tetra-hydropyrimidines on A T P Formation in Isolated Spinach Chloroplasts G . V A N D E N B E R G , M . B R A N D S E , and J . T I P K E R 651

A Cyanobacterial ATPase Distinct from the Cou-pling Factor of Photophosphorylation W . L O C K A U and S. P F E F F E R 658

The Anatomy of the Sarcoplasmic Reticulum in Vertebrate Skeletal Muscle: Its Implications for Excitation Contraction Coupling J . R. S O M M E R , M . D . 665

Sodium-Potassium Movement and the Regulation of Cardiac Muscle Activi ty J . D A U T and R. R U D E L 679

Regulation of the Sarcoplasmic Reticular C a 2 +

Transport ATPase by Phosphorylation and Dephosphorylation L . M . G . H E I L M E Y E R , Jr . and M . V A R S A N Y I 682

Equilibrium and Kinetic Studies of Calcium Trans-port and ATPase Activi ty in Sarcoplasmic Re-ticulum G . I N E S I , M . K U R Z M A C K , D . K O S K - K O S I C K A , D . L E W I S , H . S C O F A N O , and H . G U I M A R A E S -M O T T A 685

Comparative Studies on the ATP-Bindung Sites in Ca2+-ATPase and (Na+ + K+)-ATPase by the Use of ATP-Analogues W . S C H O N E R , E . H . S E R P E R S U , H . P A U L S , R . P A T Z E L T - W E N C Z L E R , H . K R E I C K M A N N , and G . R E M P E T E R S 692

Electromechanical Coupling I. Introduction H . C H . L Ü T T G A U 706

Electromechanical Coupling I L The Effect of Per-chlorate Upon Excitation-Contraction Coupling in Frog Skeletal Muscle Fibres M . G O M O L L A , G. G O T T S C H A L K , and H . - C H . L Ü T T -G A U 707

Electromechanical Coupling III . Estimation of the Ca Storage Capacity of the SR by Analysing the Time Course of Caffeine-Induced Tension Tran-sients of Skinned Muscle Fibres R . T H I E L E C Z E K 709

Intracellular Membranes as Boundaries for Ionic Distribution. I n S i t u Elemental Distribution in Guinea Pig Heart Muscle in Different Defined Electro-Mechanical Coupling States M . F . W E N D T - G A L L I T E L L I , R . J A C O B , and H . W O L B U R G 712

Notes

New Flavonol Acetates from the Frond Exudate of the Fern N o i h o l a e n a a s c h e n b o r n i a n a M . J A Y , M.-R. V I R I C E L , J . F A V R E - B O N V I N , B . V O I R I N , and E . W O L L E N W E B E R 721

Contents

Isolation and Identification of Lathycarpin, a New Pterocarpan Phytoalexin from L a t h y r u s sativus J . L . I N G H A M and K . R . M A R K H A M 724

Irregularities in the Circular Dichroism of Oligo-ribonucleotides W . Z A C H A R I A S and H . F O L L M A N N 727

Adult T-Cell Leukemia-Associated Antigen ( A T L A ) : Detection of a Glycoprotein in Cell- and Virus-Free Supernatant N . Y A M A M O T O , J . S C H N E I D E R , Y . H I N U M A , and G. H T J N S M A N N 731

Conten t s of N u m b e r 9


Nonlinear Polarisationoscillations in a Biophysical Model. System I I : External Dynamics Z . S Z A B O and F . K A I S E R 733

The Phytochemistry of South American N e p h r o m a -Species (In German) B . R E N N E R , A . H E N S S E N , and E . G E R S T N E R 739

Detection of Glyceollin on the Cellular Level in In-fected Soybean by Laser Microprobe Mass Analysis P. M O E S T A , U . S E Y D E L , B . L I N D N E R , and H . G R I S E B A C H 748

Oxygen-Isotope Effect in Enzymatic Cleavage Re-action of 13-L-Hydroperoxylinoleic Acid to Hexa-nal and ll-Formyl-d.s-9-undecenoic Acid A . H A T A N A K A , T. K A J I W A R A , J . S E K I Y A , and T. F U K U M O T O 752

On the Biosynthesis of C30 Carotenoic Acid Gluco-syl Esters in Pseudomonas r h o d o s . Analysis of c a r -Mutants H . K L E I N I G and R. S C H M I T T 758

Biosynthesis of Photosynthates and Taxonomy of Algae B . P. K R E M E R and G. 0 . K I R S T 761

Specific Localization of ß-D-Glucoside Conjugates of 2,4-Dichlorophenoxyacetic Acid in Soybean Vacuoles R . S C H M I T T and H . S A N D E R M A N N , Jr . 772

IX

Synthesis of Galloyl-Coenzyme A Thioester G. G. GROSS 778

Nitrite Inhibition of Bacterial Dinitrogen Fixation F . C A S T L L L O and J . C A R D E N A S 784

Energy Transfer Inhibition Induced by Nitrofen B . H U C H Z E R M E Y E R 787

Natural Inhibitors of Germination and Growth, I. Development of a Quantitative Biotest and Ap-plication upon Extracts from Husks of Avena s a t i v a L . (In German) R. K A R L and W . R Ü D I G E R 793

Natural Inhibitors of Germination and Growth I I . Isolation and Structure of Inhibitors from Avena s a t i v a L . (In German) E . L O H A U S , I. B L O S , W . S C H Ä F E R , and W . R Ü D I G E R 802

5-Methylcytosine Content in Erythroleucemic Cells of Rats after Induction with Dimethylsulfoxid (In German) R . B A U R , N . K L U G E , H . K R Ö G E R , and H . W O H L E R T 812

Catabolic Properties of 5',5"-Linked Dinucleoside-phosphates in Rat Liver Nuclei (In German) S. B O R N E M A N N and E . S C H L I M M E 818

The Interaction of Calf Thymus D N A with Mercuric Acetate and 3,6-Bis-(acetatomercurimethyl)-dioxane. Small-Angle X - R a y Scattering and Viscosity Studies P. Z I P P E R , G. R I B I T S C H , J . S C H U R Z , and H . B Ü N E M A N N 824

Growth Characteristics of Anaerobically Treated Ehrlich Ascites Tumor Cells after Reaeration as Studied by Combination of Flow Cytometry and Centrifugal Elutriation R. M E R Z and F . S C H N E I D E R 833

Inverse Relationships between Ecdysteroid Titres and Total Body Metabolism in Insects K . S L A M A 839

Notes

Yeast Aminopeptidase I I : Rapid Purification Using Affinity Chromatography of the Periplasmic En-zyme J . K N Ü V E R and K . - H . R Ö H M 845

X Contents

Conten t s of N u m b e r 10


On the Biogenesis of Aroma Compounds in Plants and Fruits, X V I I . Anabolie Properties of Straw-berry Fruit Tissue for the Biosynthesis of Aroma Compounds (In German) F . D R A W E R T and R. G . B E R G E R 849

Indole Alkaloids from Cell Suspension Cultures of Stemmadenia t o m e n t o s a and V o a c a n g a a f r i c a n a J . STÖCKIGT, K . - H . P A W E L K A , A . R O T H E R , and B . D E U S 857

Degradation of the Isoflavone Biochanin A by F u s a r i u m j a v a n i c u m U . W I L L E K E and W . B A R Z 861

Changes of C-Phycocyanin in Synechococcus 6301 in Relation to Growth on various Sulfur Compounds A . S C H M I D T , I. E R D L E , and H. -P . K Ö S T 870

State of Iron in the Archaebacterium M e t h a n o s a r -c i n a b a r k e r i Grown on Different Carbon Sources as Studied by Mössbauer Spectroscopy P. S C H E R E R and C H . SATTER 877

Effect of Aging on the Fluorescence Lifetime of Chloroplasts S. S. B R O D Y 881

On the Role of Hydrogen Peroxide in Peroxidase Catalyzed Metabolism of Indole-3-acetic Acid H . J . G R A M B O W 884

Distribution and Effects of Bentazon in Crop Plants and Weeds H . K . L I C H T E N T H A L E R , D . M E I E R , G . R E T Z L A F F , and R. H A M M 889

Mechanism of Bleaching in Leaves Treated with Chlorosis-Inducing Herbicides J . F E I E R A B E N D , T H . W I N K E L H Ü S E N E R , P . K E M M E R I C H , and U . S C H U L Z 898

Rotating-Field-Induced Rotation and Measurement of the Membrane Capacitance of Single Mesophyll Cells of A v e n a s a t i v a W . M . A R N O L D and U . Z I M M E R M A N N 908

The Triton X-100 and High Salt Resistant Residue of Saccharomyces c e r e v i s i a e Nuclear Membranes K . M A N N and D . M E C K E 916

Malonate and Krebs Cycle Intermediates Utilization in the Presence of other Carbon Sources by B h i z o -b i u m j a p o n i c u m and Soybean Bacteroids D . W E R N E R , W . D I T T R I C H , and H . T H I E R -F E L D E R 921

Alanine Dehydrogenase from Bacteroids and Free Living Cells of B h i z o b i u m j a p o n i c u m P. M Ü L L E R and D . W E R N E R 927

The Tautomerism of Cytosine and Hydroxycyto-sine. A Quantum-Mechanical Study J . S. K W I A T K O W S K I , B . L E S Y N G , M . H . P A L M E R , and W. S A E N G E R 937

The Reversibility of the Vitamin C Redox System: Electrochemical Reasons and Biological Aspects H . S A P P E R , S.-O. K A N G , H . - H . P A U L , and W. L O H M A N N 942

Coupled Gas Chromatography — Single Cell Re-cording: a New Technique for Use in the Analysis of Insect Pheromones L . J . W A D H A M S 947

Evaluation of (Z)-5-Decen-l~ol as an Attractant for Male Larch Casebearer Moths, C o l e o p h o r a l a r i c e l l a E . P R I E S N E R , W . A L T E N K I R C H , W. B A L T E N S -W E I L E R , and H . B O G E N S C H Ü T Z 953

A Steroid-Binding Protein from Insect Haemolymph Isolated by Photoaffinity Labelling and Immuno-adsorption L . R E U M , G. K Ä U S E R , U . E N D E R L E , and J . K o O L M A N N 967

Structure Investigations of Agonists of the Natural Neurotransmitter Acetylcholine, I I I [1]. X - R a y Structure Analysis of (2-Ethoxyethyl)trimethyl-ammonium Chloride A . G I E R E N and M . K O K K I N I D I S 977

Polycation-Bacterium Interactions and Wal l Sub-units as Endocytosis Factors. Topoisomeraselike Action of Basic Polypeptides Suggesting a 7th Class of Enzymes: The Stereases S. A N T O H I 985

Tl + -Ions: Influence on Cardiac Contractility R. Z I S K O V E N , C. A C H E N B A C H , J . W I E M E R , and U . W I N T E R 995

Contents

Tl+-Ions: Effects on the Automaticity of Sinoatrial Tissue and on d F / d £ m a x and ^K2 of Cardiac Pur-kinje Fibres C. A C H E N B A C H , J . W I E M E R , R . Z I S K O V E N , and U . W I N T E R 1006

Tl + -Ions: Comparison of the Effects on the Slow Inward Current and Contractility of Ventricular Tissues J . W I E M E R , R . Z I S K O V E N , and C . A C H E N -B A C H 1015

Differentiation in V o l v o x Carter'i: Study of Pattern Variation of Reproductive Cells R. G I L L E S and L . J A E N I C K E 1023

Thermodynamic Approach to a Possible Theory of the Evolution of a Genetic Code M . F . M A C C H I A T O and A . T R A M O N T A N O 1031

Notes

B-Decachloro-o-carborane Derivatives Suitable for the Preparation of Boron-Labeled Biological Macromolecules D. G A B E L and R. W A L C S Z Y N A 1038

Temperature-Dependent Colour Change in Larvae of the Green Spider M i c r o m a t a r o s e a (Sparassidae) (In German) A . H O L L 1040

"Memory" of First Interaction with Physiological or Biologically Active Foreign Molecules (Benz-pyrene, Gibberelline) in a Unicellular ( T e t r a -hymena) Model System G. C S A B A , G. N E M E T H , and P . V A R G H A 1042

On the Nerve Growth Factor (NGF) from Human Glandula submandibularis (In German) R . R I E M S C H N E I D E R , F . D E M B E L E , and I. A . K . G H O U R I 1045

Spatio-Temporal Visual Receptive Fields as Revealed by Spatio-Temporal Random Noise E . H I D A and K E N - I C H I N A K A 1048

On the Taxonomic Position of Hypocomidae (Ciliata) (In German) K . G. G R E L L and A . M E I S T E R 1050

XI



Composition of Epicuticular Waxes from Fruits of J o j o b a ( S i m m o n d s i a chinensis [Link] Schneider) P . - G . G Ü L Z 1053

Biliverdin I X a, Intermediate and E n d Product of Tetrapyrrole Biosynthesis H.-P . K Ö S T and E . B E N E D I K T 1057

The Oxidative Degradation of L-Ascorbic Acid via an a-Ketoaldehyde S.-O. K A N G , H . S A P P E R , and W . L O H M A N N 1064

Further Observations on the Source of Nitrogen in Phenazine Biosynthesis A . R Ö M E R and R . B . H E R B E R T 1070

Photosynthetic Adaptation in Synechococcus Cells G . D Ö H L E R and J.-C. L E C L E R C 1075

Interrelationship between Quinolizidine Alkaloid Producing Legumes and Infesting Insects: E x -ploitation of the Alkaloid-Containing Phloem Sap of C y t i s u s s c o p a r i u s by the Broom Aphid A p h i s c y t i s o r u m M . W I N K , T. H A R T M A N N , L . W I T T E and J . R H E I N H E I M E R 1081

Biological Activities of Sesquiterpene Lactones from H e l i a n i h u s annuus: Antimicrobial and Cytotoxic Properties; Influence on D N A , R N A , and Protein Synthesis O. S P R I N G , J . K T J P K A , B . M A I E R , and A . H A G E R 1087

Bleaching Activity of New 2-Phenylpyridazinones: Structure-Activity Relationship G . S A N D M A N N and P . B Ö G E R 1092

Some Observations on the Saponin Accumulation in Oat Seedlings and on the Transformation of the Avenacosides to the Antibiotic 26-Desgluco-avenacosides I L L A U D E N B A C H and J . K E S S E L M E I E R 1095

Naramycin B , an Antibiotic from Streptomyces griseus Strain 587 with Herbicidal Properties-Fermentation, Isolation, and Identification D . B E R G , M . S C H E D E L , R . R . S C H M I D T K . D I T G E N S and H . W E Y L A N D 1100

Contents XII

The Intracellular Distribution of Enzymes of the Glycerol Cycle in the Unicellular Alga D u n a l i e l l a p a r v a H . G I M M L E R and G. L O T T E R 1107

Osmoregulation in D u n a l i e l l a . Intracellular Distri-bution of Enzymes of Glycerol Metabolism A . D . B R O W N , R . McC. L I L L E Y , and T. M A R E N G O 1115

Multicomponent Mandibular Gland Secretions in Three Species of Andrena Bees (Hym., Apoidea) G. B E R G S T R Ö M , J . T E N G Ö , W . R E I T H , and W . F R A N C K E 1124

The Sex Pheromone of the Silver Y Moth C h r y s o -d e i x i s e r i o s o m a (Doubleday) in New Zealand M . H . B E N N , R . A . G A L B R E A T H , V . A . H O L T , H . Y O U N G , G. D O W N , and E . P R I E S N E R 1130

Structure-Activity-Relation of Analogues and Ho-mologues of Blepharismone, the Low-Molecular Conjugation Hormone of B l e p h a r i s m a j a p o n i c u m (In German) M . E N T Z E R O T H and L . J A E N I C K E 1136

Conformational Analysis of Benzyloxycarbonyl-Protected Peptides I. K N A C K 1141

Immunochemical Characterization of P o r p h y r i d i u m c r u e n t u m B-Phycoerythrin: Proof of Cross-Reac-tion between Chromophore-Free Apoprotein and Holoprotein-Specific Antibodies E . S E P P , G. W A N N E R , J . E D E R , and H. -P . K Ö S T 1146

X - R a y Studies on Phospholipid Bilayers. II . Poly-morphie Forms of Dipalmitoyl Phosphatidyl-ethanolamine M . S U W A L S K Y and E . K N I G H T 1157

Statistical Description of Isotope Exchange Pro-cesses: A Multisite Model for the 1 8 0 Exchange P. R Ö S C H 1161

Fluorescence Studies on N-(3-Pyrene)Maleinimide-Labeled Sarcoplasmic Reticulum ATPase in Na-tive and Solubilized Membranes H . L Ü D I and W . H A S S E L B A C H 1170

Localization of C a 2 + at the Plasma Membrane of Bullfrog Myocardial Cells R. Meyer, M . SCHMITZ, W . S T O C K E M , and H . G. H A A S 1180

Release of K + and H + from Poly U in Aqueous Solution upon y and Electron Irradiation. Rate of Strand Break Formation in Poly U E . B O T H E and D . S C H U L T E - F R O H L I N D E 1191

Model Reactions for the Degradation of D N A - 4 ' Radicals in Aqueous Solution. Fast Hydrolysis of a Alkoxyalkyl Radicals with a Leaving Group in ß-Position Foliowed by Radical Rearrange-ment and Elimination Reactions G. B E H R E N S , G. K O L T Z E N B T X R G , and D . S C H U L T E - F R O H L I N D E 1205

Induction of Single- and Double-Strand Breaks in Linear and Superhelical D N A by Phleomycin (In German) F . S C H M Ü L L I N G and W. K Ö H N L E I N 1228

Effect of Water Soluble Polymer, Polyethylene-glycol, and Glass-Forming Compounds on Cell Fusion F . Y O S H I I and I. K A E T S U 1234

System Analysis of the Circadian Rhythm o i E u g l e n a g r a c i l i s , I. Linearities and Non-Linearities in the Response to Temperature Signals W . L O R K , T. K R E U E L S , W. M A R T I N , and K . B R I N K M A N N 1240

Exogenous and Endogenous Control of Swimming Activi ty in A s t y a n a x mexicanus (Characidae, Pisces) by Direct Light Response and by a Circadian Oscillator. I. Analyses of the Time-Control Systems of an Epigean River Population W. E R C K E N S and W. M A R T I N 1253

Exogenous and Endogenous Control of Swimming Activi ty in A s t y a n a x mexicanus (Characidae, Pisces) by Direct Light Response and by a Circadian Oscillator. II . Features of Time-Con trolled Behaviour of a Cave Population and their Comparison to a Epigean Ancestral Form W. E R C K E N S and W . M A R T I N 1266

Functional Regeneration of the Visual System in Teleosts. Comparative Investigations after Optic Nerve Crush and Damage of the Retina R. K Ä S T N E R and H . W O L B U R G 1274

Notes

Monoterpenes from the True Bug H a r p o c e r a t h o r a -c i c a (Hemiptera) H . -P. H A N S S E N and J . J A C O B 1281

Contents

Diterpenes of C h e i l a n t h e s a r g e n t e a , a Fern from Asia E . W O L L E N W E B E R , P . RTTEDI,

and D . S. S E I G L E R 1283

Composition of Phospholipids in Seed Oil of J o j o b a ( S i m m o n d s i a c h i n e n s i s [Link], Schneider) P. G . G Ü L Z and C. E I C H 1286

Adenine Phosphoribosyltransferase Activity in Mitochondria of C a t h a r a n t h u s roseus Cells F . H I R O S E and E L A S H I H A R A 1288

Inactivation of the Sarcoplasmic Reticulum Cal-cium-Transport-ATPase by Lasolocid in Com-bination with Triton X-100 W . H A S S E L B A C H , H . L Ü D I , and A . M I G A L A 1290

Small- and Large-Angle X - R a y Scattering Studies of Counter Ion Influence on t R N A Conformation P. W I L H E L M , I. P I L Z , G . D E G O V I C S , and F . V O N D E R H A A R 1293

XIII

Isolation and Synthesis of Hemoregulatory Peptide W . R . Paukovits and 0 . D . Laerum 1297

Erratum

To N . Yamamoto, J . Schneider, Y . Hinuma, and G . Hunsmann (37c, 731-732 [1982]) 1300

Subject Index 1301

Authors Index 1327

Errata in this volume

To A . Hager and M . Heimle (36 c, 997 [1981]) 144

To J . Kesselmeier and D . Strack (36c, 1072 [1981]) 350

To N . Yamamoto, J . Schneider, Y . Hinuma, and G . Hunsmann (37c, 731 [1982]) 1300

Rubins and Rubinoid Addition Products from Phycocyanin * W. Kufer and H . Scheer

Botanisches Insitut der Universität München, Menzinger Straße 67, D-8000 München 19

Z. Naturforsch. 37c, 179-192 (1982); received December 15,1981

Phycocyanin, Phycorubin, Bile Pigments, Biliverdin, Bilirubin The verdin-type chromophore of denatured C-phycocyanin (1) from Spirulina platensis is

reduced to the corresponding rubin (2 a) by sodium borohydride. The structure assigned is in agreement with the uv-vis spectroscopic properties of the product and was deduced from model studies with free bile pigments.

Analogous model studies using sodium dithionite demonstrated a two-fold reactivity for this reagent, leading to products which are both of the rubin spectral type under the conditions tested. True rubins (10,22-dihydrobilindions) are formed in low yield only if an excess of reagent is used in methanol/water mixtures. It is accompanied by polar addition product(s) of the same spectral type, which are generally formed exclusively. In particular, no bili rubin was formed under the reaction conditions previously applied for the chemical modification of phycobiliproteins and phytochrome. From this finding and from the strikingly different properties of the borohydride and dithionite products, of phycocyanin upon renaturation, the dithionite product is suggested to be a rubinoid addition product (2 b) rather than a hydrogenation product.

In contrast to the dithionite addition product 2 b of phycocyanin, the chromophore of the true phycorubin (2 a) remains stable upon renaturation. The uv-vis spectral properties of the chromophore are not markedly different whether the apoprotein is in its native or denatured State. The different electrophoretic mobilities of native (renatured) phycocyanin compared to the renatured borohydride product suggest that these two have different protein conformations.

The preparation of these phycorubins renders the extensive techniques of bilirubin chemistry applicable in the study of biliproteins.

Introduction

Bile pigments, open-chain tetrapyrrolic Com-pounds, serve important functions in plants. In contrast to mammalian bile pigments derived from heme degradation, they are biosynthesized as pho-toreceptors which are covalently bound to apopro-teins, thus forming "biliproteins" (for a recent review, see [1]). Two functionally different groups can be distinguished: Firstly the phycobiliproteins as light harvesting pigments of photosynthesis in cyanobacteria, red and cryptophytan algae, and secondly phytochrome and the phycochromes as photomorphogenetic receptor pigments of higher plants and some algae.

The physico-chemical properties of the bile pig-ment chromophores are profoundly influenced by the native proteins. This is mainly due to non-covalent protein chromophore interactions which are essential in biliproteins to optimize the proper-

Abbreviations: PC, phycocyanin; tlc, thin layer chromato-graphy; mob, electrophoretic mobility relative to Standard. * Studies on Plant Bile Pigments 12; part 11 of this series: W. Kufer and H. Scheer, Angew. Chem., in press. Reprint requests to Prof. Dr. H. Scheer. 0341-0382/82/0300-0179 $01.30/0

2a R = H 2c R = SO; 2b R = SOr 2d R = S - C H 2 - CH 2 OH

180 W. Kufer and H. Scheer Rubins and Rubinoid Addition Products from Phycocyanin

ties of the chromophores in their function as photo-receptors. This can be easily seen from a com-parison of native and (reversibly) denatured pig-ments (see ref. [1] for a survey). Recently, we devel-oped a technique for the selective chemical modifi-cation of biliprotein chromophores [2, 3]. The ex-periments reported here have been performed to yield more detailed information about the structure of the chemically modified products, and the non-covalent protein chromophore interactions in bili-proteins. The previous experiments of this type were carried out with the reducing agents dithionite, sulfite and 2-mercaptoethanol, and pigments of the rubinoid spectral type were obtained [3]. Here, we wish to report the reaction of C-phycocyanin (1) (C-PQ and some free bilins as model Compounds with sodium borohydride. This reagent has been used [4, 5] for selective reduction of bilindiones ("biliverdin" type chromophores like (3)) to 10,22-dihydrobilindiones ("bilirubin" type chromophores like (4))*.

Results

R e a c t i o n of p h y c o c y a n i n w i t h s o d i u m b o r o h y d r i d e

The reactions are summarized in Scheme 1. When PC (concentration ränge 30-130 U M chromo-phores**) denatured by 8 M urea in 50 mM sodium phosphate buffer, p H 7.5, was treated at 0 °C with solid sodium borohydride (25-40 mM***), the bands in the uv-vis spectrum at 602 nm ( e = 15400 per chromophore) and 355 nm ( e = 37000) [31 dis-appeared completely after 20-30 min and a new band at 416 nm arose simultaneously. The p H of the Solution increased to about 9, which could be avoided without significant changes in reactivity by using a buffered Solution of sodium borohydride. Using PC (48 U M chromophores) and 13 mM N a B H 4 , a 90% decrease of the 602 nm band was observed within 30 min. The reaction went to completion

within additional 30 min after a further addition of the same amount of borohydride (Fig. 1)*. Assum-ing a 1:1 stoichiometry with no by-product formed, £ 4 1 6 = 24200 (per chromophore) was calculated from the spectral changes, and with £ 6 0 2=15400 [3] for the educt chromophores**. The vis-spectrum remained unaltered when the reducing agent was removed by gel Filtration on a column equilibrated with 8 M urea. The yellow colour of the borohydride product also remained unchanged after simultanous removal of urea and borohydride by gel filtration (/.max = 418 nm). The extinction coefficient of the pigment thus obtained was determined by unfolding the protein again, by addition of solid urea to a final concentration of 8 M . With £ = 24200 as reference value for the denatured pigment (see above) and allowing for the dilution caused by addition of urea, e418 - 21900 was obtained for "native"phycorubin.

The electrophoretic mobility of the product ob-tained after removal of urea and borohydride dif-fered from that of native PC in tris-glycine electro-phoresis. With equal protein concentrations applied, the yellow product had a higher mobility (mob = 0.72 vs. bromophenolblue as reference Compound) as compared to PC (mob = 0.59) (Fig. 2). They were identical, however, in SDS-gel electrophoresis hav-ing a Single band corresponding to M W = 19900 in coelectrophoresis using the System of Weber and Osborn [6] (a Separation into subunits was generally not obtained with PC from S p i r u l i n a p l a t e n s i s ) . Reaction of the yellow pigments with diazotized ethyl anthranilate (5-10 fold molar excess) yielded uv-vis spectroscopically identical pigments with a long wavelength band at 485 nm, irrespective of the removal of only N a B H 4 or both N a B H 4 and urea before the reaction.

Addition of solid sodium borohydride (43 mM) to native PC (6.6 U M chromophores, calculated from £ 6 2 0=98700/chromophore [3]) led to only partial con-version to the yellow pigment. After 10 min reaction

* See ref. [43] for the nomenclature of bile pigments, and formulas for the numbering System. In addition, the non-systematic terms "rubin" and "verdin" are used to characterize pigments bearing the conjugation Systems of bilirubin and biliverdin, respectivley.

** Concentrations throughout are given for chromo-phores. Since monomeric (

W. Kufer and H. Scheer • Rubins and Rubinoid Addition Products from Phycocyanin • ca. U OmM Na B H 6

P C n a t i v e • "Phycocyanorub»n M n a t i v e

181

(A)

\ « « « = 620nm A. mox 3 type A chromophores

\ =620, 418 nm * max ' type A • type D chromophores

• 8M urea

' Phycorubin" r e n a t u r e d

Xmax = i , 8 n m

3type D chromophores

PC denatured • 2U mM NaBH, "Phycorubin" denatured Xmax = 6 0 2 n m

3type B chromophores

• 5-1fJ- 3M Dithionite

= 418 nm

PC native + \ = 620 nm ^max 3type A chromophores

-D i th ion i te

Amax 3 type D chromophores

"Phycocyanosulfinat"nat jve \ = 620, 418nm

type A +type C chromophores

(B)

• 8M urea

-u rea ; 0 or 5 -10"* M Dithionite

+ 8 M urea

5-10' 3 M Dithionite

-urea j

5-lO" 3 M Dithionite

PC • 5-10~4 M Dithionite

denatured ' P C - Sulfinat'

- Dithionite denatured v max 3 type B chromophores

: L 18nm ^max 3 type C chromophores

Type A TypeB TypeC: R = SO; TypeD: R = H

Scheme 1. Reaction scheme of the reversible renaturation of PC from Spirulina platensis, and of the reactions with sodium borohydride (A) and sodium dithionite (B). The chromophore structures A - D are schematic representations of the chro-mophore geometry. Structure A is representative of an extended conformation without major steric hindrance of the /?-pyr-rolic substituents. Structure B is the cyclic-helical conformation found for biliverdin in Solution [22, 23]. Structures C and D have been drawn with the two dipyrromethenone units nearly perpendicular to each other, similar to the crystal structu-re of bilirubin [46, 47]. The reactions of PC with sodium sulfite and 2-mercaptoethanol principally follow Scheme 1B as well but different concentrations of the reagents are needed.

194 H. Hüdig and G. Drews • Isolation of a 6-Type Cytochrome Oxidase from Rps. capsulata

P u r i f i c a t i o n of c y t o c h r o m e o x i d a s e

A l l Operations were performed at 4 °C unless stat-ed otherwise. The membrane fraction was re-suspended in TRIPE buffer at a concentration of about 6 mg protein per ml. Triton X-100 (10% stock Solution) in TRIPE buffer was added dropweise to the Suspension under stirring to a final concentration of 1.3% (w/v, protein to detergent ratio of 1 : 2). Af-ter incubation for 30 min insoluble material was pre-cipitated at 145,000 xg , 90 min, 4 °C. The super-natant contained about 90% of the cytochrome oxi-dase. The enzyme extract was diluted with TRIPE buffer to a final concentration of 1% Triton X-100.

10 ml of the supernatant (4 mg protein/ml) were applied to a column (1x8 cm) of D E A E Sepharose CL-6B (Pharmacia Freiburg) equilibrated with TRIPE buffer plus 1% Triton. The column was wash-ed with 30 ml of TRIPE buffer plus 1% Triton and eluted with a linear gradient of 0 - 1 M K C l (2 x 40 ml). Fractions of 3 ml were collected and test-ed for cytochrome oxidase activity.

The peak fractions eluted at 0.3 M K C l were pool-ed and desalted as described in [10]. 9 ml of the de-salted enzyme Solution (5.6 mg protein) was then ap-plied to a cytochrome c-thiol-activated Sepharose 4B column (1x4 cm; Pharmacia) equilibrated with TRIPE buffer plus 1% Triton X-100. The coupling of cytochrome c from Saccharomyces c e r e v i s i a e (Sigma, München, type VIII) and the preparation of the cys-teine inactivated column were performed as describ-ed in [12]. The binding capacity of the column was 7.7 mg cytochrome c per ml of packed material. The column was washed with 20 ml of TRIPE buffer and then eluted with a linear gradient of 0-0.5 M K C L Solution (2 x 40 ml).

D e t e r m i n a t i o n of e n z y m a t i c a c t i v i t y

Cytochrome oxidase was assayed at 30 °C by fol-lowing the decrease of absorption at 546 mn in an Eppendorf photometer (type 1101 M) using dithion-ite reduced horse heart cytochrome c as electron donor (6 = 21 x 103 cmVmmol). The reaction mix-ture contained 50 mM TRIPE buffer (pH 8), 1 mg ferrocytochrome c and enzyme Solution in a final volume of 1 ml. The reaction was started by addition of 10 to 100 u] enzyme Solution. When inhibitors were used, the enzyme preparation was preincubated for 5 min with the inhibitor. The reaction was then started by addition of reduced cytochrome c. Horse

heart ferrocytochrome c was prepared as described in [10] with the modification that the p H of the fer-rocytochrome c Solution was adjusted to p H 8 with 1 MTris.

N a t i v e P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (charge- s h i f t - e l e c t r o p h o r e s i s )

Electrophoresis in 5% Polyacrylamide slab gels containing 0.1% Triton X-100 was carried out ac-cording to the method of Simons et a l [13] with the modification that D T T was added to the gel and the buffers in 1 mM concentration. The samples were preincubated in the sample buffer for 15 min at 4 °C. Electrophoresis was performed at 4 °C with 2 mA/gel track. After the run one track was cut into 2 mm slices. The slices were incubated over night with 200 ul of TRIPE buffer plus 0.1% Triton and tested afterwards for activity in the enzyme assay. The slices of the second track were extracted for 4 h with 50 ul of sample buffer (4% SDS) at 60 °C. The extracts were then subjected to SDS-gel electrophor-esis. The third track was stained with 0.04% Coo-massie brillant blue in 25% isopropanol and 10% acetic acid (v/v) and destained with 10% acetic acid and 10% methanol.

Sodium dodecylsulfate P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s

Slab-SDS-gel electrophoresis was performed ac-cording to the method of Laemmli [14] using 11.5-16.5% acrylamide gradient gels of 1 mm thick-ness. The samples were dialyzed for two days, lyo-philized and resuspended in sample buffer. A l l sam-ples were preincubated at 60 °C for 30 min or at 100 °C for 10 min, respectively.

D e t e r m i n a t i o n of p r o t e i n

Protein concentration was determined by the method of Lowry et a l . [15]. The presence of Triton X-100 was corrected by addition of 0.5% (w/v) sodi-um dodecylsulfate to the alkali Solution of the Lowry reagent [16]. Bovine serum albumin was used as Standard.

M e a s u r e m e n t of difference a b s o r p t i o n s p e c t r a

Difference spectra were obtained with a Perkin-Elmer split beam spectrophotometer model 330 (Überlingen) using 1 cm light-path cuvettes at room

H. Hüdig and G. Drews • Isolation of a 6-Type Cytochrome Oxidase from Rps. capsulata 195

Table I. Purification of cytochrome oxidase from Rhodopseudomonas capsulata strain 37 b4. The purification procedure and the enzyme assay are described under Materials and Methods.

Activity Protein Specific Purification Yield Activity Activity

[umol/min • ml] [mg/ml] [umol/min mg protj

(= fold) [%]

Membranes 2.3 6.15 0.37 1 100 Triton extract 3.4 3.9 0.87 2.4 148 DEAE-Sepharose eluate; 1.19 0.19 6.7 17 51 peak fraction Cytochrome c-thiol- 0.63 0.011 59.7 161 29 activated Sepharose eluate; peak fraction

temperature. 150 JIM potassium ferricyanide was added to the reference cuvette and 100 U M sodium dithionite to the sample. Before recording the spec-trum the cuvettes were kept for 2 min in the dark.

Results and Discussion

S o l u b i l i z a t i o n of c y t o c h r o m e c o x i d a s e

Ninety seven percent of the enzyme was solubi-lized at a Triton concentration of 1.3% and a protein to detergent ratio of 1 : 2. The activity of the enzyme was enhanced about 30-50%. Lowering the Tris concentration from 50 mM to 20 mM reduced the solubilization of the enzyme. A n increase of de-tergent concentration to more than 1.3% resulted in a loss of activity. Addition of K C l up to 1 M stimulated the activity about 10% (in contrast to NaCl) but had no effect on the solubilization of the cytochrome oxi-dase activity.

The detergent N,N-dimethyllaurylamineoxide (LDAO) solubilized the enzyme activity at a concen-tration of 1.3% nearly 100% but the half life of the activity dropped to 3 - 5 h. A mixture of L D A O and other detergents or increasing the ionic strength by addition of K C l or NaCl caused a complete loss of the activity. The detergents Brij-58, Tween 20, L u -brol-PX and SDS applied at 4 °C and at room tem-perature solubilized the enzyme only partially or re-sulted in inactivation of cytochrome oxidase. Sodi-um cholate solubilized cytochrome oxidase activity up to 90% but with a lower yield than Triton. In the presence of 1.3% Triton X-100 the cytochrome oxi-dase had a half-life of 24 h at 4 °C. Freezing of the solubilized enzyme even in the presence of 50% gly-cerol destroyed the activity. The crude Triton-extract lost approx. 20% of activity at 4 °C in 24 h.

P u r i f i c a t i o n

Triton-extract from membranes was applied to a DEAE-Sepharose C L 6B column. Enzyme activity eluted shortly after a dark red band at 300 mM K C l (Fig. 1). The specific activity was increased 17-fold (Table I).

The enzyme fraction which eluted from the DEAE-Sepharose column contained considerable amounts of cytochrome c besides cytochrome b. The desalted fraction was loaded on a cytochrome c-thiol activated Sepharose 4B column. The cytochrome c was covalently linked through its cysteine residue lo-cated close to the N-terminus [17] thus leaving free the important lysine residues for binding cy-tochrome c oxidase and reductase [12]. The enzyme was not bound to the column when cystein was inac-tivated.

A 2 9 0

FRACTION NUMBER

Fig. 1. Ion exchange chromatography on DEAE-Sepharose Cl-6B-column (1x8 cm; Pharmacia) in presence of 1% Tri-ton X-100 and Standard buffer (0.05 M Tris-HCl pH 8, 0.1 mM PMSF, 0.1 mM EDTA). • • Cytochrome oxidase-activity; - -O—O— A 2 9 0 ; KCl-gradient 0 -1 M KCl , 2 x 40 ml; fractions containing 3 ml.

184 W. Kufer and H. Scheer • Rubins and Rubinoid Addition Products from Phycocyanin

Table I (continued).

Compound and reagent Absorptions A m a x [nm]

A m a x after extraction in CHC13

tlc: ÄrvaluesxlOO Absorptions A m a x [nm]

A m a x after extraction in CHC13 a b c d e f g h

Mesobilirubin, 4 b

MixtureofIII,IXand XIII a-isomers of mesobilirubin (5b,4b,6b)

424,398 shj), 419m)

66-74

68 74 78

60-69

60

31

31

65

Octaethylbilindion, 7 7 + NaBH 4 !) 7 + 2-mercaptoethanoli,s)

646l) 400,420 sh 426

47 11

2,3-Dihydrooctaethyl-bilindion, 8 8 + NaBH,1) 8 +dithionitej) 8 + 2-mercaptoethanol*» s)

596 *)

400 398c) 410

65

05

The octaethylbilindion 7 and its 2,3-dihydro-deri-vate 8 similarily yielded yellow products upon treat-ment with solid borohydride in methanol. 7 reacted smoothly at ambient temperature, 8 only at elevated temperatures (50 °C, under nitrogen). The conjuga-tion System of 8 is identical to that of C-PC.

Using sodium dithionite instead of borohydride, the results depended critically on the reaction con-ditions. In an experiment in methanol/water (1:1, v/v) analogous to the one described above, a yellow product different from 4 a was obtained from 3 a with 1 mM dithionite. It migrated with an Äp-value of or close to zero on silica gel-tlc, developed with neutral or acidic solvent Systems (a, e, f in Table I). On polyamide-tlc with a basic solvent System, it mi-grated close to the front (g in Table I). No bilirubin (4 a) could be detected under these conditions. Upon rising the dithionite concentration to 24 mM, 4 a and its isomers (5, 6) could be identified as by-products besides the aformentioned polar product(s). The product mixture was analyzed quantitatively after reaction on the preparative scale (3a = 10~ 4M, di-thionite = 2 x 10~2 M). After work up, esterification with diazomethane and preparative tlc, 4 c was isolated in 4% yield and identified by uv-vis and tlc comparison with authentic bilirubin dimethylester (4 c). The results obtained with mesobiliverdin (3 b) were analogous (see Table I).

In another series of experiments, biliverdin was treated with dithionite in the absence of organic solvents. In particular, the reaction of 3 a (9 x 10~6) was studied in phosphate buffer (50 mM sodium phosphate, pH 7.5) containing urea (8 M ) , e. g . under

conditions identical with those used for the reaction of PC [3]. With dithionite concentrations ranging from 2xl0~ 4 to 2 X 1 0 " 2 M , no bilirubin could be identified in the yellow product mixtures. Chro-matography revealed only polar product(s) (RF = 0 in System a, as compared to 0.85 for 4 a). The same results were obtained using buffer without urea.

The conformationally restricted pigments phorca-bilin (9) and isophorcabilin (10) reacted smoothly, too, with dithionite (see Table II). Treatment of 9 (13 x 10 - 6 M) with sodium dithionite (5 x 10~5 M) in a buffer/methanol mixture led to decrease of the

Table II. UV-vis spectral data of the biliverdin 11, the ver-dins 9 and 10 with an extended conformation similar to the one suggested for the chromophores of native PC [25], and of their reaction products with reducing agents.

Compound A m a x [nm] in methanol

Phorcabilin-dimethylester, 9 555 562 b)

9+NaBH 4 417 9 + dithionite (0.05 mM) 412 b) Isophorcabilin-dimethylester, 10 606,690 (sh)

614b) 10+NaBH4 417 10 + dithionite (0.5 mM) 435,455 (sh),

405 (sh)b) Biliverdin IX y-dimethylester, 11 645 l l+NaBH 4 408 11 +dithionite5) c) a) See footnote (s), Table I. b) 50 mM sodium phosphate buffer, pH 7.5/methanol = 1:1 c) Not determined due to precipitation.

W. Kufer and H. Scheer • Rubins and Rubinoid Addition Products from Phycocyanin 185

60

Ü0

20

700 600 500 400

« X[nm] Fig. 3. Titration of isophorcabilindimethylester (10) (9.6X10~6M) in a 1:1 mixture of methanol and phosphate buffer (50 mM, pH 7.5) with sodium dithionite. The absorption spectra were recorded at the following concentrations of dithioni-te curve 1: 0 M, 2: 5xl0~6 M, 3: 5xl0~ 5 M, 4: 5xl0~ 4 M, and have been recorded each 5 min after the respective additions of the reagent.

long-wavelength band ( A m a x = 562 nm) by 95%, and the appearance of only one product band ( X M A X = 412 nm). Titration of 10 with dithionite (Fig. 3) led to 96% bleaching with 5 x 10~4 M dithionite, again forming a yellow product ( A m a x = 435, Shoulders at 435 and 405 nm).

In addition to the results described here, some earlier results [3] of the reaction of model Com-pounds for the PC chromophore with sodium sulfite and 2-mercaptoethanol have been incorporated in Table 1. They have already been published, to-gether with preliminary results obtained with sodium dithionite.

Discussion

The reaction product of denatured PC with sodium borohydride has an absorption maximum at 416 nm. Spectroscopically similar products were found with other reducing agents, viz. sodium dithionite, sodium sulfite and 2-mercaptoethanol [2, 3] (see also Scheme 1). This suggests for all these products an interruption of the conjugated System of the PC chromophore (la) at the C-10 position. For the rings C and D , this leads to the chromophoric System of mesobilirubin (4 c, / L m a x = 419 nm in phos-phate buffer containing 8 M urea). A l l four products have similar extinction coefficients: £ 4 1 6 = 24200 with borohydride, £ 4 1 8 = 25500 with dithionite [3], £ 4 1 8 = 27900 with sulfite and £ 4 1 8 = 23600 with 2-mercaptoethanol. They amount to roughly 50% of the value reported for mesobilirubin (4 c) (e4Z3 (CHC13) = 54600 [10]); thus supporting the general structure 2. For rings A and B, the interruption of

the conjugation at C-10 leads to a vinylpyrrole chromophore not absorbing in the visible ränge (e.g. 313 nm for a series of 3,4-dihydro-6(l H)-pyr-romethenons [8, 9] closely related to the ring A , B-fragment of 2 a). The borohydride product has an absorption in this spectral ränge ( A m a x = 310 nm) which appears as a Shoulder of the protein band (̂ max = 280 nm), the other products could not be investigated in this region due to strong absorption of the reagents.

Although the phycocyanin reaction products with these four reductants are uv-vis spectroscopically nearly identical, they differ in their structure con-cerning the residue R in formula 2. From uv-vis and tlc-data of the reaction products of model Com-pounds with sulfite, and from the acid reversible product formation with 2-mercaptoethanol, the structures 2 c and 2d were suggested [3] for these pigments arising from addition of the respective reagents. The pronounced reactivity of C-10 in bilindiones towards nucleophiles had been inferred first from M O calculations [12]. After circumstantial evidence [3, 13 a, b], NMR-data of adducts of thioles with bilindiones in agreement with the proposed structure 2d have been reported [14, 15], and the thermodynamic data for related equilibria have been obtained [15].

Reaction of the same model Compounds with sodium borohydride led via addition of hydride at C-10 to true reduction products, e.g. bilirubins, as revealed by comparison of their uv-vis and tlc-data with those of the authentic corresponding rubins (see Table I). Reduction of verdins to rubins with borohydride in methanol has been reported in the

186 W. Kufer and H. Scheer • Rubins and Rubinoid Addition Products from Phycocyanin

R

3a R = C 2 H 3 R ' = H R = R'= 5a R = C 2 H 3 3b R = C 2 H 5 R '=H 4a C 2 H 3 H 5b R = C 2 H 5 3c R = C 2 H 3 R ' = C H 3 4b C 2 H 5 H

4 c C 2 H 3 C H 3

C O O C H , C O O C H j

11


literature [4, 5]. The appearance of III- and XIII- a isomers 5 and 6 besides bilirubin (4 a) and mesobili-rubin (4 c) in methanol/water can be explained by the isomerisation ("scrambling") reaction, which has been observed with bilirubin in moderately basic aqueous Solutions [11]. Structure 2 a was thus assigned to the reaction product of phycocyanin with borohydride, and since it is a true hydrogena-tion product is should be named "phycorubin".

Conflicting results have been reported for the reaction of verdins with dithionite. Bilirubin has been isolated in low yield from the reaction of sodium dithionite with biliverdin, in aqueous al-caline Solution [16], and the reduction of a bil i-verdin-iodine complex to bilirubin has been re-ported [17]. On the other hand, a product different from bilirubin has been identified as well [18]. The model studies reported here support this differential reactivity of bilindiones towards dithionite. In the neutral pH-range studied, rubins have only been identified in low yield, and only in Solutions con-taining methanol. In particular, rubins have in no case been observed under the conditions used for the reactions of PC with sodium dithionite. A product with similar Chromatographie mobilities as the sulfite product (see Table I) arose instead. Deri-vatives of nicotinamide are reduced by dithionite in a complex mechanism. Addition of sulfinate, the homolytic fission product of dithionite, has been suggested as the first step, followed by a slower and pH-dependent elimination and a final true reduc-tion step with HSOj rather than S 207 as the reagent proper for hydride transfer [19]. Whereas this se-quence eventually leads to reduction of nicotin-amide in good yields, this is apparently not the case with bilindiones, possibly due to the increased stability of the first addition products *. Even pro-longed treatment of the product at p H 2.7 during workup did not result in bilirubin formation. Struc-ture 2 b is thus suggested for the dithionite reaction products of PC and A P C , and analogous structures

* Caughey and Schellenberg [20] have identified sul-finate addition products of nicotinamides by JHmr spec-troscopy. Attempts to identify the corresponding product of biliverdin failed. 3 c in C 2HC1 3 (0.02 M) was treated with an aqueous Solution of sodium dithionite, and the yellow Solution formed was thoroughly washed with water and dried. The Solution exhibited only broad Signals. When the reaction was carried out with sodium borohydride instead, the spectrum of the product was identical to that of bilirubin dimethylester (4 c) (see experimental part).

for the reaction products with other biliproteins, e.g. phycoerythrin, and phytochrome P r . They had earlier been tentatively identified as true reduction products [2, 3], which has to be corrected in view of the comparison with the rubins proper obtained with borohydride. Thus, the stability of phycorubin (2 a) upon renaturation (removal of urea e. g . by gel filtration), was strikingly different as compared to the products obtained with sulfite (2 c), 2-mercapto-ethanol (2d) and dithionite (2 b) [3] see Scheme 1. The yellow colour of the borohydride product remained unchanged, while the products with sul-fite, 2-mercaptoethanol and dithionite were con-verted back to native PC.

The reactions summarized in Scheme 1B, can read-ily be explained by a reversible addition to the central methine bridge, with the equilibrium strong-ly dependent on the State of the protein. In the denatured State with the chromophores still bound but uncoupled from the protein, the addition-elimi-nation equilibrium is shifted to the addition pro-duct, whereas the eduet is favored in the native or renatured pigments even in the presence of a large excess of dithionite. Also, the equilibrium is dif-ferentially affected by the different environments of the chromophores of identical molecular structure (e.g. three in PC). Reversible addition reactions are thus a useful tool to probe these different environ-ments [3].

The different stabilities of the native and dena-tured pigments may originate from a different con-formational freedom of the chromophores in the two states. Biliverdin dimethylester (3 b) and related bilindions are rather flexible molecules, which exist in Solution in several rapidly interconverting forms [21] of predominantly cyclic conformation [22, 23]. The nearly identical spectroscopic properties of denatured biliproteins make a similar mobility like-ly for their chromophores. The comparably narrow absorption bands of native biliproteins, as well as their high fluorescence yield indicate that the chro-mophores are rigidly fixed in (an extended [24, 25]) conformation. The nucleophilic addition results in the conversion from an sp2 to sp3 hybridised C-10, and thus necessarily a conformational change of the chromophores. A conformational fixation could then shift the equilibrium between the biliprotein chromophore proper, and its addition product. We have attempted to test this hypothesis by studying the reaction of phorcabilin-dimethylester (9) and


isophorcabilin dimethylester (10), bilins held in an extended conformation by additional bridges be-tween the pyrrole rings. Unfortunately, however, the central seven membered ring is still flexible enough to allow both sp2- and sp3-hybridization of C-10 without steric restrictions. Thus, 9 and 10 react with both dithionite and borohydride as well as does biliverdin-IX y-dimethylester 11 (see Table II).

The slow formation of denatured PC from the dithionite product [3] after removal of the reducing agent (gel filtration, see Scheme la) is then ex-plained by a shift in the equilibrium to the side of phycocyanin.

According to the structure of the phycorubins obtained with N a B H 4 as true reduction products, their properties are quite different. The major dif-ference between the addition of nucleophiles to and the hydrogenation of biliverdin is, that the former reaction is in thermodynamic equilibrium, whereas the latter h essentially irreversible under the reac-tion conditions. Biliverdin can be reduced to bi l i -rubin only with rather strong reductants (e.g. NaBH 4 ) , whereas the oxidation of bilirubin to biliverdin requires high-potential quinones* [4, 14]. Recent electrochemical studies have shown, too, that the biliverdin-bilirubin redox couple is not an equilibrium System [26]. The striking difference between phycorubin and the addition products of phycocyanin summarized in Scheme 1 can readily be explained on this basis. The yellow phycorubin chromophore remains stable upon renaturation of the protein and removal of the reducing agent, sodium borohydride. Moreover, the absorption maximum remained roughly in the same position and the extinction coefficient was the same within experimental error. The geometry of the phyco-cyanin chromophores is profoundly changed when the protein is renatured and v i c e versa, which is reflected by pronounced spectral changes [25]. The absence of similar effects in phycorubin could then indicate, that the altered chromophores do no longer fit the binding sites, and remain — although co-valently attached — only loosely coupled to the protein. It should be noted, though, that the uv-vis-

* We have achieved the reoxidation of phycorubin with benzoquinone to products identical with PC if judged from absorption, fluorescence and electrophoretical mobilities. Details of this procedure and the reaction of PC with quinones [27] are to be published separately.

spectra of bilirubins [28] appear less conformation dependent than those of biliverdins [29].

The different binding Situation of the chromo-phores in native phycocyanin and phycorubin can also be the reason for their different electrophoretic mobilities (Fig. 2). Phycorubin migrates faster to-wards the anode, which could be due to an increase of the net negative charge by the free carboxylic acid side chain(s) at or close to the surface. The ready reaction of renatured phycorubin with diazot-ized ethyl anthranilate leading to a product which cannot be distinguished uv-vis spectroscopically (Amax = 485 nm) from the denatured form, is compat-ible with this proposal. In SDS-gel electrophoresis, PC and phycorubin migrated identically, indicating the absence of proteolytic artefacts during the prepa-ration of the latter pigment.

In view of the irreversible reactions of N a B H 4 , the treatment of native PC may be used to discriminate between thermodynamic and kinetic effects in the reactions of biliprotein chromophores. In first order, one may assume that (i) the chromophores react with N a B H 4 i f only their methine bridges are acces-sible to the reagent, and that (ii) this reaction may be slowed down by the influence of the protein, but is irreversible once the chromophore has been re-duced. The influence of the State of the protein on the reaction kinetics with N a B H 4 has already been stud-ied by Crespi et a l . [45], and interpreted as a change in accessibility of the cromophores to the reagent. A n alternative mechanism of the protein to slow down the reaction with sodium borohydride would be a conformational change of the chromophore. The similar reactivity of biliverdin and isophorcabi-lin does not prove this assumption, but indicates that a mere conformational change without the coplanar fixation suggested for PC has no dominant in-fluence.

The reaction of native PC with dithionite is incomplete even at high concentrations of the re-agent. In this case the reaction is determined by thermodynamics. Sodium borohydride reduces na-tive PC slowly, but completely. This demonstrates, that C-10 of all chromophores is accessible by the reagent dissolved in the aqueous phase. Similar conclusions have been reached by Troxler [30] for the terminal 0-1 and 0-19, and hence practically the entire chromophore must be accessible to the aqueous phase. It requires further work to deter-mine, whether the chromophores are actually at the


surface, or rather in interior hydrophilic parts of the protein.

The possibility of the preparation of a phycobili-protein with a rubin-type chromophore described here makes further modifications feasible, since bi l i -rubins are accessible to a variety of reactions. A first example is the use of the diazoreaction of bilirubin as a novel degradation technique for biliproteins [31].

Materials and Methods

PC was isolated from S p i r u l i n a p l a t e n s i s as de-scribed previously [3].

P r e p a r a t i o n of P h y c o r u b i n

10.0 ml of a Solution of denatured P C (5 x 10~5 M chromophores) in 50 mM sodium phosphate/8 M urea-buffer (containing 5 mM E D T A and 5 mM NaN 3 ) , pH 7.5, were cooled with ice and 500 ul of a Solution of 10 mg/ml sodium borohydride in the same buffer were added. The Solution was left Standing for 1/2 h and then again 500 ul of the borohydride Solution were added (2.4 x 10"2 M final concentration). With-in again 1/2 h the reaction followed spectrophoto-metrically went to completion.

R e n a t u r a t i o n of p h y c o r u b i n

For removal of urea (and borohydride) 1.0 ml of the Solution obtained by the reaction described above was passed through a 1.7 x 12 cm biogel P 2 column (Bio-Rad, Richmond, California) which was previously equilibrated with 0.05 M sodium phosphate buffer, pH 7.5. The procedure was car-ried out in a cold room at 4 °C.

P r e p a r a t i o n of m o d e l b i l i n s f o r t h e P C - c h r o m o p h o r e a n d of a u t h e n t i c r u b i n s

Bilirubin (4 a), biochemical grade, was purchased from Merck, Darmstadt. Mesobilirubin (4 b) was prepared from 200 mg of 4 a by catalytic hydro-genation [4, 32] (40 mg of 10% Pd on charcoal) in 20 ml 0.1 N NaOH. The reaction was followed spec-trophotometrically, adding aliquots from the reac-tion mixture to methanol (A m a x (4 a) = 452 nm, X M A X (4b) = 428nm). The mixture was worked up with Chloroform after addition of glycine/HCl-buffer, pH 2.7, saturated with ammonium sulfate. Yield: 98%. Biliverdin (3 a) was prepared from 50 mg bili-

rubin (4 a) dissolved in 50 ml dimethylsulfoxide by oxidation with a Solution of 50 mg 2,3-dichloro-5,6-dicyanobenzoquinone in 10 ml of the same solvent in a nitrogen atmosphere [4]. After work-up with Chloroform and water (yield: 38%), the reaction product was purified by preparative tlc on silica using the upper phase of a mixture of toluene/acetic acid/water 5:5:1 (v/v/v) as solvent System. The green band (ÄF-value 0.1) was eluted with acetic acid and worked up by partitioning between Chloro-form and water. The Chloroform phase was washed with water and 1% aqueous NaHC0 3 -solution until neutral (yield: 26%). For crystallization the product was dissolved in 3 ml Chloroform (containing a small amount of methanol). 12 ml of «-hexane were added and the mixture was left Standing at - 20 °C over night (yield: 9%).

Mesobiliverdin (3 b) was prepared by an analo-gous procedure from mesobilirubin (4 b).

10 mg of crude (3 a) dissovled in 150 ml methanol were esterified to the corresponding dimethylester (3 c) by treatment with 60 ml methanol containing 20% (w/w) B F 3 [4] under reflux for 10 min in a nitrogen atmosphere. After work-up with Chloro-form and water, the crude product was purified by tlc on silica with chloroform/acetone 95:5 (v/v) as solvent [33]. The green zone was eluted with acetone (yield: 85%).

Bilirubin dimethylester (4 c) was prepared from 30 mg (4 a) suspended in 10 ml CHC1 3 by esterifica-tion with diazomethane over night [34, 35]. The Chloroform phase was washed with an aqueous 10% NajjCOa Solution and the product subsequently chro-matographed on neutral A 1 2 0 3 (2x6 cm; super ac-tivity I, Woelm, Eschwege) [36]. By-products were eluted with CHC1 3 , (4c) with C H C l 3 / M e O H 9:1 (yield: 65%).

Octaethylbilindiones (7) and (8) were synthesized from octaethylporphyrin [37, 38]. The products were purified by tlc on silica with CCl 4/acetone 9:1 (v/v) as solvent.

Biliverdin-IX y-dimethylester (11): a mixture of the isomeric biliverdin-IX dimethylesters was ob-tained by coupled oxidation (02/hydrazine) of hemin with subsequent esterification [33]. Biliverdin-IX dimethylester was freed from the ß and 5-isomers by tlc on silica with chloroform/acetone 97:3 (v/v) [39], and subsequently from the a-isomer by tlc on silica with toluene/ethylmethylketone/acetic acid 10:5:0.5 (v/v/v) [40].


Phorcabilindimethylester (9) was prepared by heating biliverdin-IX y-dimethylester (11) for 1 h at 100 °C in dimethylsulfoxide under nitrogen [39]. Isophorcabilindimethylester (10) was obtained from phorcabilindimethylester (9) in 20% M e O H / H 2 S 0 4 , the mixture being kept under reflux for 1/2 h [39]. The products were purified by tlc on silica with chloroform/acetone 8:2 (v/v) as solvent System and crystallized from CHCl 3 /«-pentane 1:25 at - 20 °C.

Mixture of the III-, IX- and XHI-a-isomers of bi l i -rubin (4 a, 5 a, 6 a) and mesobilirubin (4 b, 5 b, 6 b): bilirubin (4 a) or mesobilirubin (4 b) were dissolved in 0.5 ml 0.1 N N a O H and added to 5.5 ml 50 mM sodium phosphate buffer, pH 7.5, containing 8 M urea. The samples were purged with nitrogen and kept at 34-38 °C for 2 h. The rubins were extracted with Chloroform after addition of glycine/HCl buf-fer, pH 2.7, saturated with ( N H 4 ) 2 S 0 4 . For prepara-tion of pure isomers of bilirubin, the mixture was chromatographed on silica with C H C l 3 / H O A c 97:3 (v/v) [7 c] and the products eluted with Chloroform.

The model Compounds were reacted with sodium borohydride (Merck-Schuchardt, Hohenbrunn, p.s.) and sodium dithionite (technical grade, Merck, Darmstadt). The reaction mixtures were worked up by extraction with 1.5 vol of Chloroform after addi-tion of 1.5 vol. glycine/HCl-buffer, p H 2.7, satu-rated with ammonium sulfate in the case of free acids [5]. The Chloroform phase was briefly dried over NaCl and after filtration on cotton, uv-vis spectra were taken. The samples were dried in a stream of nitrogen for tlc.

P r e p a r a t i v e s c a l e r e a c t i o n of b i l i v e r d i n d i m e t h y l e s t e r (3 c) w i t h d i t h i o n i t e

A Solution of 10 mg 3 c in 10 ml methanol was added to a Solution of 50 mg sodium dithionite in 10 ml doubly glass distilled water under a nitrogen atmosphere. After 10 min, 20 ml glycine/HCl-buf-fer, pH 2.7 [5] were added and nitrogen was bubbled through the Solution for further 30 min. The mix-ture was extracted twice with 10 ml Chloroform, with some yellow pigment remaining in the aqueous phase. Preparative tlc of the organic extracts on silica gel (0.75 mm) with benzene/ethanol = 25:2 (v/v) yielded four zones: 1, yellow, Rf = 0 ; II, yellow, i ? F = 0.3-0.5; III, yellow with greenish and brownish components, R F = 0.65; IV, blue, R F = 0.70 (identified as the starting material by analytical tlc

with System "a" of Table I). Bands II—IV were elut-ed with acetone, elution of zone I was impossible. Zones II and III were each fractionated by column chromatography (2 x 5 cm valumina, neutral, activi-ty super 1; Woelm, Eschwege): zone II: the main fraction was eluted with Chloroform, a minor frac-tion with chloroform/methanol 9:1, zone III: two fractions were separated with Chloroform, one with chloroform/methanol 9:1, a further zone could not be eluted with methanol.

P r e p a r a t i v e s c a l e r e a c t i o n of b i l i v e r d i n (3 a) w i t h d i t h i o n i t e

7.8 mg biliverdin were dissovled in 50 ml hot methanol and after cooling, added to a Solution of 500 mg sodium dithionite in 50 ml water under a continous stream of nitrogen. 400 ml glycine/HCl-buffer, saturated with ammonium sulfate, p H 2.7 [5], were immediately added and the aqueous phase was extracted four times with a total volume of 300 ml of Chloroform. The Chloroform phase was concentrated in vacuo to 30 ml and fractionated by tlc on silica gel plates (0.25 mm) with benzene/ ethanol = 25:2 (v/v). A zone with green, brown and violet coloured substances with a yellow margin re-mained at the Start. The only major zone (orange R F = 0.4) was eluted with 130 ml Chloroform to yield 0.8 mg crude bilirubin (4 a); it was esterified with C H 2 N 2 in CHC1 3 . Chromatography on A1 2 0 3 yielded the following fractions: I, yellow substance eluted with Chloroform; II, main fraction, yellow substance eluted with C H C l 3 / M e O H 9:1= yield 0.3 mg; A m a x (CHC13) = 403 nm, Shoulder at 435 nm; identical in cochromatography with bilirubin-dimethylester (4 c) with System a of Table I; III, yellow-greennish sub-stance, eluted with CHC1 3 acetic acid =1:1.

*HMR-spectra of biliverdin dimethylester (3 c), its reaction products with borohydride and dithionite, and of authentic bilirubin dimethylester (4 c):

Preparation of the reactions products: 3 c /NaBH 4 and 3c/dithionite: A Solution of 6 mg biliverdin dimethylester (3 c) in 2 ml CHC1 3 was shaken with 1.0 ml of an aqueous Solution of 50 mg/ml di-thionite until the colour changed to yellow-green. The CHC1 3 phase was washed with water, dried on NaCl and evaporated under a stream of nitrogen. An analogous experiment was carried out using sodium borohydride instead of dithionite.


Biliverdin dimethylester (3 c): 1.80 (exo-CH 3); 2.01, 2.04, 2.10 ( e n d o - C R ^ 2.56 t, 2.93 t (8.12-C H 2 - C H 2 ) ; 3.61 (OCH 3 ) ; 5.28-6.61 (vinyl-protons); 5.94,6.00 (5.15-CH); 6.73 (10-CH).

Bilirubin dimethylester (4 c): 1.68 (exö-CH 3); 1.91, 202 ( e n d o - C H ; 2.39 t, 2.84 t (8.12-CH 2-CH 2); 3.63 (OCH 3 ); 4.10 (10-CH 2); 4.65-6.66 (vinyl protons); 5.84, 613 (5.15-CH); 10.04,10.12,10.42,11.14 (NH).

3 c / N a B H 4 : 1.68 (exo-CH 3); 1.91, 201 ( e n d o - C H 3 ) ; 2.38 t, 2.83 t (8.12-CH 2-CH 2); 3.63 (OCH 3 ) ; 4.09 (10-CH2); 4.65-6.66 (vinyl protons); 5.84, 6.12 (5.15-CH); 10.03,10.12, 10.41,11.11 (NH).

3c/dithionite: Broad Signals in the ranges 1.5-3.6 ppm and 5.1-6.4ppm; no signal detectable in the ränge 3.7-5.0 ppm.

G e n e r a l methods:

UV-vis spectra were taken with a model D M R 22 spectrophotometer (Zeiss, Oberkochen), the spectra of Compounds 9-12 with a model Superscan spec-trophotometer (Varian, Palo Alto). x Hmr spectra were recorded in C 2 HC1 3 with a model H F X 9 0 spectrometer (Bruker, Karlsruhe). Chemical shifts in ö (ppm) are given relative to S i (CH 3 ) 4 as internal Standard. Signals appeared as singlets, unless other-wise noted. t = triplet. The vinyl protons appeared as A B X System.

Tris-glycine electrophoresis was done by the meth-od of Davies [41] as modified by Wagenmann [42], SDS-electrophoresis with the System of Weber and Osborn [6]. Densitograms were obtained with a type T L D 100 densitometer (Vitatron). Analytical tlc was carried out on silica H P T L C plates and polyamide 11 F 254 plates (both Merck, Darmstadt). Prepara-tive separations were performed on self-made 20 x 20 cm plates covered with 0.75 mm silica H (Merck, Darmstadt).

A c k n o w l e d g e m e n t s

This work was supported by the Deutsche For-schungsgemeinschaft, Bonn-Bad Godesberg. We are indebted to Prof. W. Rüdiger for continuing Sup-port. We thank Prof. R. Gautron (Grenoble) for sti-mulating discussions and Dr. C. Petrier (Grenoble) for aquainting one of us (W.K.) with the prepara-tion of isophorcabilin. The visit to Grenoble was made possible by a travel grant from the European Photochemistry Association. We thank Mrs. G . Schild for the measurement of the *HMR spectra, Mrs. H . Wieschoff and Mrs. C. Bubenzer for valu-able technical assistance in the preparation of the phorcabilins and C-PC, respectively.

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Physiol. Chem. 274,231 (194

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