310

Biochimica et Biophysica Acta, 5 3 7 ( 1 9 7 8 ) 3 1 0 - - 3 1 9 © Elsevier/North-Holland Biomedical Press

BBA 38057

THE COMPLETE AMINO ACID SEQUENCE OF THE a-SUBUNIT OF PEA LECTIN, PISUM SATIVUM

C H A R L E S R I C H A R D S O N *, W. D A V I D B E H N K E , J A M E S H. F R E I S H E I M a n d K E N N E T H M. B L U M E N T H A L **

Department of Biological Chemistry, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, Ohio 45267 (U.S.A.) (Received May 10th, 1978)

Summary The complete primary structure of the a-subunit of the lectin from the pea

(Pisum sativum) has been determined using a combination of tryptic and staphylococcal protease digestion, purification using Sephadex gel filtration and high-voltage electrophoresis followed by either manual or automated Edman degradation. The molecular weight of the a-subunit from sequence data and gel filtration in guanidine-HCl is close to 5800, which is lower than that determined by sedimentation equilibrium techniques. The sequence reveals considerable homology to concanavalin A and near identity to the a-subunit of the lentil lectin (Lens culenaris). As in the case of the lentil a-subunit, the a-methyl glucose binding site(s) are not present in this region, nor are the $1 and $2 metal ion binding sites as judged by homology consideration, though the residues for the $3 lanthanide binding (Glu 87 and Asp 136) are conserved from the available data on the a- and fi-subunits. Preliminary metal exchange experiments on the intact pea lectin indicate some differences in the metal exchange properties of this lectin compared to concanavalin A, and therefore possible ligand variations in this region of the fi-subunit.

Introduction Pea lectin (Pisum sativum), like concanavalin A, is a mitogen [1] and preferentially agglutinates tumor cells [2]. Studies of the inhibition of hemagglutinaSupplementary data to this article, giving details of amino acid c o m p o s i t i o n of a-subunit, tryptic peptides and staphylococcal protease peptides, are deposited with, and m a y b e obtained from: Elsevier S c i e n t i f i c Publishing Company, BBA Data Deposition, P.O. Box 1345, 1000 BH Amsterdam, T h e Netherlands, R e f e r e n c e s h o u l d b e m a d e to No. RBA/DD/092/38057/537 (1978) 310. * In partial fulfillment of t h e r e q u i r e m e n t s for the degree of Ph.D., University of Cincinnati. ** To w h o m correspondence should be directed.

311 tion demonstrate its saccharide binding specificity to be similar to concanavalin A [3]. Pea lectin has been purified by affinity chromatography on Sephadex [4,5]. The lectin has a reported molecular weight of 49 000 and consists of two non¢ovalently linked subunits of approx. 7000 and 17 000. The subunit structure is a2f12. Unlike concanavaiin A, chemical modification such as succinylation or acetylation does not disrupt the quaternary structure [1]. The amino acid composition of pea lectin has been reported [5]. The protein contains high amounts of threonine, serine and aspartic acid and no methionine or cysteine. The purified protein contains both Mn 2+ and Ca2+ which may be related to activity [6]. The addition of Mn 2+ and Ca2+ enhance the precipitating activity of pea lectin. The presence of EDTA inhibits both hemagglutination and precipitating activities. The N-terminal sequence of both units has been reported [ 7]. There is a striking homology between portions of the a-subunit and concanavalin A. Upon modification with 2-nitrophenylsulfenyl chloride one tryptophan is modified with simultaneous loss of biological activity [8]. A peptide containing the modified tryptophan is homologous to a region where carbohydrate may bind in concanavalin A. Any understanding of the biological effects of lectins must be based upon the understanding of the molecular structure of the proteins, themselves. The characterization of the pea lectin with emphasis on its primary structure and its comparison with concanavalin A provide an important additional approach to the molecular structure of lectins. Materials and Methods Pea lectin was isolated as described by Trowbridge [1]. The protein was denatured by dissolving in 6 M guanidine hydrochloride and the large and small subunits separated by gel filtration on a column of Sephadex G-50 fine equilibrated with 6 M Gdn-HC1. Purified subunits were dialyzed exhaustively against distilled water and lyophilized. Trypsin treated with L-l-tosylamido-2-phenylethylchloromethylketone and carboxypeptidase Y were purchased from Worthington, staphylococcal protease (Staphylococcus aureus, strain V8) from Miles, and aminopeptidase M from Sigma. Reagents used for Edman degradation were either Sequenal grade products of the Pierce Chemical Co., or highly purified products from Beckman Instruments. 57% HI was obtained from Fisher and was used without further purification. Silica gel thin-layer sheets with fluorescent indicator were purchased from Eastman. All other reagents were the purest grades commercially available. Enzymic methods The sample to be hydrolyzed was suspended in 0.1 M NH4HCO3 buffer (pH 8) at a concentration of 0.5--1.0 pmol/ml and treated with 2--4% by weight of the desired protease. Hydrolyses were carried out for either 4 or 16 h at 40°C and were terminated by acidification to pH 3.0 followed by lyophilization. For the longer times of hydrolysis, the enzyme : substrate ratio never exceeded 1:40. For determination of amides, 10--20 nmol peptide was dissolved in 0.1 ml

312 0.1 M NH4HCO3 buffer (pH 8) and hydrolyzed with 200 munits aminopeptidase M for 16 h at 40°C. Free carboxylates and amides were quantitated by amino acid analysis. COOH-terminal sequences of the intact protein and certain peptides were examined by hydrolysis with carboxypeptidase Y as described by Hayashi et ai. [9]. The amino acids released were identified and qu.antitated by amino acid analysis. Molecular weights. The molecular weights of the large and small subunits were assessed by gel filtration in the presence of 6 M Gdn-HC1 [10]. Standard proteins employed (all reduced and alkylated) were chicken lysozyme, bovine pancreatic trypsin inhibitor, insulin B chain, and the hexapeptide Leu-Trp-MetArg-Phe-Ala. A plot of log Mr versus elution position was linear for these standards. Peptide purification. Peptides were initially fractionated on a column (1.5 X 100 cm) of Sephadex G-25 superfine equilibrated with 50 mM NH4HCO3, and were detected in the eluate by ultraviolet absorption at 230 nm. Further purification was by preparative paper electrophoresis at pH 1.9 (90--100 kV × min). The purity of the peptides was assessed by thin-layer chromatography in 1-butanol/pyridine/acetic acid/water (15 : 10 : 3 : 12, v/v, [11] ). Edman degradations. Amino-terminal sequences of the intact subunit or the C-terminal tryptic fragment derived therefrom were derived by use of the Beckman Sequenator using DMBA program I; silylated phenylthiohydantoins were identified by gas-liquid chromatography [12]. Manual degradation of small peptides was carried out as described by Peterson et al. [13]. The anilino-thiazolinone derivatives were either hydrolyzed directly to the free amino acid in HI [14] and identified by amino acid analysis or converted to the phenylthiohydantoins and identified by thin-layer chromatography on silica gel sheets [15,16]. Amino acid analysis and related procedures. Samples were hydrolyzed at 110°C for 22--96 h in evacuated glass tubes containing 1 ml 6 M HCI and 1 drop 1% aqueous phenol. Analyses were performed with a Durrum D-500 analyzer, and all results are expressed as molar ratios. Unless otherwise specified, values given in the tables are uncorrected either for the partial destruction of certain amino acids during hydrolysis, or for incomplete liberation of isoleucine and valine after 22 h. Subunit tryptophan content was determined by N-bromosuccinimide titration [ 17]; the presence of tryptophan in peptides was ascertained by ultraviolet absorption between 300 and 270 nm and by reactivity with Ehrlich's reagent

[11]. Results

The complete amino acid sequence of the a-subunit of pea lectin is shown in Fig. 1. Also shown are the tryptic and staphylococcal protease peptides derived from the intact subunit. Peptides are numbered sequentially from the NH2 terminus of the protein.

Amino acid composition and molecular weight The amino acid composition of ~-subunit calculated from the sequence is in

313 10

20

VAt-THR-SER-TYH-THR-LEU-StR-~SP-VAL-VAt-SER-LEU-tYS-~SP-VAt-VAt-PRO-Gt~-TRP-VAt-ARG-lLESP-las SP-lb‘ ---_-sm-----cTI-a---+ l -?-,------yJ-,---

-

TI-b-------,

n

.t-----pJMe----.lqJ

-

-

GLY-PHE-SER-ALA-THR-THR-GLY-ALA-GLU-TYR-ALA-AL~~-HlS-Gt~-VAt-tEU-SER-TRP-SER-PHE-~iS-SER-SP-2 l csp-3-ssp-4----.-.--.-0 7 7--------v----------T-3-----Z-Z -----10 CLU~LEU-SER-FLY-TI'R-SER-SER-LYS-GL~i-Coot! . SP-5t--r--s---* -Fig. 1. Amino acid sequence of pea lectin o-subunit. Peptides are designated as either tWPtic (T) or &Iphylococcal protease (SP). Residues positioned by Edman degradation (-_) or hydrolysis with carboxypeptidwe y (.-) are indicated. Residues included within the sewenator run performed On intact subunit are also shown. (-)

good agreement with that we obtained by analysis, but at variance with the composition reported in the literature [5]. This variance can be largely attributed to differences in the subunit molecular weight, reported to be 7000 by Trowbridge [ 51. In our hands, molecular weight estimates obtained by gel filtration in 6 M Gdn-HCl [lo] have given molecular weights of 5800 and 15 500 for the cy- and P-subunits, respectively; the molecular weight of 5800 for the a-subunit is in excellent agreement with the value of 5753 obtained by summation of the sequence. Purification of tryptic and staphylococcal protease peptides Initial fractionation of each of these peptide mixtures was accomplished by gel filtration on a column (1.5 X 100 cm) of Sephadex G-25 superfine equilibrated with 50 mM NH4HC03. Either four (tryptic) or three (staphylococcal protease) peaks of A 230nm absorbing material were separated (Figs. 2 and 3). Peptide T3 was obtained in a pure state from fraction 1. All other fractions in

E

1

l.O--

no l-J

.

E

E

O.B--

I

2

.6--

e

.6--

5 g

.4--

.

E 0.6.z

(\

z 0.4-:: : 0.2--

0 : a

*

*2--

/ 2'0

do FRACTION

do

io NUMBER

I60

20

40 FRACTION

60

BO

100

12C

NUMBER

Fig. 2. Fractionation of tryptic peptides of a-subunit on a column (1.6 X 100 cm) of Sephader G-26 superfine esuilibmted with 60 mM NH4HCOg. Fractions (1.6 ml) were collected at a flow rate of 18 ml/h and monitored by their absorbance at 230 nm ( -). Pooled fractions are indicated by solid bars. Fig. 3. Fractionation of staphylococcal protease peptides of o-subunit on Sephadex G-25 superfine. The column and elution conditions are identical to those in Fig. 2. Pooled fractions are indicated by solid bars.

314

both digests were heterogeneous and were purified further by preparative paper electrophoresis at pH 1.9.

Sequence determinations A u t o m a t e d Edman degradation of 270 nmol intact a-subunit was performed as described elsewhere [12]. Although gaps appeared at positions 3, 19, 25, 27 and 28, the unambiguous identification of phenylthiohydantoin-amino acids liberated at the remainder of the first 30 cycles allowed alignment of the three tryptic peptides. Valine was the sole NH2-terminal residue present and there was no evidence for sample heterogeneity at any step. The repetitive yield was 9370 (Table I). Peptide T3 was likewise submitted to automated Edman degradation with a repetitive yield of 9470 (Table II). The sequence information obtained extended the known structure through position 38 and allowed alignment of the four staphylococcal protease peptides used to complete the C-terminal sequence. Gaps encountered in the automated degradation were filled by manual Edman degradation of small tryptic or staphylococcal protease peptides as described elsewhere [13]. In most cases, the amino acid derivative released at each step was identified qualitatively after conversion to the phenylthiohydantoin [8,9] or b y high-voltage electrophoresis after hydrolysis in 5770 HI [7]. Neutral amino acids in HI hydrolyzates were identified and quantitated by amino acid analysis.

Carboxy-terminal analysis Quantitative determination of the C-terminal residue was complicated b y the extreme insolubility of purified a-subunit, even in the presence of 4--6 M urea. Yields of amino acids released by carboxypeptidase Y were variable when calculated on a mol to tool basis with protein. In one experiment, however, 0.8 mol glutamine per mol protein were released after 4 h treatment with carboxypeptidase Y in the presence of 4 M urea, suggesting that it was the C-terminal residue. This placement was supported b y Edman degradation of peptide SP-5. Seven Edman cycles accounted for all amino acids present, except for Glx.

TABLE

I

AMINO-TERMINAL DEGRADATION

SEQUENCES

OF

INTACT

~-SUBUNIT

DERIVED

BY

AUTOMATED

EDMAN

N u m b e r s g i v e n b e n e a t h e a c h residue r e f e r t o t h e n m o l o f p h e n y l t h i o h y d a n t i o n - a m i n o acid o b s e r v e d at t h e g i v e n s t e p . I n t h o s e cases w h e r e q u a n t i t a t i o n w a s d i f f i c u l t , t h e s y m b o l + i n d i c a t e s qualitative i d e n t i f i c a tion.

Residue identified by Gas-liquid c h r o m a t o g r a p h y s p o t t e s t

NH 2 -Val-Thr-Ser-Tyr-Thr-Leu-Ser-Asp-Val-Val 120 + + 92 + 80 + 63 6 5 6 5

Gas-liquid c h r o m a t o g r a p h y s p o t t e s t

Ser-Leu-Lys-Asp-Val-Val-Pro-Glu-Trp-Val-Arg-Ile + 80 6 0 58 55 + 35 26 + 20

Gas-liquid chromatography

Gly-Phe21 21

-Ala25

-Gly-Ala-Glu-Tyr-Ala 17 13 8 7 9

315 T A B L E II AMINO-TERMINAL TION

SEQUENCE OF PEPTIDE T3 DERIVED BY AUTOMATED EDMAN DEGRADA-

N u m b e r s g i v e n b e n e a t h each residue refer to the n m o l o f p h e n y l t h i o h y d a n t i o n - a m i n o acid o b s e r v e d a t the given s t e p . In t h o s e cases w h e r e q u a n t i t a t i o n w a s difficult, the s y m b o l + i n d i c a t e s q u a l i t a t i v e identification. Residue identified by Gas-liquid chromatography spot test

NH2-ne-Gly-Phe-Ser-Ala-Thr-Thr-Gly-Ala-Glu-Tyr-Ala 295 330 315 + 300 + + 272 264 185 124 210

Gas-liquid chromatography spot test

Ala-His-Glu-V~-Leu-Ser195 + 120 108 126 +

Gas-liquid chromatography

Gly9

Phe-His-Ser-Glu-Leu43 + + 15 12

-Set+

Quantitative hydrolysis with aminopeptidase M [11] failed to release any glutamic acid and indicated the presence of four serine residues. Since glutamine and serine were not resolved in this analysis, this supported the assignment of glutamine rather than glutamic acid. Finally peptide SP-5 was shown to have a charge of +1 at pH 6.5; if residue 53 were glutamic acid, the peptide would have been neutral. Discussion The molecular weight of pea lectin a-subunit has been reported to be 5200 based on sedimentation equilibrium in 6 M guanidine-HC1 [5]; size heterogeneity in these experiments was evidenced by upward curvatures in semilogarithmic plots of concentration against r 2. Other methods gave a subunit molecular weight of 7000 with no evidence of heterogeneity, from which Trowbridge concluded that this latter value represented the true subunit molecular weight. We have obtained a value of 5800 by gel filtration in 6 M guanidine-HC1, which is consistent with the structural studies reported herein. Two-dimensional fingerprint analysis of the tryptic and staphylococcal protease digests of a-subunit revealed the presence of five and seven ninhydrin-positive spots, respectively. All of these peptides have been isolated and sequenced, and these sequences are accommodated by the structure shown in Fig. 1. The fact that no peptide was lost during purification, and that the molecular weight calculated from this sequence, 5753, is in excellent agreement with the value obtained by gel filtration, argues conclusively that the earlier molecular weight estimates were in error. Certain aspects of the sequence determination itself are noteworthy. One of two aspartyl peptide bounds in the subunit was cleaved by staphylococcal protease under conditions whereby this enzyme is reported to be specific for glutamyl bonds [18,19]. This cleavage may be due to the extended time (16 h) employed for this digest. More surprising was the cleavage by trypsin at the Tyr4-Thrs bond. The trypsin used for this digestion had been treated with L-l-tosylamido-2-phenylethylchloromethyl ketone by the supplier to block

316 I(i

ZU

P[ P

PR'] 'S[ IJ TF:P VAL

LBiTIL

PR,3-SEll

/z PEA LDJT]L C,I;'J A

IRP.V,^L -

30

3@ ARG-ILE-Giv PHE SER f~L,", The IF~R GLY-ALA 5LU ~Q!5-I LE-Qv-PHE-SF R-A1A- II :~- IFP-SLY-AL,",-ELU,

q0 $EP TRP SH- l!~l'

~SFP,-h&A~II:R-',qLYIL!:LF[YR '~O

,~EF .kP if)} bLl

lENTIL

set< PHEIASN)SERIGU41LEU~:ILY-HIS~TPR%ER.]Iys SFiICOOll

CON A

SEIVDHEITIIRS~LS~JLYSL~LL.~_]LY~ SER A$*JS [ ~ I H R HIS ,3Lr4-

I

llO

I

I

I

I

~

I

12[)

Fig. 4. C o m p a r i s o n o f t h e a m i n o a c i d s e q u e n c e s of c o n e a n a v a l i n A ( C o n A), l e n t i l l e c t i n ~ - s u b u n i t , a n d p e a l e c t i n ~ - s u b u n i t . I d e n t i c a l r e s i d u e s &re e n c l o s e d in b o x e s ; a single gap h a s b e e n i n t r o d u c e d in t h e conc a n a v a l l n A s e q u e n c e b e t w e e n S e t - 7 4 a n d V a l - 7 5 to m a x i m i z e h o m o l o g y . T h e p e a a n d l e n t i l n u m b e r i n g s y s t e m is s h o w n a b o v e a n d c o n c a n a v a l i n A n u m b e r i n g b e l o w t h e a c t u a l s e q u e n c e s .

residual c h y m o t r y p t i c activity. As an added precaution, the enzyme was dissolved in 1 mM HC1 to inactivate any residual chymotrypsin if present [20]; nonetheless, a single c h y m o t r y p t i c site was cleaved. It should be pointed o u t that a similarly anomalous cleavage has recently been reported in the sequence of lentil lectin a-subunit [21], a protein highly homologous to pea lectin {Fig. 4). Great difficulties were encountered in determination of the carboxyl terminus of a-subunit, in part due to peptide insolubility. A high degree of quantitative variation was observed in carboxypeptidase Y digestions; our best results indicated a b o u t 0.8 mol glutamine released per c o l protein after 4 h in 4 M urea. Glutamine was assigned as the C-terminal residue of peptide SP-5 by subtractive Edman degradation, by aminopeptidase M digestion, and by the net charge of SP-5 (+1) at pH 6.5. In retrospect, the quantitative variability observed is most likely due to differential cyclization of released glutamine to 2-pyrrolidone carboxylic acid generated u p o n termination of carboxypeptidase Y hydrolysis b y acidification to pH 2.4. Trowbridge [5] has presented evidence for charge heterogeneity in pea lectin a-subunit. Attempts were made to resolve these forms on DEAE-ceUulose columns at pH 8.8 [5]. In no case were we able to detect the late-eluting component A. Since this c o m p o n e n t represents no more than 5% of the total a-subunit we would suggest that it either represents an impurity in the earlier preparations, or that ~its relative a m o u n t varies according to the batch of peas used. Whatever the case, no c o m p o n e n t A is present in our preparations; the sequence reported is that of the B form. Moreover, we have observed no amidation heterogeneity at any position in the sequence determination. A comparison of the sequence obtained for the pea lectin to that determined for concanavalin A indicates considerable h o m o l o g y between the two proteins and particularly between the a-subunits of the pea and lentil lectins (Fig. 4). A single t w o residue deletion has been introduced to maximize the homology, identities of amino acids are b o u n d b y a solid border. The homology between the first 30 residues was reported earlier by van Driessche et al. [7]. This sequence includes the chemically modified peptides

317 isolated by Cermakova et al. [8] which they reported were obtained from modification of only the large subunit. Attempts to reproduce this experiment in this laboratory (unpublished data) have resulted in the modification of both subunits. Therefore, the disposition of this sequence in the pea lectin is not, as yet, clear. The second region of homology runs from valine 37 through leucine 46. These two regions are connected by a stretch of amino acids which have no apparent relationships with the corresponding residues in concanavalin A. An alternative method for expressing these results is to calculate a minimum base change per codon for these regions. The minimum base change per codon is equal to the summation of the nucleotide base changes necessary to transpose a given sequence into another divided by the number of residues involved. The minimum base change per codon for the entire pea ~-subunit sequence as presented is 0.62, demonstrating a considerable homology between it and concanavalin A. Even more striking are the different values obtained for particular regions of the pea a-subunit sequence. The minimum base change per codon for the two homologous regions are: 0.40 for residues 36--46 and 0.29 for residues 8--28. The connecting region from residues 29--35 has a value of 1.57, clearly not homologous. Since the crystallographic structure of concanavalin A has been well established [22,23], it is important to examine the position and role of the homologous sequences in the structure of concanavalin A. Consider first the homologous region from, using concanavalin A numbering, residues 106--115 (Fig. 4). In concanavalin A this region is part of the 'back' fl-structure described by Becket et al. [22]. This pleated sheet forms almost the entire back surface of the molecule and is involved with most of the interactions involved in dimer and tetramer formation. Of particular interest are lysine residues 114 and 116, which in concanavalin A form salt bridges with glutamic acid 192 to stabilize the tetrameric species at neutral pH [23]. It has been suggested that modification of these lysines is responsible for maintaining succinylated concanavalin A as a dimer at neutral pH. In both the pea lectin small subunit and the lentil ~-subunit [21], these two lysines are not conserved, but are replaced by glutamic acid and serine residues. In addition, the C-terminal region of pea ~-subunit which is not greatly conserved, contains residues in concanavalin A that provide many tetramer contact points. Therefore, one might predict that the pea lectin is incapable of forming a similar tetrameric molecule. Both the C-terminal region of pea a-subunit and the internal non-homologous region (concanavalin A numbering 9 9 - 1 0 6 ) are exposed to solvent and serve as connecting loops for ~-pleated sheet regions. There apparently has been no evolutionary pressure to preserve homologous sequences in these regions. In contrast, the region from residues 78 to 98 in concanavalin A have been strictly conserved. As noted earlier, the minimum base change per codon for this region is 0.29. In concanavalin A, the latter portion of this sequence is part of the front ~-pleated sheet. Residues 80--90 serve as a bridge between the two fl-pleated sheet regions and form the opening of the hydrophobic binding site [24]. In fact, the hydrophobic binding region of concanavalin A contains the side chains of leucine 81 and 85, valine 89 and 91, phenylalanine 111 and

318 serine 113; all of which are conserved in the pea lectin with the exception of a leucine to valine and a valine to isoleucine replacement. It should be noted that this hydrophobic binding region in concanavalin A contains residues which are conserved in both regions of the pea a-subunit sequence. The homologies evident in the sequence of pea a-subunit are, in concanavalin A, a portion of both major fi-pleated sheet regions. This implies that pea lectin, like concanavalin A, has considerable fi-structure. This would be consistent with the remarkable similarity in the far ultraviolet circular dichroic spectra of concanavalin A and the pea lectin [25]. There are none of the ligand elements of the concanavalin A $1 or $2 metal binding sites in the a-subunit of the pea lectin; if present, these sites must be confined to the ~-subunit. From the sequence data that is available for both the a- and ~-subunits of the pea lectin, the $3 lanthanide binding site ligands (Glu 87 and Asp 136) appear to be conserved suggesting that an additional paramagnetic locus may be present as in the case of concanavalin A [26]. Both fluorescence and additional sequence studies are currently in progress to address some of the remaining important unanswered questions (vide infra). There is certainly considerable biological significance in the sequence currently available. Chemical modification of concanavalin A by succinylation has produced a derivative of concanavalin A with differential mitogenic and agglutination properties [27]. Some of these differences have been attributed to a change in valency, since the modified concanavalin A cannot form tetramers at neutral pH. The pea lectin may be considered to have the same valence as succinyl-concanavalin A, y e t the mitogenic and agglutination properties of pea lectin are similar to those of concanavalin A [1--3]. Factors other than valency may be responsible for the differential effects of modified concanavalin A derivatives as suggested by Trowbridge [ 1]. It is noteable that residues involved in the formation of the hydrophobic binding site of concanavalin A are so highly conserved in the pea lectin. It is possible that this is coincidental, y e t it is tempting to postulate a functional role for this site. Poretz and Goldstein [28] have given evidence for a hydrophobic binding region near the saccharide site. Until recently, it was thought that the hydrophobic cavity contained a saccharide site adjacent to the hydrophobic region [22]. Perhaps the hydrophobic cavity is an extended binding region on concanavalin A that interacts in a complex manner with the cell surfaces. The a-methyl-D-glucopyranoside binding site near the top of the molecule may then serve as an effector site. Such a proposal is consistent with the suggestion by Cuatrecasas [29] that the a-methyl-D-glucopyranoside binding site was distinct from the membrane binding site. Clearly, there is n o t y e t enough evidence to support this same model in the pea lectin with the limited data available. Strong evidence should include a combination of distance measurements, such as were utilized for determination of the a-methyl-D-glucopyranoside site and structural modification attempts such as affinity labeling. It would also be important to compare the sequence of the large subunit of pea lectin to concanavalin A for the presence or absence of the postulated ~-methyl-D-glucopyranoside site(s). Such studies are currently in progress.

319 Acknowledgements We are pleased to acknowledge the assistance of Mr. Dale Blankenship for amino acid analyses and automated Edman degradations. This work was aided by grants CA-11666 (J.H.F.) and NS-14368 (K.M.B.) from the National Institutes of Health. K.M.B. is a Research Career Development Awardee, National Institutes of Health (1 K04 NS 00304). References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

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The complete amino acid sequence of the alpha-subunit of pea lectin, Pisum sativum.

310 Biochimica et Biophysica Acta, 5 3 7 ( 1 9 7 8 ) 3 1 0 - - 3 1 9 © Elsevier/North-Holland Biomedical Press BBA 38057 THE COMPLETE AMINO ACID SE...
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