160 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 25 579

DETERMINATION OF T H E CARBOXYL-TERMINAL AMINO ACID R E S I D U E

OF T H E /~-GALACTOSIDASE OF E S C H E R I C H I A COLI K12"

STANLEY G. KORENMAN**, GARY R. CRAVEN AND CHRISTIAN B. ANFINSEN Laboratory of Chemical Biology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md. (U.S.A.)

(Received January 24th, 1966)

SUMMARY

I. The carboxyl-terminal amino acid residue of the fl-galactosidase (EC 3.2.1.23) of Escherichia coli Klz has been shown to be lysine by both hydrazinolysis and carboxypeptidase B (EC 3.4.2.2) digestion. One mole of lysine per mole of monomer was found on the basis of a monomer molecular weight of 135 ooo.

2. Further evidence is presented suggesting that the monomer may be composed of more than one peptide chain and the suggestion is made that unusual covalent bonds may be present in the molecule.

INTRODUCTION

Chemical questions arising from genetic studies of the Lac operon of Escherichia coli KI~ have led us to investigate the conformation and pr imary sequence of the product of the Z gene, fl-galactosidase (EC 3.2.1.23). The active molecule is believed to be a tetramer of molecular weight 540 ooo (see refs. I, 2). Peptide maps of trypsin digests and determination of the number of peptides that are formed during cleavage with cyanogen bromide both indicate the plesence of unique pr imary sequence sufficient for a molecular weight of at least IOOOOO (see ref. 3). Treatment of reduced alkylated fl-galactosidase with sodium hydroxide at pH I2.5 for o to 48 h, with 7o % formic acid, or with 0.5 to I.O % sodium dodecylsulfate resulted in material with a substantially lower molecular weight by ultracentrifugal analysis 8. Similar results have been obtained by WALLENFELS, SUND AND WEBER 4 using native fl-galactosidase in acetic acid-formic acid mixtures or performic acid oxidized protein in sodium dodecylsulfate. Since the presence of these lower-molecular-weight fragments sug- gested that the monomer units of fl-galactosidase might be composed of a number of different subunits, carboxyl-terminal analysis was carried out in an a t tempt to define the number of these subunits. The present results confirm and extend the findings of KOORAJIAN AND ZABIN 5 who recently reported that digestion with carboxy-

Abbreviations: CM-fl-galactosidase, carboxymethylated/~-galactosidase; DFP, diisopropyl- fluorophosphate.

* Presented in part at the Forty-ninth Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N.J., 1965 .

** Present address: Endocrinology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

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CARBOXYL-TERMINAL RESIDUE OF ~-GALACTOSIDASE 161

peptidase B (EC 3.4.2.2) resulted in the release of a single carboxyl-terminal lysine residue and that treatment with carboxypeptidase A did not cause the release of free amino acids.

MATERIALS AND METHODS

Preparation of [3-galaetosidase The enzyme was prepared from E. eoli Kx2 , strain 33oo i - z +, and carboxy-

methylated as previously described 2. Carboxymethylated ~-galactosidase was used exclusively for the studies reported below.

Carboxypeptidase A digestion DFP-treated carboxypeptidase A, obtained from Worthington Biochemical

Corporation, was dissolved at a concentration of IO mg/ml in 2 M ammonium bi- carbonate and stored in the frozen state in 2oo-t~l aliquots. The enzyme, when tested against oxidized rihonuclease, liberated approximately molar quantities of the six carboxyl-terminal amino acids with no evidence of internal peptide bond cleavage. Digestion was carried out at 23 ° in either o.2 M ammonium bicarbonate (pH 8.5) or in o.I M trimethylamine acetate (pH 9.8) for periods up to 24 h using 1:5 to 1:25 molar ratios of carboxypeptidase A, at a substrate concentration of 5 to IO mg/ml. The reaction was stopped by precipitation, by adjustment of the pH to 5 with acetic acid. After centrifuging, the amino acids were determined e in the supernatant fraction.

Carboxypeptidase B digestion DFP-treated carboxypeptidase B, obtained from Worthington Biochemical

Corporation, was found to be heavily contaminated with carboxypeptidase A activity. Therefore, the commercial material was further purified by a modification of the method of FOLK et a/ft. DFP-treated carboxypeptidase B (30 mg) was applied to a i cm × 20 cm column of DEAE-ceUulose (California Corporation for Biochemical Research) equilibrated at 4 ° with o.oo5 M Tris-HC1 buffer (pH 6.0). Carboxypeptidase B was readily eluted by a linear gradient of NaC1 to o.i M while carboxypeptidase A remained on the column. Activity was measured by a modification of the method of FOLK et alY using hippuryl-L-arginine. The active fractions were combined and precipitated with 65 % (NH4)~SO4, taken up in distilled water, dialyzed exhaustively at 4 ° against distilled water and stored frozen. The recovered material, 20 mg, was assayed for carboxypeptidase B and carboxypeptidase A activity by Dr. J. FOLK and was found to be highly active against hippuryl-L-arginine and to have slight residual carboxypeptidase A activity. Carboxypeptidase B was assayed for trypsin and chymotrypsin activities using N-benzoyl-L-arginine ethyl ester and N-benzoyl- L-tyrosine ethyl ester as substrates (Determatube, Worthington Biochemical Corpo- ration). On the basis of these assays, contamination was calculated to be less than o.ooi % by weight for both substrates.

Digestion was carried out at a 1:2o molar ratio of carboxypeptidase B to CM- fl-galactosidase. The protein was taken up in 1% NH4OH and dialyzed against o.I M trimethylamine (pH 9.8) at 4 ° for I6 h. Digestions were carried out at 37 ° at a protein concentration of 6-1o mg/ml. The reaction was stopped by the addition of I vol. of IO % trichloroacetic acid and the resulting supernatant was washed twice with

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I62 S .G. KORENMAN, G. R. CRAVEN~ C. B. ANFINSEN

ether, lyophilized and applied to the amino acid analyzer. The precipitates were taken up in water and the residual trichloroacetic acid was removed by partition into ether. Aliquots of this protein residue were taken up in 8 M urea and analyzed by polyacryl- amide-gel electrophoresis as previously described ~.

Hydrazinolysis Anhydrous hydrazine was obtained from the Matheson Coleman, and Bell

Chemical Company and used without further purification. Nuclear magnetic-resonance spectroscopy performed on an aliquot of the hydrazine through the kindness of Dr. H. FALES showed, by analysis of the spectral shift resulting from the stepwise addition of water, that less than 0.5 % contamination was present.

Hydrazinolysis was carried out essentially as described by WlNSTEAD AND WOLD 8. CM-fl-galactosidase was added to a hydrolysis tube (and in some instances an aliquot of uniformly labelled [14C]arginine or uniformly labelled [14C]lysine) and dried at 7 °0 over P205 in vacuo for 3 to 5 days. Anhydrous hydrazine, o. 5 ml, was added, the solution was frozen, and the tube was evacuated and sealed. After incu- bation at either IiO or 7 °0 the tubes were opened and the excess hydrazine removed by evacuation over H2SO4. After adding i ml of H,O and 0.3 ml of redistilled benz- aldehyde the contents of the tube were stirred vigorously for 2 h at 23°. The upper aqueous layer was removed and stirred with an additional 0.2 ml of benzaldehyde for IO min and the second aqueous layer was washed with 2 ml of peroxide-free ether. The final aqueous layer was sampled for radioactivity (see m~SULTS) and the remainder was lyophilized and applied to the amino acid analyzer.

Amino acid analysis Amino acid analyses were performed using a Spinco amino acid analyzer,

Model ieoB, equipped with a Minneapolis Honeywell recorder containing a No. 365 928-999 resistor card for range 4 to 5 mV. This modification allowed the detection of as little as 0.005 ~mole of an amino acid.

For analysis of samples after hydrazinolysis a "basic" column 2o cm long was u s e d to achieve good separation of lysine from ornithine, the principal reaction product formed from carboxyl-terminal arginine.

Liquid scintillation counting Radioactive samples were counted in a Tri-Carb liquid scintillation spectrometer

in BRAY's solution 9. A sufficient number of counts were collected to yield a counting error of less than 2 %.

RESULTS

Carbozypeptidase digestion Digestion of CM-fl-galactosidase occurred to a minor extent with carboxy-

peptidase A. No more than o.13 mole of any amino acid per 135ooo g of protein was liberated even after 24 h and, in general, the appearance of other amino acids paralleled the release of lysine. These data suggest that there are no carboxypeptidase A sensitive C-terminal residues. Digestion with carboxypeptidase B for I to 4 h, on the other hand, invariably resulted in the early liberation of a single lysine residue.

Biochim. Biophys. Acta, I24 (x966) x6o-x65

CARBOXYL-TERMINAL RESIDUE OF ~-GALACTOSIDASE 163

Af te r longer periods of digestion, leucine, ty ros ine and phenyla lan ine appeared. After 2 4 h 4 moles of ty ros ine and 2 moles of pheny la lan ine per mole of lysine, leucine, a n d arginine were present .

Po lyac ry lamide-ge l e lectrophoresis (in 8 M urea) of the pro te in remain ing af ter c a r b o x y p e p t i d a s e B digestion, has cons is ten t ly shown the fo rmat ion of 4 new bands and the concomi tan t d i sappea rance of the f l-galactosidase b a n d (Fig. I). These bands are ba re ly visible in control samples and are not formed on t r e a t m e n t wi th ca rboxy- pep t idase A. These d a t a suggest t h a t enzymat ic t r e a t m e n t of c a r b o x y m e t h y l a t e d f l -galactosidase m a y also resul t in the fo rmat ion of species of lower molecular weight .

In order to es tabl ish the number of C- terminal lysine residues per molecule of CM-fl-galactosidase and to eva lua te the poss ib i l i ty of enzyme-res i s tan t C- terminal residues, hydraz inolys i s was carr ied out. The react ion took place at I iO ° for from

Fig, I. Polyacrylamide-gel electrophoresis of carboxypeptidase treated reduced alkylated fl- galactosidase. Carboxymethylated fl~galactosidase was treated for 4 h with a I : 20 molar ratio of carboxypeptidase A or carboxypeptidase B at pH 9.8 in o.I M trimethylamine acetate buffer. The reaction was stopped by the addition of i volume of Io % trichloroacetic acid. After removal of the supernatant fluid, the precipitate was dissolved in 8 M urea, neutralized, and applied to polyacrylamide-gel for electrophoresis.

TABLE I

HYDRAZINOLYSIS OF CARBOXYMETHYLATED fl-GALACTOSIDASE

Time Amino acid moles/mole monomer Corrected* (h) moles lysine]

L ysine Serine Glycine mole monomer

4 0.32 o 0.07 0.64 6 0.30 0.09 o.i 4 0.60 8 o.51 0.07 o I.o2

Io 0.47 o . i i o.13 0.94

* Corrected for an estimated 50 % recovery on the basis of the recovery of [14C]lysine which had been added to two of the samples.

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164 S. G. KORENMAN, G. R. CRAVEN, C. B. ANFINSEN

4 to IO h (Table I). In each case lysine was the principal amino acid liberated. The only other amino acids detected were glycine and serine which have been observed by others with this method s. In two experiments [a4C]lysine was added to the protein before treatment. Recovery, estimated from the total radioactivity in the final aqueous fraction, was 40 % and 6o %. Assuming an average recovery of 5o %, only a single C-terminal residue lysine per I35 ooo g protein was found (Table I).

Since hydrazinolysis at IIO ° may result in substantial destruction of arginine beyond the ornithine stage, we used the milder conditions developed by BENNETT AND DRE'eER (personal communication) to evaluate the possibility that C-terminal arginine may be present. To human hemoglobin and CM-fl-galactosidase a tracer amount of [a~C]arginine was added and hydrazinolysis was carried out for 48 h at 7 o°. The data in Table I I show that approx. 2 moles of histidine and ornithine were liberated per 68000 g of hemoglobin and that only lysine was recovered from the CM-fl-galactosidase.

TABLE II

RECOVERY OF C-TERMINAL AMINO ACIDS AFTER HYDRAZINOLYSIS Ol ~ HUMAN HEMOGLOBIN AND CARBOXYMETHYLATED fl-GALACTOSIDASE FOR 48 h AT 70 °.

Theoretical Recovery Recovery of . % of (l~M) ( I ~ M ) [x~C]arginine theoretical

(%)

Hemoglobin Ar~nlne o. 119 0.0457 71.6 54 Histidine o. 119 0.0580 68

/~-Galactosidase Arginine 0.030 o 54-5 o Lysine 0.03 ° 0.0099 61 Glycine o.o3o o.oo3I I9 Serine 0.030 0.0021 13

* Recovery of added radioactivity in the final aqueous: extract before amino acid analysis corrected for the loss of One labelled carbon in the conversion of arginine to ornithine during hydrazinolysis.

DISCUSSION

The presence of a single C-terminal lysine residue per 135 ooo-molecular-weight monomer of fl-galactosidase has been demonstrated in these studies, in agreement with the findings of KOORAJIAN AND ZABIN s. The liberated neutral amino acids which were found in both studies must be non-terminal since they are released neither by carboxypeptidase A nor by hydrazinolysis.

The presence of a single C-terminal amino acid is in accord with the findings of COHN 1°, and WALLENFELS AND ARENS n of a single NH~-terminal threonine residue per 135 ooo-molecular-weight monomer. However, the formation of lower-molecular- weight components after reaction with dilute alkali, formic acid, or sodium dodecyl- sulfateS, 4, t reatments which would not ordinarily be expected to break peptide bonds, has suggested the presence of subunits within the monomer. Furthermore, there is genetic evidence lz obtained from studies of deletion mutants in the Z gene suggesting

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CARBOXYL-TERMINAL RESIDUE OF ~-GALACTOSIDASE I 6 5

that there are at least two distinct complementation units within the gene, one of which codes for a peptide fragment termed omega (to). The discovery of to supports the view that the monomer of/3-gaiactosidase is composed of more than one chain.

Glutamine and asparagine, which form monohydrazides when C-terminal, would not have been detected by the hydrazinolysis procedure but should have been released by carboxypeptidase A.

In view of the genetic and chemical evidence for subunity in the monomer of ~-galactosidase, it is puzzling that only one carboxyl- and one amino-terminal amino- acid has been found. The presence of "masked" terminal residues cannot be excluded. However, the possibility that the monomer unit of ~-galactosidase may be composed of more than one peptide chain held together by covalent bonds which are extremely labile, cannot be excluded.

R E F E R E N C E S

i H. SUND AND K. WEBER, Biochem. Z., 337 (r963) z4. z G. CRAVEN, E. STEERS AND C. B. ANFXNSEN, J. Biol. Chem., 240 (I965) 684. 3 E. STEERS, G. R. CRAVEN, C. B. ANFINSEN AND J. L. BETHUNE, J. Biol. Chem., 240 (I965) 2478. 4 K. WALLENFELS, H. SUND AND K. WEBER, Biochem. Z., 338 (x963) 714 • 5 S. KOORAJIAN AND I. ZABIN, Biochem. Biophys. Res. Commun., I8 (x965) 384. 6 D. H. SPACEMAN, S. MOORE AND W. H. STEIN, Anal. Chem., 30 (x958) xI9O. 7 J. E. FOLK, K. A. PIEZ, W. R. CARROLL AND J. A. GLADNER, J. Biol. Chem., z35 (I96o) 2272. 8 ] . A. WXNSTEAD AND F. WOLD, Biochemistry, 3 (I964) 79I. 9 G. A. BEAY, Anal. Biochem., I (I96o) 279.

IO M. COliN, Bacteriol. Rev., 2i (I957) I4o. IX K. WALLENFELS AND A. ARENS, Biochem. Z., 333 (I96O) 395. IZ A. ULLMANN, D. PEERrN, F. JACOB AND J. MONOO, J. Mol. Biol., i2 (i965) 918.

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