ARCHIVES

OF BIOCHEMISTRY

Crotalus

AND

BIOPHYSICS

adamanteus

NH ,-Terminal

FRANCIS

H. C. TSA0,2 Department

167, 706717

(1975)

Phospholipase

A,-cu: Subunit

Sequence, And Homology Phospholipases’ PAMELA

of Biochemistry,

S. KEIM,

The University Received

AND of Chicago,

November

Structure,

With Other

ROBERT Chicago,

L. HEINRIKSON3 Illinois

60637

25, 1974

Automated Edman degradation of reduced and carboxymethylated phospholipase A,-a from Crotalus adamanteus venom revealed a single amino acid sequence extending 30 residues into the protein from the amino terminus. The singularity of the sequence and the yields of the phenylthiohydantoin amino acids thus obtained indicate that the subunits comprising the phospholipase dimer are identical. Further chemical evidence in support of subunit identity was obtained by cleavage of phospholipase Al-o with cyanogen bromide. Compositional analysis of the protein revealed one residue of methionine per monomer and the sequence determination placed this amino acid at position 10 in the sequence of 133 amino acids. Cyanogen bromide cleavage of the protein, followed by reduction and carboxymethylation afforded the expected 2 fragments: an NH,-terminal decapeptide (CNBr-1) and a larger COOH-terminal fragment of 123 residues (CNBr-II). Automated Edman degradation of the latter has extended the sequence analysis to 54 residues in the NH,-terminal segment of the monomer chain. Comparison of this sequence with those derived for phospholipases from other snake venoms, from bee venom, and from porcine pancreas has revealed striking homologies in this region of the molecules. As expected on the basis of their phylogenetic classification, the phospholipases from the pit vipers C. adamanteus and Agkistrodon halys blomhoffii are more similar to one another in sequence than to the enzyme from the more distantly related viper, Bitis gabonica. Furthermore, the very close similarities in sequence observed among all of these phospholipases in regions corresponding to residues 24 through 53 in the C. adamanteus enzyme suggest that this segment of the polypeptide plays an important role in phospholipase function and probably constitutes part of the active site.

Phospholipases are important enzymes in lipid metabolism and in investigations of phospholipid structure and lipid-protein interactions, especially with regard to deciphering structure-function relationships of biological membranes and lipoproteins (1). Of these enzymes, the phospholipases A, (EC 3.1.1.4), which hydrolyze selectively ‘This work was supported by grants HD-07110 and HL-15062 from the United State Public Health Service and grant GB 29098 from the National Science Foundation. *Present address: Department of Pediatrics of the University of Wisconsin and the Perinatal Center at St. Mary’s Hospital, Madison, WI 53715. *To whom reprint requests should be sent. 706 Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

the 2-acyl groups from sn-3-phosphoglycerides (2), have been studied most extensively. Phospholipases A, possess a number of interesting properties in common, including an absolute requirement for Ca2+ (3), a remarkable heat stability (4, 5) and a high degree of constraint in their tertiary structures imposed by numerous disulfide bonds (6). Their capacity to catalyze reactions in micelles, emulsions, and monolayers has made them objects of considerable interest in studies on the mechanism of heterogeneous catalysis (7). Although most of these enzymes are active as monomers with molecular weights of about 15,000, the phospholipases A, from the rattlesnakes C.

C. ADAMANTEUS

PHOSPHOLIPASE

adamanteus (8) and Crotalus atrox (9), and from bee venom (Apis mellificu) (10) appear to be present in active dimeric forms composed of monomers of about this same size. Volwerk, Pieterson, and de Haas (11) have published evidence for the catalytic importance of histidine-53 in the phospholipase A, zymogen from porcine pancreas. Lysine has been implicated in the function of bee venom phospholipase A (10) and Wells (12) has shown that chemical modification of either a single lysine or of two tryptophan residues per dimer leads to inactivation of the enzyme from C. udumunteus. No direct evidence for the participation of serine in phospholipase catalysis has been published thus far although the reaction has been shown to occur by 0-acyl cleavage (13). The complete amino acid sequences of bee venom (14) and porcine pancreatic (15) phospholipases A, have been determined as well as the placement of disulfide bonds in the latter enzyme (16). In spite of the fact that phospholipases A, have been purified to homogeneity from numerous snake venom sources, (8, 9, 17-27), relatively little information is available presently regarding their primary structures. The complete amino acid sequence of the enzyme from B. gabonica was reported recently by Botes and Viljoen (28), and a partial structure for the phospholipase A, of A. halys blomhoffii was published by Samejima et al. (29). Venom from C. adumunteus contains two forms of phospholipase A, (a! and 0) that have identical amino acid compositions but which differ in chromatographic and electrophoretic behavior (8). Both (Y and /3 forms of this enzyme are dimers which dissociate under certain conditions to subunits of molecular weight approximately 15,000. In view of the fact that monomers from either (Y or p were indistinguishable by chemical and physical means, Wells (30) suggested that they were, in all likelihood, identical. However, nonidentical subunits might be inferred from the fact that the (Y and @ monomers are inactive (30, 31)1, especially since most phospholipases A, are active as monomers (30).

AZ-o

STRUCTURAL

STUDIES

707

The present investigation was undertaken in an attempt to characterize the subunits of C. udumunteus phospholipase A,-cr dimer. The results reported herein indicate that the enzyme monomers are identical single chain polypeptides of 133 residues. Primary structural analysis by automated Edman degradation of the intact reduced and alkylated subunit and a cyanogen bromide fragment derived therefrom has elucidated the complete sequence of the first 54 residues in the monomer. Comparison of this structure with those published for phospholipase A, from porcine pancreas and from venoms of other snakes and the honey bee (A. mellifica) has revealed a number of interesting homologies in the NH,-terminal segments of the molecules which reflect phylogenetic and functional relationships. EXPERIMENTAL

PROCEDURES

Materials. Lyophilized C. adamanteus venom (Lot No.‘s CA2-1, CA43-1) was obtained from the Miami Serpentarium Laboratories and phospholipase AZ-a was purified from this source according to the procedures of Wells and Hanahan (8). Iodoacetic acid from Sigma Chemical Co. was recrystallized from petroleum ether (30-60°C) and diethyl ether. Cyanogen bromide and N,O-bis (trimethylsilyl) acetamide were purchased from Pierce Chemical Co. Fluorescamine (Fluram) was obtained from Roche Diagnostics, Hoffmann-LaRoche Inc., and guanidine hydrochloride (Ultra Pure grade) from Schwartz Bioresearch. [2“C]Iodoacetic acid was the product of Amershami Searle Corp. Reagents employed during the course of automated Edman degradation of polypeptides were the highly purified products of Beckman Instruments Inc., Palo Alto, CA. All other reagents were of the purest commercially available grade. Amino acid analysis. Amino acid compositional data were obtained by automated ion-exchange chromatography on a single column according to the general procedures of Spackman, Stein and Moore (32) and on a Durrum D-500 analyzer. Desalted and lyophilized protein and peptide samples were hydrolyzed in uucuo at 110°C in 6 N HCl for 24,48, and 72 h. Recoveries of serine, threonine, and S-carboxymethylcysteine were corrected for decomposition by extrapolation to zero time. The quantitation obtained by analysis of samples hydrolzyed for 24 h served as a reference for the precise determination of protein or peptide concentrations in samples subjected to automated Edman degradation (33). Amino acid analysis was also employed, together with gas chromatogra-

708

TSAO, KEIM

AND HEINRIKSON

phy, as a means of quantitating the phenylthiohydantoin (PhNCS’) amino acids sequentially released during Edman degradation. For this purpose, the PhNCS derivatives were converted to amino acids by hydrolysis in concentrated hydroiodic acid as described by Smithies et al. (34). Determination of molecular weight. The molecular weights of native phospholipase Al-a dimer and the monomeric subunit were determined, respectively, by ultracentrifugation and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Meniscus depletion sedimentation equilibrium was carried out as described by Yphantis (35). Phospholipase AZ-a (3 mg) was dissolved in 1 ml of mM Tris-HCI buffer, pH 8.0, containing 0.1 M NaCl and this solution was dialyzed against the same buffer. Sedimentation equilibrium ultracentrifugation was performed in a Beckman Model E analytical ultracentrifuge with a sample diluted in the same buffer to a final concentration of 0.5 mg/ml. The analysis was carried out at 188°C for 24 h at 34,000 rpm. Conclusions relative to both the size and the purity of the phospholipase subunit were based upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol (36). Reduction and carboxymethylation. The disulfide bonds in phospholipase AZ-a were reduced with 2-mercaptoethanol and the cysteinyl residues were S-alkylated with iodoacetate by a modification (37) of the general procedures described by Crestfield, Moore, and Stein (38). Lyophilized protein (lo-100 mg) was dissolved in 10 ml of denaturing solvent (0.25 M Tris-HCl, pH 8.6, containing 6 M guanidine hydrochloride and 0.003 M disodium EDTA). Following deaeration for 5 min under a stream of nitrogen, the solution was treated with 100 pl of 2-mercaptoethanol. The reaction vessel was tightly sealed under nitrogen and the solution was incubated at 50°C for 1 h. To the solution at room temperature was added 1.0 ml of a freshly prepared solution of iodoacetic acid in 1.0 N NaOH (270 mg/ml). After reaction at room temoerature for 30 min in the dark. an additional 50 ~1 of mercaptoethanol was added to consume any excess reagent. The solution was then dialyzed exhaustively against distilled water and the modified protein was recovered by lyophilization. When gel filtration was required for removal of salts and reaction byproducts, as was the case described below for the preparation of reduced and S- [“C lcarboxymethylated cyanogen bromide fragments, the reaction volume was reduced to 2.5 ml and glacial acetic acid rather than mercaptoethanol was added to terminate the alkylation. Cleavage mide. The

of phospholipase

original

‘The abbreviation ohydantoin.

AZ-a

by cyanogen

bro-

procedure of Gross and Witkop used is: PhNCS, phenylthi-

(39) for the cleavage of polypeptides at methionine residues with cyanogen bromide was modified somewhat as described by Sterner, Noyes and Heinrikson (40). Desalted, native phospholipase A,.(Y (20 mg; 1.3 rmol of monomer) was dissolved in 3 ml of 70% formic acid containing a 100-fold molar excess of cyanogen bromide. The reaction vessel was sealed under nitrogen and maintained at room temperature for 24 h in the dark. At this time, a second quantity of CNBr equal to the first was added to ensure completion of the reaction. After an additional 24-h period, excess cyanogen bromide, solvent, and volatile reaction byproducts were removed by lyophilization, and the peptides were subjected to reduction and carboxymethylation with [2-“Cliodoacetic acid (3 x 10’ dpm/gmol) as outlined above. The reaction mixture (3 ml) containing the reduced and carboxymethylated cyanogen bromide fragments was diluted with an equal volume of glacial acetic acid and the solution was applied to a column (1.5 x 90 cm) of Sephadex G-50 eluted with 50% acetic acid. Fractions of 2 ml were collected at a flow rate of 12 ml/h, and the elution of fragments was monitored by absorbance at 280 nm, by radioactivity, and by the fluorescence generated following reaction of the peptides with fluorescamine (41). In the fluorescamine reaction, 100 rl of each fraction was added to 3.5 ml of 0.2 M sodium borate buffer, pH 8.0-8.5. Fluorescamine (50 ficl of a solution containing 0.15 mg of reagent/ml of acetone) was added with vigorous mixing and the fluorescence of the solutions was determined in a fluorometer (Turner Model 111) with a Wratten Filter No. 8. The radioactivity of the fractions was measured with a Nuclear-Chicago liquid scintillation counter (ISOCAP/SOO). Based upon the elution profile thus obtained (cf. Fig. l), appropriate fractions were pooled and lyophilized. Automated Edman degradation. Procedures for the automated Edman degradation (42) of reduced and carboxymethylated phospholipase AZ-a and the larger of the two fragments generated therefrom by cleavage with cyanogen bromide (CNBr-II) were essentially as described earlier (33). A Beckman protein-peptide Sequencer (Model 890-C) was employed for these operations with the Quadro15 program supplied by the manufacturer (program MKII-8). The PhNCS amino acids liberated after each cycle of degradation were identified and quantitated by gas chromatography (43) and by amino acid analysis following hydrolytic back conversion in hydroiodic acid (34). Identifications were confirmed in some instances by thin layer chromatography of the PhNCS-derivatives (44). The positions of S-carboxymethylcysteine residues were 6Quadrol is a registered trademark of the Wyandotte Chemical Corp., Wyandotte, Mich., for the substance N,N,N’,N’-tetrakis-(2.hydroxypropyl)ethylenediamine.

C. ADAMANTEUS

PHOSPHOLIPASE

Ap-a STRUCTURAL

709

STUDIES

gle sequence, together with the recovery of Leu-2 in 72% yield based upon the monomeric molecular weight, lend strong support to the conclusion that the subunits in RESULTS phospholipase A, dimer are identical. Both the initial yield (>72%) and repetitive Molecular weight studies. Before undertaking chemical studies relative to the yield (99% calculated with respect to Leu-2 subunit structure and partial sequence and Leu-18) are in harmony with our usual experience with intact polypeptides. Moreanalysis of C. adamanteus phospholipase AZ-~, it was deemed appropriate to charac- over, there is no evidence that any of the constituent chains are blocked at the NH,terize our preparation in terms of monomer terminus. and dimer molecular weight. Meniscus Noteworthy in the NH*-terminal sedepletion sedimentation equilibrium (35) quence of 30 residues is the occurrence of a of the native phospholipase dimer gave a methionine at position 10. The composimolecular weight of 28,800. In this calculaanalysis of phospholipase AZ-~ tion, the partial specific volume (a) of the tional protein was determined from amino acid dimer published by Wells and Hanahan (8) Amino compositional data (45) normalized to a gives two residues of methionine. acid analysis of reduced and carboxymethcontent of 10 valine residues per dimer (uide infra). Polyacrylamide gel electrophoresis of the phospholipase (20 pg) in the TABLE I presence of sodium dodecyl sulfate and IDENTIFICATION AND QLJANTITATION OF 2-mercaptoethanol (36) indicated the PHENYLTHIOHYDANTOINS SEQUENTIALLY REMOVED BY presence of a single band, the mobility of AUTOMATED EDMAN DEGRADATION OF REDUCED AND which, relative to known standards, correCARBOXYMETHYLATED PHOSPHOLIPASE A,-& sponded to a subunit roughly’ 14,000 in PhNCS RePosition PhNCS Fkmolecular weight. These findings are in Position in sederivacovery” in sederivacovery” good agreement with the molecular weight q”C?IlCe tive (nmol) quence tive (nmol) values for the dimeric and monomeric 1 Ser 65 16 Ser 5 protein previously reported (30) and cor2 Leu 99 17 Gly 5 roborate the earlier observations that the 3 Val 105 18 Leu 18 protein is dissociated under denaturing Gln 38 19 4 Leu 25 conditions. 5 Phe 40 20 6 Tw

assigned during the sequence analysis of reduced and S- [‘Elcarboxymethylated CNBr-II by monitoring each PhNCS derivative for radioactivity.

Demonstration of subunit identity and partial sequence analysis of the monomer.

Having confirmed the existence of dissociable monomers in native phospholipase, we were still faced with the question as to whether or not the subunits are identical. Identical monomers are easily documented both by the yields of the PhNCS derivatives sequentially liberated by automated Edman degradation, and by the singularity of the sequence thus generated. Although it was reported that the NH,-terminus of phospholipase AZ-~ is blocked (8), we found this not to be the case. As may be seen in Table I, automated Edman degradation of 2 mg of the reduced and carboxymethylated Iprotein led to the elucidation of a single amino acid sequence extending 30 residues i:nto the polypeptide. This sin-

6 7 8 9 10 11 12 13 14 15

Glu Thr Leu Ile Met Lysc Val Ala Lysc Arf

37 38 48 43 33 30 30 32 30 30

21 22 23 24 25 26 27 28 29 30

Tyr Serc Ala TyF Gly CMCys Tyr CMCys Gly Trp

4 -3 6 -3 4 4 7 2 4 3

n Automated Edman degradation of reduced and carboxymethylated phospholipase A*-LY (2 mg, 133 nmol) was carried out for 30 cycles using the Quadrol program MKII-8. * Except where otherwise indicated, quantitation was obtained by gas chromatography of the PhNCSamino acid or its silylated derivative (43). c Quantitated and identified by hydrolytic back conversion to amino acids followed by amino acid analysis (34).

710

TSAO,

KEIM

AND

ylated phospholipase was performed after varying times of hydrolysis and the data given in Table II are expressed in terms of the subunit composition. As expected, our analysis of the monomer gave values for most of the amino acids which were approximately half those reported earlier (8). Our results indicate a single methionine residue in the subunit of 133 amino acids and the molecular weight of the monomer calculated from these data (14,782) is in good agreement with values obtained by physical characterization (14,400). In order to corroborate our contention of identical subunits, native phospholipase AZ-a was cleaved with cyanogen bromide and the products were reduced and S-carboxymethylated with radioactive iodoacetate. If the subunits are identical, this procedure should yield a nonradioactive decapeptide containing homoserine and having no uv absorbance at 280 nm, and a COOH-terminal fragment of 123 residues accounting for all of the ‘C-label in the mixture of fragments. As may be seen in Fig. 1 and Table II, these suppositions were fully realized by experimentation. Two peaks were obtained by gel filtration of the reduced and S- [“Clcarboxymethylated cyanogen bromide fragments. Compositional analysis of the first peak, labeled CNBr-II in Fig. 1, was consistent with that expected for the COOH terminal fragment (Table II). Homoserine and its lactone were absent in the analysis and all of the radioactivity incorporated into the fragments was accounted for by this fraction. The nonradioactive second peak, CNBr-I, analyzed for the decapeptide expected on the basis of the sequence determination (Table I). As shown in Table II, this fragment alone contained homoserine. No other fragments were observed and CNBr-I and CNBr-II were isolated in yields of 85% and 80%, respectively. These findings, together with the results of sequence analysis of the intact, alkylated polypeptide chain provide chemical evidence that native phospholipase AZ+ is a dimer composed of identical subunits. In an attempt to extend the sequence of 30 residues obtained by analysis of the

HEINRIKSON

intact subunit (Table I), automated Edman degradation was performed on 14 mg (-930 nmoles) of “C-labeled CNBr-II which extends from Lys-11 to the end of the monomer chain. Stepwise degradation was carried out over 44 cycles with unambiguous identification of the single PhNCS derivative at each step (Table III). A single sequence was generated in the determination, and the repetitive yield calculated from the recoveries of Ala-13 and Ala-39 was 97%. The initial yields of the first three PhNCS amino acids were roughly 53-76%, in line with our usual observations. Since the cysteine residues were converted to the S- [“C lcarboxymethyl derivatives, their location in the sequence was confirmed by radioactivity measurements. Sequence analysis of CNBr-II confirmed the identifications made on the intact chain (Table I) of residues 11 through 30, and extended the known sequence of the phospholipase Al-a! monomer to 54 residues in the NH,-terminal segment of the molecule. This 54-residue sequence is presented in Fig. 2 for purposes of comparison with those reported earlier for phospholipases from the venoms of A. halys blomhoffii (29), B. gabonica (28), and A. mellifica (14), and from porcine pancreas (15). DISCUSSION

Automated Edman degradation is a tool that has been applied successfully in our laboratory to questions regarding the subunit structures of yeast inorganic pyrophosphatase (33), glutamine synthetase (46), and phytohemagglutinin isomitogenic proteins (47). The observation of single or multiple sequences and the quantitative nature of the means employed in the identification of the PhNCS derivatives as they are released sequentially during each degradative cycle serve as a basis for conclusions regarding the identity or nonidentity of subunits in the sample under analysis. In the case of the C. adamanteus phospholipase A*-(Y, we have shown that the subunits are identical through the first 54 amino acids in the monomer chain of 133 residues. Of course, an unequivocal claim for subunit identity must await elucidation

C. ADAMANTEUS

PHOSPHOLIPASE

As-o

TABLE AMINO

ACID COMPOSITION

Amino

acid

STRUCTURAL

711

STUDIES

II

OF REDUCED AND CARBOXYMETHYLATED PHOSPHOLIPASE AZ-~ AND FRAGMENTS THEREFROM BY CLEAVAGE WITH CYANOCEN BROMIDE Phospholipase Relative molar quantities”

CNBr-I

A,-cu Residues

(per subunit, M, 14,400)

=

Relative molar quantities”

DERIVED

CNBr-II Residues

Relative molar quantitie&

Residues

Carboxymethylcysteine

13.2

14

0.0

0

13.8

14

Aspartic

15.7

16

0.3

0

15.6

16

Threonine

6.8

7

0.9

1

5.3

6

Serine

7.1

7

0.9

1

5.7

6

12.7

13

2.1

2

10.6

11

Proline

8.7

9

0.1

0

9.5

9

Glycine

12.5

13

0.2

0

12.5

13

Alanine

8.0

8

0.1

0

8.0

8

Valine

5.3

5

1.0

1

4.1

4

Methionine

0.9

1

0.0

0

0.0

0

Isoleucine

5.3

5

1.0

1

4.2

4

Leucine

5.2

5

1.9

2

3.4

3

Tyrosine

7.9

8

0.1

0

8.0

8

Phenylalanine

4.5

5

1.0

1

3.6

4

Histidine

2.2

2

0.0

0

2.1

2

Lysine

7.4

7

0.1

0

7.5

7

Arginine

5.3

5

0.1

0

5.1

5

Tryptophan’

2.5

3

0.0

0

2.5

3

0.6

1

0.0

0

acid

Glutamic

acid

Homoserine lactone Total

residues

+ asparagine

+ glutamine

+ homoserine

-

-

133

10

123

0 Reduced and carboxymethylated protein was hydrolyzed in 6 N HCl at 110°C in uucuo for 24,48, and 72 h. Values for amino acids stable to acid hydrolysis are an average of three analyses with Ala set at 8.0 residues. Threonine and serine were corrected for decompositional losses by back extrapolation to zero time. h Native enzyme was cleaved with cyanogen bromide followed by reduction and carboxymethylation and the fragments were isolated as described in Fig. 1. Analyses were performed on 24 h hydrolyzates without correction for decomposition. CNBr-I, Val = 1.0; CNBr-II, Ala = 8.0. ( Determined spectrophotometrically (51).

712

TSAO, KEIM AND HEINRIKSON

10 20 30 40 FRACTION

NUMBER

FIG. 1. Gel filtration of the products obtained by cleavage of native phospholipase A1+ with cyanogen bromide followed by reduction and alkylation. Native enzyme (20 mg) was treated with cyanogen bromide and, after reduction and S-[“Clcarboxymethylation, the products were separated on a column (1.5 x 90 cm) of Sephadex G-50 eluted with 50% acetic acid (cf. Experimental Procedures). Peaks designated CNBr-I and CNBr-II correspond to the NH,-terminal decapeptide and the COOH-terminal fragment of 123 residues, respectively. Fluorescence observed by reaction of portions of each fraction with fluorescamine is expressed in arbitrary units.

of the entire covalent structure (work which is currently in progress). An earlier report by Wells and Hanahan (8) gave evidence based upon dansylation that the NH,-terminus of C. adamanteus phospholipase A,-a is blocked. The results of the present investigation clearly establish that serine is the NH,-terminal residue in this enzyme. That the dansyl derivative of serine might have been overlooked in the previous study is probably due in part to the difficulties inherent in the application of this method to proteins. Moreover, dansyl serine is sensitive to the conditions of acid hydrolysis required for its release from the polypeptide chain (48). By all criteria applied, such as chromatographic and electrophoretic behavior, physical characterization, specific enzyme activity, and compositional analysis, our enzyme is identical to the preparation of Wells and Hanahan (8). In a strategic sense, the sequence determination performed on the S-alkylated phospholipase provided the key for another experiment whereby subunit identity could be documented, namely, the identification of methionine at position 10. Phospholi-

pase AZ-~ contains two methionines per dimer (8). If the subunits are identical, only two products should result from cleavage with cyanogen bromide, and indeed, this procedure liberated the expected NH,terminal decapeptide (CNBr-I) and the COOH-terminal fragment of 123 residues (CNBr-II) (Fig. 1, Table II). These fragments were isolated in yields exceeding 80% based upon the monomeric molecular weight, and no other peptides were observed. In view of the evidence for subunit identity, and the fact that compositional analyses published heretofore have been based upon the enzyme dimer, the amino acid analysis of the monomer was determined (Table II). The residue numbers obtained for each amino acid correspond to roughly half those determined earlier for the dimer (8) and the compositional data indicate a monomer chain of 133 amino acids containing seven disulfide bonds. The molecular weight calculated from the analysis (14,782) is in accord with the results of physical measurements cited herein and elsewhere (8). With the sequence of the 30 NH*-termi-

C. ADAMANTEUS

PHOSPHOLIPASE

nal residues in the phospholipase monomer in hand, it was of interest to obtain further sequence information by Edman degradation of the COOH-terminal cyanogen bromide fragment (CNBr-II) which begins with Lys-11 and extends to the end of the chain. In so doing, 44 residues of CNBr-II were placed, thus confirming residues 11-30 and extending the known sequence to a total of 54 residues into the monomer chain (Fig. 2). Six of the 14 half cystine residues were located in this region of the molecule, together with three of the five arginines. The latter finding is of importance to the completion of the structure by automated Edman degradation since the fragments generated by tryptic cleavage at TABLE IDENTIFICATION

Cycle

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

no.

AND QUANTITATION DEGRADATION Residue

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

no.

A,-a!

STRUCTURAL

arginine in lysine-blocked protein should be large and amenable to this approach. Moreover, all of the six arginine fragments can be placed immediately in sequence, thus eliminating the problem of fragment alignment. Of more immediate significance, however, is the comparison of this sequence with those obtained for phospholipases from another pit viper (A. halys blomhoffii), an Old World viper (B. gubonica), honey bee venom (A. mellifica), and porof these secine pancreas. Alignment quences as depicted in Fig. 2 reveals a truly striking homology in the regions corresponding to residues 1 through 10 and 24 through 53, numbered with respect to the III

OF PHENYLTHIOHYDANTOINS SEQUENTIALLY OF REDUCED AND S- [“CICARBOXYMETHYLATED

PhNCS derivative

Recoverp (nmol)

LYS’ Val Ala LYSC Argc SeP GlY Leu Leu Tw TV SeP Ala TV GUY CMCy& TV CMCy+ GUY Trp ‘JY GUY

493 625 708 500 402 369 415 472 518 317 444 250 449 395 333 250 (350) 379 250 (320) 230 254 190 197

c *

713

STUDIES

Cycle

23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

no.

Residue

33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

REMOVED BY AUTOMATED CNBr-II” no.

EDMAN

PhNCS derivative

Re-;)P

GUY GUY Argc Pro Glnc Asx’ Ala Thr Ser iw CMCys’, CMCy& Phe Val His Asx CMCys CMCys Tyr GUY LYS Ala

190 200 150 169 100 141 89 95 24 76 52 60 78 61 60 50 45 41 15 10 20

p *

(70) (85)

(53) (66)

u Automated Edman degradation was performed on CNBr-II (14 mg, 930 nmoles) from phospholipase AZ-a. This fragment comprises residues 11-133 in the monomer chain. Program = Quadrol MKII-8. o Except where otherwise indicated, recoveries were determined by quantitative gas chromatography (43) of the PhNCS amino acid or its silylated derivative. Figures in parentheses are recoveries calculated on the basis of quantitative radioactivity measurements of PhNCS-S-[‘*C]carboxymethylcysteine. c Identified and quantitated by hydrolytic back conversion to amino acids (34) followed by amino acid analysis. d Confirmed by thin layer chromatography (44). p Identified and quantitated by radioactivity measurements (see footnote b).

I

adamanteus

ha1

s blomhoffii

H2N t

Il.?

Ile-Tyr-Pro-Gly-

&r-Ala---

FIG. 2. Sequence homologies in the NH,-terminal segments of phospholipases from snake and bee venoms and from porcine pancreas. Numbering of residues in the phospholipases from A. halys blomhoffii (29), B. gabonica (28), pancreas (15), and A. mellifica (14) is made with reference to the C. adamanteus sequence reported herein. Dashes (- - -) indicate gaps introduced by the authors to provide the highest degree of homology; brackets denote the beginning ([) or termination (I) of unknown sequences. The sequence shown for the A. halys blomhoffii enzyme is a revision of that published by Samejima et al. (29) based upon structural comparisons with the other phospholipases (cf. Discussion). Arg-42 in this enzyme is enclosed in parentheses since the following peptide was derived by tryptic hydrolysis; this residue could be a lysine, but the homology argues for its assignment as arginine. The sequence of this tryptic fragment from A. halys blomhoffii phospholipase is based in large part upon homology. The Asp, Asn, and Asx residues at positions 38, 41, and 49 are considered homologous without regard to their state of amidation.

A. mellifica -___

P0rcine pancreatic

-B. gabonica

-.A

-C. adamanteus

A. mellifica -___

pancreatic

C.

C. ADAMANTEUS

PHOSPHOLIPASE

C. adamanteus phospholipase. This comparison has revealed what we believe to be an error in the A. halys blomhoffii sequence of Samejima et al. (29). These workers applied manual Edman degradation to their protein and found no NH,-terminal residue. They then cleaved with cyanogen bromide and found the tripeptide Ser-LeuHSer and the blocked heptapeptide, (PCA)-Phe-Glu-Thr-Leu-Ile-HSer. They concluded that the sequence was PCAPhe-Glu-Thr-Leu-Ile-Met-Ser-Leu-Met, However, when the fragments are reversed as shown in Fig. 2, the sequence Ser-LeuMet-Glx-Phe-Thr-Leu-Ile-Met is exactly that obtained for the C. adamanteus enzyme with the exception of Val-3. It is our contention, based upon experience with the manual Edman degradation of polypeptides, that the procedure did not work with the intact protein and that cyanogen bromide cleavage liberated a heptapeptide (residues 4410) with Gln as the NH,-terminal residue which then cyclized to pyroglutamate. This provides a reasonable explanation for the proposed misalignment of the cyanogen bromide fragments by Samejima et al. (29). The amended sequence for the A. halys blomhoffii phospholipase shows a strong homology to the C. adamanteus enzyme and a lesser but convincing homology t.o the phospholipase from B. gabonica and porcine pancreas. The phylogenetic relationships between the Viperidae and Crotalidae (Solenoglypha) based upon morphology (49) are thus corroborated by tbe degree of similarity in their phospholipase sequences. It is of interest that Leu-2, Gln-4, Phe-5, and Ile-9 are conserved in all the enzymes listed in Fig. 2. The close similarities in these phospholipase sequences are most dramatically visualized in the region extending from residue 24 to 53. In this segment of the molecule the C. adamanteus enzyme is nearly identical to that from B. gabonica and the few differences, such as the existence of an arginine at position 35 in the former rather than a lysine, are conservative. It is unfortunate that we do not as yet have the sequence in this region for the A. hal.ys blomhoffii enzyme, but one of the tryptic

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peptides (T-6-2) reported by Samejima et al. (29) fits very nicely, with one amendment, in the region from residue 43 through 53. On the basis of the homology with the other snake venom phospholipases, it seems likely that the sequence is His,,Asp,, rather than the reversed sequence reported (29). It is often observed that histidine residues cyclize and cleave during the coupling stage of the Edman degradation (50) thus giving rise to “preview” of the next residue in sequence. The structure shown in Fig. 2 for the tryptic undecapnptide from A. halys blomhoffii reflects this alteration in sequence and the previously unassigned residues (29) are positioned with respect to homology with the known sequence in the other snake venom enzymes. The strong homology exhibited by the snake venom phospholipases from residues 24-53 is evident to a lesser degree in the enzymes from bee venom and porcine pancreas. In their comparison of the latter two enzymes with the phospholipase from B. gabonica, Botes and Viljoen (28) aligned Ile-1 of the bee venom enzyme with Pro-14 and Lys-11 of the pancreatic and viper enzymes, respectively. This operation yielded little evidence of homology between the bee venom enzyme and the other two. However, when the sequences are aligned as shown in Fig. 2, Cys-28, Gly-29, Gly-31, Asx-38, Arg-42, and Cys-49 are conserved in all of the phospholipases, and the bee venom protein shows a much closer structural similarity to the pancreatic phospholipase. After residue 53, the striking similarities evident in this region disappear, at least for the proteins of known sequences (14, 15, 28). It will be of interest to have the complete structures of the two pit viper enzymes from A. halys blomhoffii and C. adamanteus to further this comparison. For the present, however, it is more than idle speculation to infer that the phospholipases have homologous active sites and that this portion of the molecule must comprise in part the highly invariant region between residues 24 and 53. Our information regarding active site residues is scant; the only specific amino acid

716

TSAO,

KEIM

AND

implicated thus far is His-53 (11) in the pancreatic phospholipase zymogen (His-45 in Fig. 2). Although a histidine is present two residues removed in the snake venom enzymes, the bee venom protein, aligned as shown in Fig. 2, has a histidyl residue corresponding exactly to that in the pancreatic enzyme. Lysine and tryptophan have been suggested to be of functional significance in the C. adamanteus phospholipase A, (12). The only tryptophyl residue in the region of interest is that at position 30, and it is present also in the gaboon viper enzyme. It is noteworthy that this amino acid is “locked in” between three residues that are conserved in all the enzymes. Lys-53 is present in all the snake venom phospholipases and it is the only lysine in the sequence between 24 and 53 in the rattlesnake phospholipase. These clues provided by sequence homology as to the identification of the lysine, tryptophan, and possibly histidine residues of functional importance may facilitate their verification by chemical modification. As mentioned above, comparison of the known phospholipase sequences from residue 53 to the end of the chains reveals much less homology. In their comparative analysis, Botes and Viljoen (28) pointed out the existence of the sequence Ala-AlaIle-Cys-Phe in the COOH-terminal regions of both the pancreatic and B. gabonica phospholipases. Since the disulfide bond pairings in the pancreatic prophospholipase A, are known (16), it is of interest to see whether or not the half cystine residue in the aforementioned sequence (Cys- 111) is bonded to a half cystine in the region of high homology, i.e., in residues 24 through 53 (Fig. 2). Indeed, the partner for Cys-111 in the porcine pancreatic proenzyme is Cys-57 which corresponds to Cys-49 in Fig. 2. Therefore, this region of homology in the COOH-terminal half of the phospholipases lies in close proximity in the three-dimensional structure to the highly homologous sequence comprising residues 42-53 (Fig. 2) and may constitute another portion of the active site. It is interesting, however, that Shipolini et al. (14) did not find the sequence Ala-Ala-Ile-Cys-Phein bee

HEINRIKSON

venom phospholipase. This enzyme, in contrast to the B. gabonica and pancreatic phospholipases which are active in the monomer form, depends upon the dimeric structure for activity (30). If this sequence were absent from C. adamanateus phospholipase, which is also catalytically active as the dimer, but present in the A. halys blomhoffii enzyme (active monomer) it could be inferred that this segment of the structure is important in regard to monomer or dimer functionality. It is clear that more phospholipase structures must be determined in order to establish with confidence the phylogenetic and functional interrelationships suggested in this paper. In the meantime, our contention that the active site of the phospholipases comprises, in part, some segment of the sequences between residues 24 and 53 (with respect to the C. adamanteus protein) is supported by these comparative studies of enzymes from widely divergent species. Thus far, the hypothesis is consistent with the results of chemical modification studies and it will serve as a model for future experimentation. ACKNOWLEDGMENTS The authors wish to thank Ferenc J. Kezdy for helpful course of this investigation.

Drs. John discussions

H. Law during

and the

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