Planta (Berl.) 118, 133--144 (1974) �9 by Springer-Verlag 1974

A Procedure for the in vivo Determination of Enzyme Activity in Higher Plant Tissue*

David Rhodes and G. R. Stewart

Department of Botany, The University, Manchester, M13 9PL, U. K.

Received March 18 / April 22, 1974

Summary. Rapid freezing of higher plant tissue in liquid nitrogen renders the cells permeable to a wide range of substrate molecules. Tissue permeabilized by repeated freeze-thaw treatment can be used for the measurement of several enzymes. With most of the tissues examined maximum in vivo activities were obtained using a combination of freeze-thaw treatment followed by vacuum infiltration. The activities and properties of enzymes determined with this procedure are very comparable with those obtained for enzymes prepared by conventional extraction procedures.

Introduction

Syrett (1973) has recently described a procedure for the in vivo determination of nitrate reductase and other enzymes in the unicellular alga Chlorella. The assay employs freezing of the cells at - -15 ~ for 5 h; this t reatment, followed by thawing, renders the cells permeable to nitrate and other substrates. In this assay, not only is nitrite production linear with time and proportional to cell number, but the K m for nitrate is similar whether measured with intact cells or cell-free extracts.

A variety of assays are currently used for the determination of nitrate reductase activity in intact cells of higher plants. En t ry of the substrates is achieved by vacuum infiltration (Klepper, Flesher and Hageman, 1971) or chemical t reatment which alters membrane integrity (J~worski, 1971). More recently, a combination of vacuum irrfiltration and chemical t reatment have been used (Streeter and Bolser, 1972; Stewart, Lee and Orebamjo, 1973). Although these procedures are simple and rapid the in vivo activities are generally lower than those measured in vitro and the high concentrations (100-150 mM) of nitrate required for maximum activities limit the scope of the physiological interpretations which are possible from such assays. These high nitrate concentrations contrast with tha t used in the freeze-thaw procedure of Syret t and with the substrate concentrations used by Delmer and Mills (1969) for the in vivo determination of t ryptophane syathetase in

* Abbreviations: DPT, Thiamin pyrophosphate; FMN, Flavin mononucleotide; ~qAD, nicotinamide adenine dinucleotide.

134 D. Rhodes and G. R. Stewart

tobacco callus cells. T ryp tophane synthetase can be measured in cells permeabil ized with d imethyl sulphoxide and the K m values are similar whether measured in such cells or cell-flee extracts.

Recen t ly Ferguson and Sims (1974) have described an in vivo pro- cedure for the de te rmina t ion of g lu tamine syathetase in which yeast cells are rendered permeable by rapid freezing in l iquid ni trogen. The results described in this paper show tha t the freeze-thaw procedure of Ferguson and Sims can be modified for use with a wide var ie ty of higher p lan t tissues. The activit ies and properties of enzymes measured by this method are very comparable with those of enzymes prepared by s tandard ext rac t ion procedures.

Materials and Methods Plant tissue was prepared for the in vivo assay procedure by slicing it into

segments no greater than 3-4 mm in any one plane. Where the tissue was only a few cells thick, no slicing was found to be necessary (e.g. with Lemna, Azolla and bryophyte tissue). The weights of tissue used in any one assay varied between 5-100 mg fresh weight. As will be shown in the results, activity is proportional to incubation time and to the weight of tissue. After weighing, the tissue was placed in a thin-walled test-tube which was then immersed in liquid nitrogen for 30-60 s and then thawed for 1-2 rain at 30 ~ :Freeze-thawing was repeated for the optimum number of cycles (see results). After the final cycle of freeze-thaw, substrate solu- tion was added and the sample incubated at 30 ~ In some experiments freeze-thaw treatment was followed by vacuum infiltration with the reaction mixture. The reaction was stopped either by filtering off the tissue or by the addition of reagent to measure the product. The tissue was then removed by filtering. Appropriate blanks were run with treated tissue, incubated without substrate.

Details of the substrate concentrations and methods for measuring enzyme activity are sho~m in Table 1. For the assay of enzyme activity in cell-free extracts the substrate concentrations were the same as those used for the in vivo assay. Enzyme extraction was as described previously (Stewart et al., 1973). Details of the extraction buffers are shown in Table 1. All extraction buffers contained 1.5% Polyclar AT.

Vacuum infiltration was carried out as described by Klepper et al. (t971). In some experiments 1% Propan-l-ol was included in the vacuum infiltration reaction mixture.

Most of the experiments were carried out oll plants of Lemna minor. The con- ditions for the growth of axenic cultures were as described previously (Stewart, 1972). Other plant species were grown from seed or obtained from the university Botanic Gardens.

Results

Cell Permeabilization by Freeze-thaw Treatment

Pre l iminary exper iments indica ted t ha t when plants of Lemna were rapidly frozen in l iquid ni t rogen, thawed and incuba ted with the ap- propriate substrates, i t was possible to detect the ac t iv i ty of several enzymes. Increas ing either the period of freezing above 1 rain, or the period of thawing above 2 min, had li t t le effect on the level of ac t iv i ty

in, vivo Determination of Enzyme Activity 135

Table 1. Enzyme extraction and assay procedures

Enzyme Extraction Assay mixture Comments buffer

(1) Acetolactate 100 mM phosphate, 150 tzmol phosphate; acetoin synthetase pH 7.5 contain- 150 ~mol pyruvate; formation

ing 0.5 mM EDTA 0.75 ~zmol MnC12; (Westerfield, 0.75 ~mol DPT; 1945) p i t 6.5; 1.5 volume

25 ~mol Tris-HC1; 20 ~mol arginine; 0.5 ~mol MnC12; pH 9.0; 1 ml volume

~-galacto- 4 ~mol p-nitro- sidase phcnyl a-D-galaeto-

ornithine formation (Denes, 1970)

(2)

(3)

(4)

(5)

46)

<7)

(8)

Arginase E.C. 3.5.3.1

E.C. 3.2.1.22

Glutamate dehydro- genase E.C. 1.4.1.2

Glutamine synthetase (synthetase reaction)

E.C. 6.3.1.2

Glutamine synthetase (transferase reaction)

:Nitrate reductase

E.C. 1.6.6.1

Nitrite reductase

E.C. 1.6.6.4

50 mM Maleic acid, pH 7.8, contain- ing 2 mM dithio- threitol

100 mM Tris-ace- rate, pH 5.5

100 mM phosphate, pH 8.5

100 mM Imidazole- HC1, pH 7.2, containing 1 m~I dithiothreitol

as for (5)

as for (1)

as for (1)

side; 50 tzmol Tris- acetate, pH 5.5; 2 ml volume

200 ~zmol Tris; 200 9mol glutamate; 20 ~mol NAD; pH 9.5; 2 ml volume

50 tzmol Imidazole- HC1; 20 ~mol MgC12; 10 ~mol ATP; 100 ~zmol glutamate; 20 ~mol hydroxy- lamine; pH 7.2; 2 ml volume

50 ~zmol Tris- as for (5) acetate; 140 9tool glutamine; 9 ~mol MnC12; 35 ~mol hydroxyl- amine; 1.5 ~mol ADP; pH 6.6; 2 ml volume

150 ~mol phosphate; 5 ~lmol KNOa; 0.3 ~mol FMN; 1 nag sodium dith- ionite; pH 7.5; 1 ml volume

100 ~mol phosphate; nitrite loss 0.4 ~mol NAN02; 0.8 ~mol benzyl viologen; 0.3 mg sodium dithionite; pH 7.2; 1 ml volume

nitrophenol formation (Dey and Pridham, 1969)

~-oxoglutarate formation (Greenberg, 1962)

glutamyl- hydroxamate formation (Ferguson and Sims, 1971)

nitrite formation (Filner, 1966)

136 D. Rhodes and G. 1~. Stewart

Table 1 {continued)

Enzyme Extraction Assay mixture Comments Buffer

(9) Ornithine 100 mM Tris-ttC1 4 ~mol ornithine; citrulline Transcarbamy- 26 ~mol carbamyl formation lase phosphate; (Prescott and

100 ~mol Tris-ttC1; Jones, 1969) E.C. 2.1.3.3 p i t 8.5; 1 ml volume

(10) Phosphatase as for (3) 4 ~mol p. nitro- nitrophenol phenyl phosphate; formation

E.C. 3.1.3.2 100 ~mol Tris- (Reid and acetate; Bieleski, 1970) pH 5.5; 2 ml volume

Table 2. Effect of number of freeze-thaw cycles on enzyme activity

Enzyme Number of freeze-thaw cycles

0 1 2 4 6 8

Acetolaetate synthetase N.D. 0.53 a 0.60 0.72 0.74 0.73 Arginase N.D. 4.2 4.3 6.8 8.7 - - Glutamine synthetase N.D. 287 385 389 610 607

(transferase reaction) Glutamine synthetase N.D. 19.8 20.6 25.4 36.4 -~

(synthtase reaction) Glutamate dehydrogenase N.D. 11.4 15.3 21.6 21.0 22.8 Nitrate reductase N.D. 3.1 4.2 5.2 5.9 6.1 5~itrite reductase N.D. 7.3 10.1 12.6 14.9 - - Phosphatase 4 76 83 100 101 104

a Enzyme activity expressed as ~mol/h/gfw. Material was nitrate adapted Lemna minor. N.D. Not detectable.

measured . I n contras t , increas ing the n u m b e r of f reeze- thaw cycles resu l ted in m a r k e d increases in a c t i v i t y (Table 2). F o r the enzymes examined the effects of r e p e a t e d f reeze- thaw were similar , four to six cycles of f reeze- thaw appea r to be op t ima l wi th Lemna t issue.

F reeze - thaw t r e a t m e n t would seem to render Lemna cells pe rmeab le to a wide range of subs t r a t e molecules, inc luding ATP , n i t r a t e and NAD. I t is possible however t h a t t he endogenous subs t r a t e levels are suffi- cient in some cases to make the a c t i v i t y in th is in vivo assay i ndependen t of exogenous ly suppl ied subst ra tes , f reeze- thaw t r e a t m e n t mere ly al lowing the release of p roduc t s in to the reac t ion mix ture . The ex t en t to which subs t r a t e dependence can be d e m o n s t r a t e d is va r iab le (Table 3). Bu t for the enzymes shown, t he a c t i v i t y in the absence of one or more of the subs t r a t e s was never g rea te r t h a n 40% of the r a t e wi th t he corn-

in vivo Determination of Enzyme Activity 137

12

E 8

"3

A

20 40 60

TiME (minuteS)

1.2

O.E

0.4

B

A

20 40 60

Weight of T;ssue (mgm f.w.)

Fig. 1 A and B. Linearity of enzyme assay with (A) duration of reaction and (B) quantity of tissue in reaction mixture. A. glutamine synthetase (transferase) tLmol/gfwX 10 -1 (B); nitrate reduetase ~tmol/gfw (e); phosphatase tzmol/gfw • 10 -1 (A); 6 cycles of freeze-thaw treatment. B. glutamine synthetase (transferase) ~mol/m (B); nitrate reductase ~mol/30m (e); phosphatase ~mol/10m (A);

6 cycles of freeze-thaw treatment followed by vacuum infiltration

Table 3. Substrate dependence in freeze-thaw assay

Omission Activity as % of that with complete mixture

Acetolaetate synthetase MnCl 2 18 DPT 37 Pyruvate 5

Glutamate 9 ATP 13 MgC12 33

Glutamate 28 NAD 13

Nitrate 35 FM_N 8 Dithionite 8

Glutamine synthetase (synthetase reaction)

Glutamate Dehydrogenase

Nitrate reductase

Plants of Lemna minor assayed using 6 cycles of freeze-thaw treatment followed by vacuum infiltration.

p le te reac t ion mix ture . These resul ts confirm the suggest ion t h a t r epea t ed f reeze- thaw renders the cells pe rmeab le to a wide range of subs t r a t e molecules.

Using the f reeze- thaw procedure the t ime course of the reac t ions are l inear (Fig. la) . There is no evidence of a lag phase as has been r epor t ed for the v a c u u m inf i l t ra t ion ~ssay (Klepper etal . , 1971). The

138 D. Rhodes and G. R. Stewart

r a t e of r eac t ion is also p ropor t iona l to t he weight of t issue in t he reac t ion mix tu re (Fig. lb) . I t was possible to de te rmine g lu tamine syn the t a se and n i t r a t e r edue tase on as l i t t le as 5 mg fresh weight of Lemna plants .

Comparison o/Freeze-thaw Treatment with other Assay Procedures

I t is ev iden t f rom the resul ts in Table 4 t h a t t he level of enzyme a c t i v i t y measu red b y the f reeze- thaw procedure is in all cases h igher t h a n t h a t d e t e r m i n e d using v a c u u m inf i l t ra t ion. The inclusion of p ropan- l -o l in t he r eac t ion mix tu r e increases t he a c t i v i t y in the v a c u u m inf i l t ra t ion assay b u t even so i t is stil l much lower t h a n t h a t in the f reeze- thaw assay. A c t i v i t y can be increased b y 5-15% if f reeze- thaw t r e a t e d p l an t s are v a c u u m in f i l t r a ted wi th the r eac t ion mix tu re . This t r e a t m e n t has more effect when app l ied to t issue which has been g iven a sub-op t ima l n u m b e r of f reeze- thaw cycles. The m a x i m u m act iv i t ies o b t a i n e d wi th the combina t ion of f reeze- thaw and v a c u u m inf i l t ra t ion compare ve ry closely wi th those de t e rmined on cell-free ex t rac ts . I n no case was the in vivo a c t i v i t y less t h a n 80% of t h a t in cell-free ex t rac ts .

Table 4. Comparison of in vivo and in vitro assay procedures with Lemna minor

Vacuum Vacuum 6 cycles 6 cycles Cell-free infil- infil- Freeze- Freeze- extract tration tration -~ thaw thaw

1% propanol vacuum infil- tration

Acetolactate 0.20 a 0.42 0.74 0.81 0.79 synthetase

Glutamine 29 40 610 630 654 synthetase (transferase)

Nitrate 0.8 1.5 5.9 6.3 5.8 reductase

Nitrite 1.0 2.1 14.9 15.8 17.3 reduetase

Ornithine 6.8 7.8 9.2 9.3 10.1 trans- carbamylase

Phosphatase 14 37 102 106 104

a Enzyme activities expressed as ~tmol/h/gfw.

in vivo Determination of Enzyme Activity 139

Standardization o/the Freeze-thaw Assay /or other Plant Tissues

I t is clear from the resul ts p resented above t h a t r epea ted freeze- t haw t r e a t m e n t is an effective procedure for permeabi l iz ing Lemna cells. Lemna t issue is however only a few cells th i ck compared with cer ta in o ther p l an t t issues and i t could be t h a t f reeze- thaw t r e a t m e n t is effective for th is reason. The possible app l ica t ion of the me thod to o ther p l an t t issue was inves t iga ted by de te rmin ing the o p t i m u m con- di t ions for the measu remen t of two enzymes, g lu tamine syn the tase and orni th ine t r ansca rbamylase . The results in Table 5 show the effect of r epea ted f reeze- thaw t r e a t m e n t on the ac t i v i t y of g lu tamine syu the tase

Table 5. Effect of repeated freeze-thaw treatment on measurement of Glutamine synthetase activity

Number of freeze-thaw cycles

1 2 4 6 8

Azolla [iliculoides 0.43 a 0.61 0.70 0.73 0.78 Camptothecium sericeum 1.45 1.75 1.70 1.81 1.78 Dryopteris parasitica 1.07 1.53 1.47 1.43 1.21 Ilex aqui/olium 1.66 1.73 1.81 1.31 1.18 Porella platyphylla 0.12 0.13 0.14 0.16 0.15 Puccinellia distans (roots) 3.11 4.45 4.83 4.55 4.76 Zannichellia palustris 0.69 2.01 1.86 1.41 1.20

a Enzyme activity expressed as txmol/min/gfw.

in a range of p l an t tissues. I t is ev ident from these resul ts t h a t the number of f reeze- thaw cycles necessary to permeabi l ize the cells is var iable . I n some eases, such as Ilex and Zannichellia increasing the n u m b e r of f reeze- thaw cycles above 2 or 4 resul ted in a decrease in g tu tamine syn the tase ac t iv i ty . Similar resul ts were ob ta ined wi th orn i th ine t r ansea rbamylase indica t ing t h a t the response to the number of f reeze- thaw cycles is a t issue r a the r t h a n an enzyme de te rmined character is t ic .

The resul ts in Table 6 show the ac t i v i t y of orni thine t r a n se a rba my- lase in var ious t issues de t e rmined with different assay procedures. W i t h all of the t issues examined, the f reeze- thaw procedure y ie lded act iv i t ies which were considerably higher t han those de te rmined b y vacuum inf i l t ra t ion. W i t h some tissues vacuum inf i l t ra t ion of freeze- t h a w t r ea t ed t issue increased ac t i v i t y b y up to 30 %. I n some eases, in pa r t i cu la r Dryopteris, the ac t i v i t y in the f reeze- thaw assay was con- s iderab ly higher t h a n t h a t ob ta ined with cell-flee ext rac ts . I n no in-

10 Plan~a (Bed.), Vol. 118

140 D. Rhodes and G. R. Stewart

Table 6. Comparison of in vivo and in vitro assays for ornithine transcarbamylase

Number Freeze- Freeze- Vacuum Cell- of thaw thaw A- infil- free freeze- vacuum tration extract thaw infil- cycles tration

Pisum sativum (cotyledons) 6 0.31a N.D. N.D. 0.33 Pisum sativum (roots) 6 1.73 1.82 0.33 1.98 Equisetum arvense 2 0.09 0.12 0.08 0.02 Dryopteris parasitica 2 0.24 0.39 N.D. 0.02 Cycas revotuta 6 9.09 0.09 N.D. 0.02 Trichocereus dantzii 6 0.03 0.04 N.D. 0.04 Azolla/iliculoides 4 0.05 0.07 N.D. 0.03

a Enzyme activities expressed as ~mol/min/gfw. N.D. not determined.

s tance was the f reeze- thaw a c t i v i t y less t h a n 80 % of t h a t in the cell-free ex t rac t . These resul ts show t h a t f reeze- thaw t r e a t m e n t can be used to permeabi l ize a wide range of p l an t t issues and t h a t the level of ac t iv i ty de t e rmined in th is w a y is s imilar in most cases to t h a t oY cell-free ex t rac ts .

Properties o / E n z y m e s in the Freeze-thaw A s s a y

E x p e r i m e n t s were carr ied out to compare the character is t ics of a number of enzymes in the f reeze- thaw assay wi th those of enzymes in cell-free ex t rac ts . W i t h the excep t ion of phospha tase from Lemna minor t he p H op t ima are s imilar in vivo and in vitro (Table 7). A l though i t is dif f icul t to make d i rec t comparisons of the response to suhs t ra te con- cen t ra t ions due to the presence of endogenous subst ra tes , the resul ts (Table 7) ind ica te t h a t the concen t ra t ion giving half the m a x i m u m ra te is s imilar when de t e rmined on f reeze- thaw t r e a t e d t issue and in cell-free ext rac ts . These resul ts show t h a t the ca ta ly t i c proper t ies of the enzymes examined are unaffec ted by r epea t ed f reeze- thaw t rea t - ment . F u r t h e r m o r e the s imi la r i ty of the response to subs t r a t e con- cen t ra t ion indica tes t h a t f reeze- thaw t r e a t m e n t is an efficient means of permeabi l iz ing the cells.

A fur ther check was carr ied out to de te rmine the effects of freeze- t h a w t r e a t m e n t on enzyme proper t ies . I t is well known t h a t several a l lostcr ic enzymes can be desensi t ized to feedback inhibi tors by r epea t ed f reeze- thaw (see e.g. S t a d t m a u , 1966). The resul ts in Table 8 show t h a t the sens i t iv i ty of g lu tamine syn the tase to several inhibi tors is unaf- fec ted by r e p e a t e d f reeze- thaw of t he t issue. The magn i tude of the inhibi t ions are s imilar in vivo and in vitro. Simi la r ly wi th ace to lac ta te syn the tase i t was possible to demons t r a t e inhib i t ion of the enzyme b y

in vivo Determination of Enzyme

Table 7. Comparison of pH optimum and substrate in vitro assays

Activity 141

response in the in vivo and

Source Enzyme pH optimum S. 0.5 a

in vivo in vitro in vivo in vitro b

Lemna minor nitrate 7.5 7.5 1.3 • 10 .4 M 2.3 • 10 .4 M reductase (nitrate)

Lemna minor glutamine 6.6 6.4 6.7 • 10 -3 M 4.4 • l0 -3 M synthetase (hydroxy- (transferase) lamine)

Lemna minor ~-galaeto- 5.5 5.5 5.6 • l0 -4 M 3.2 • 10 -4 M sidase

Lemna minor phosphatase 5.5 6.0 5.0 • 10 -4 M 4.4 • 10 -4 M

Pisum sativum ornithine 8.5 8.5 N.D. N.D. (cotyledon) transcar-

bamylase

Pisum sativum phosphatase 4.5 4.5 5.8 X 10 -4 M 3.4 • 20 -4 M (cotyledon)

a Substrate concentration giving half maximum velocity. b Enzyme extracts passed through Sephadex G.25, equilibrated with extraction buffer. In vivo assay procedure as for Table 3. N.D. not determined.

Table 8. Comparison of glutamine synthetase inhibition in vivo and in vitro (Lemna minor)

Inhibitor (10 mM) % Inhibition

in vivo in vitro

AMP 5 3 Alanine 63 67 Carbamyl phosphate 33 39 CTP 4 9 Glucosamine-6-phosphate 0 -b 4 Glycine 38 44 Histidine 19 17 Tryptophane 5 10 Mixture a 52 54

Assay procedure as for Table 3. a Mixture of all eight metabolites each at 1.25 mM. Reaction mixture contained: 50 ~xmol Tris-acetate, 20 ~zmol glutamine, 9 ~mol MnCle, 35 ~mol hydroxylamine and 1.5 ~mol ADP.

10"

142 D. l~hodes and G. R. Stewart

Table 9. Comparison of acetolactate synthetase inhibition in vivo and in vitro, (Zea mays shoots)

Amino acid % Inhibition

in vivo a in vitro (Miflin and Cave, 1972)

Leucine 49 67 Isoleucine 22 16 Valine 33 49 Leucine -4- Valine 83 85 Leucine + Isoleucine 58 62 Isoleucine -4- Valine 29 50 Leucine + Valine + Isoleucine 76 82

Assay procedure as for Table 3. a Reaction mixture contained: 150 DPT, at a final p i t of 6.5.

izmol phosphate, 20 ~mol pyruvate, 0.75 ~mol

leucine, isoleucine and vMine in the f reeze- thaw assay (Table 9). The p a t t e r n of inh ib i t ion ob t a ined in vivo is s imilar to t h a t r epo r t ed for the cell-free enzyme (Miflin and Cave, 1972). Leucine was the mos t inh ib i to ry amino acid when suppl ied singly, and the combina t ion of leucine and vMine gave the h ighest overal l inhibi t ion.

Discussion

R a p i d freezing of p l an t t issue in l iquid n i t rogen appears to be an efficient means of render ing the cells pe rmeable to a wide range of subs t r a t e molecules. I n order for any in vivo procedure to be used for the measu remen t of enzyme a c t i v i t y i t is necessary t h a t e i ther the d i sappea rance of the subs t r a t e or appea rance of the p roduc t can be measured . W i t h mos t of the enzymes examined here, p roduc t appear - ance has been the basis for a c t i v i t y de te rmina t ions . F o r quan t i t a t i ve measurement s of a c t i v i t y i t is essent ial t h a t the p roduc t should undergo no fu r the r metabol i sm, l%outine checks on p roduc t d i sappearance indica te t h a t for the sys tems examined this was negligible.

The enzymes examined here ut i l ize a wide va r i e t y of subs t r a t e molecules, and wi th none of t h e m was pene t r a t i on of the subs t r a t e found to l imi t the r a t e of react ion. This is in cont ras t to the f indings of Sy re t t (1973) where f reeze- thaw t r e a t m e n t of Chlorella cells was found to be ineffect ive in permeabi l iz ing the cells to A T P and N A D H . This difference in behav iour means t h a t i t is unnecessary to employ coupled assays wi th higher plants . W i t h n i t r a t e reduc tase for example , r educed F M N is able to pene t r a t e the cells mak ing i t unnecessary to couple n i t r a t e reduc t ion to the genera t ion of N A D H .

in vivo Determination of Enzyme Activity 143

The only problem encountered with plant tissue was in the deter- mination of dehydrogenase activity. In theory it should be possible to couple NADH or NADPH generation to the reduction of a tetrazolium dye. In practice it was found that although reduction of tetrazolium dyes could be observed with freeze-thawed tissue, there was a marked retention of the reduced dye within the tissue. I t is not clear whether this arises from the solubility of the reduced dye or because the cells are impermeable to reduced tetrazolium dyes. These can however be extracted from the tissue by homogenization in a methanol/chloroform/ water solvent.

I t is clear from the results obtained with different plant tissues that the number of freeze-thaw cycles required for the measurement of maximum enzyme activity is a tissue dependent characteristic. Having established, for one enzyme, the optimum number of cycles it is un- necessary to repeat this for other enzymes�9 With the majority of tissues examined vacuum infiltration of the freeze-thaw treated tissue gave some further increases in activity and we now include this treatment in the assay procedure. Comparisons of the level of activity that can be measured by the freeze-thaw method with those measured in con- ventional cell-free extracts show that very similar levels of activity are obtained by both methods. Exceptions to this are certain tissues where much higher activities were determined using the freeze-thaw procedure. These tissues contain appreciable quantities of phenolic compounds which are probably responsible for the low yields of extractable enzyme activity. I t is significant in this context, that such tissues show a re- duction in activity with repeated freeze-thaw treatment. Using this rapid freezing in liquid nitrogen we have been able to measure nitrate reductase and several other enzymes in whole cells of some unicellular algae, including Scenedesmus, Porphyra and Staurastrum. I t is not clear why these species should behave differently from the Chlorella used by Syrett (1973). The difference may lie in the nature of the cell walls since Syrett and Thomas (1973) have reported strain differences in Chlorella with respect to the effect of freeze-thaw treatment on the permeability to NADIt.

The major advantages of the freeze-thaw procedure are that it permits enzyme assays to be carried out rapidly and precisely on small quantities of tissue and gives results which are very similar to those obtained with cell-free extracts. Furthermore the method enables the determination of enzyme activity in tissue where low yields are obtained with extraction methods. The small quantity of tissue required allows studies to be made of the tissue distribution of enzymes and the pro- cedure can be applied to microtome sections of plant roots and other tissues (Rhodes unpublished). The method should be suitable in situations

144 D. Rhodes and G. R. Stewart

where e i ther small quant i t ies of tissue arc avai lable or where rapid

processing of large numbers of samples is required. The range of enzymes which can be measured by this procedure can be readi ly ex tended

th rough the use of rad ioac t ive substrates.

D. Rhodes was supported by a Science Research Council Studentship.

References Delmer, D. P., Mills, S. E. : A technique for the assay of enzymes in intact plant

cells in the presence of dimethyl sulphoxide. Plant Physiol. 44, 153-155 (1969) Denes, G.: Ornithine aeetyl transferase. In: Methods of enzymology, Colwick,

S. P., Kaplan, N. O., eds., vol. XVII, p. 273-277. New York: Aead. Press 1970 Dey, P.M., Pridham, J. B. : Purification and properties of ~-galactosidase from

Vicia/aba. Biochem. J. 113, 49-55 (1969) Ferguson, A.R., Sims, A. P. : Inactivation in vivo of glutamine synthetase and

lXTAD-specific glutamate dehydrogenase: its role in the regulation of glutamine synthesis in yeasts. J. gen. Microbiol. 69, 423427 (1971)

Fergugon, A. R., Sims, A. P. : The regulation of glutamine metabolism in Candida utilis: the role of glutamine in the control of glutamine synthetase. J. gen. Microbiol. 80, 159-171 (1974)

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