The Seed Proteins of Abrus precatorius L.

Short Communication

The Seed Proteins of Abrus precatorius 1. DAVID

R. MURRAY*) and FRANKLIN VAIRINHOS**)

*) Biology Department, University ofWol!ongong, N.5.W. 2500 Australia **) Department of Agricultural Biochemistry, Waite Research Institute, Glen Osmond, South Australia Received October 11, 1982· Accepted October 18, 1982

Summary The globulin fraction was found to occupy the minor proportion of total seed protein in

Abrus precatorius (43%). The main lectin (agglutinin) accounted for about 30% of total extractable protein and contributed to both the albumin (water-soluble) and the globulin fractions. This agglutinin consists of two disulphide-linked polypeptides: 36,000 + 34,000 = 70,000 daltons. The major globulin, considered to be vicilin, consists of non-covalent associations of polypeptide(s) of 60,000 daltons. The legumin of A. precatorius lacks a large subunit of the size typical of Vicieae and Cicereae (ca. 40,000 daltons) and instead appears to consist of disulphidelinked pairs of polypeptides of equal size, 21,000 + 21,000 = 42,000 daltons. This main component of Abrus legumin is present as a minor constituent of the seed globulins of Cicer arietinum (Cicereae) and representatives of Vicieae, the tribe to which Abrus formerly belonged.

Key words: Abrus precatorius L., albumin, embryo, globulin, lectin, reserve protein, seed.

Introduction Twice in recent history we have witnessed the expulsion of a member of the Vicieae to form a new monogeneric tribe. Abrus was excluded by Dormer (1946) and Hutchinson (1964) to form Abreae, a move supported by Kupicha (1977) and Polhill (1981). Cicerwas in turn excluded by Kupicha (1977,1981) to form Cicereae. The inheritance of seed proteins has considerable relevance to the phylogenetic classification of legumes. Earlier studies of Abrus precatorius L. suggest that the albumins (Simola, 1969) and the globulins (Boulter et aI., 1967) include proteins that are immunochemically or electrophoretically distinct compared to corresponding protein fractions from members of the Vicieae. As we have recently compared the seed globulin composition of three members of Cicereae with representatives of genera remaining in Vicieae (Vairinhos and Murray, 1982 b), we were interested in extending our studies to Abrus precatorius. The seeds of this widespread tropical Abbreviations: Con A, concanavalin A; ME, 2-mercaptoethanol; MW, molecular weight; SDS, sodium dodecyl sulphate.

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legume are well known for their scarlet and black seedcoats, and as the source of an extremely potent phytotoxin, abrin (Kingsbury, 1964; Oisnes et ai., 1974 a, b).

Materials and Methods Seeds of Abrus precatorius L. came from Florida, U.S.A., and were kindly provided by Dr. R.

J. Whelan. The seeds were cut in half longitudinally and the seedcoats were removed. Forty

embryos weighing 2.94 g were milled in a coffee grinder. 1 g portions of meal were extracted with 10 ml of the medium of ~oulter et al. (1967), 5 % (w/v) K2S04 in 0.1 M Na phosphate, pH 7.0, for 1 h at room temperature. The suspension was squeezed through two layers of cheesecloth and centrifuged at 10,000 g for 10 min at 5°C. Albumin (water-soluble) and globulin (water-insoluble) fractions were then obtained following exhaustive dialysis as before (Murray and Vairinhos, 1982a, b; Vairinhos and Murray, 1982 b). The globulin fraction was largely redissolved in 1 % K2S04 in 0.02M Na phosphate, pH 7.0, containing 40% (v/v) glycerol and 0.02 % (w/v) Na azide (Murray and Vairinhos, 1982 b). This solution could be stored frozen at -20°C without any loss of protein by precipitation on subsequent thawing. Protein solutions were subjected to electrophoresis in «Gradipore" gradient gels, type GG-l, 2.5% to 27% acrylamide, obtained from Gradient Gels Pty. Ltd., Sydney, N.S.W. Australia as previously described (Murray and Vairinhos, 1982a, b; Vairinhos and Murray, 1982 a, b). Standard proteins applied to gradient gels were: ferritin (450,000), catalase (240,000), euphaseolin (150,000), bovine serum albumin (68,000) and ovalbumin (45,000). Samples of each protein fraction were treated with ethanol to a final concentration of 80 % (v/v) to precipitate protein for analysis by the biuret reaction (Gornall et aI., 1949), or for analysis on SDS-polyacrylamide disc gels (Weber and Osborn, 1969). The ethanol-insoluble precipitates were treated as before (Murray, 1979a, b; Murray and Crump, 1979) and loaded at 50 JLg protein per gel. Standard proteins used under reducing conditions were: bovine serum albumin (68,000), catalase (60,000), ovalbumin (45,000), lactate dehydrogenase (36,000), carbonic anhydrase (29,000), ferritin (18,500) and cytochrome c (12,000). Double diffusion tests against the jack bean lectin Con A were conducted in agarose gels as before (Murray and Knox, 1977; Murray and Crump, 1979) except that the wells at the origin contained 5 JLl of each fraction. All gels were stained with Coomassie brilliant blue.

Results Distribution ofProtein between Albumin and Globulin Fractions Embryos of A. precatorius yielded extractable protein representing 14.6 % of dry matter. After dialysis, the water-soluble (albumin) fraction accounted for the major proportion of the total, 56.7 %.

Gradient Gel Electrophoresis The protein components of the water-insoluble (globulin) and water-soluble (albumin) fractions were separated by gradient gel electrophoresis as shown in Fig. 1. The most prominent globulins were those of estimated MW 160,000 and 300,000 daltons. Other prominent globulins were found to range in size from 230,000 up to 550,000 daltons, however proteins in the 60,000 to 135,000 dalton region were poorly resolved. Proteins of MW less than 60,000 were not detected in the globulin fraction. In the water-soluble fraction, proteins of MW 48,000, 53,000 and 65,000 were all Z. Pjlanzenphysiol. Bd. 108. S. 471-476. 1982.

473

Seed proteins of Abrus precatorius L.

G

A

- 48 - 53

- 65 - 88 135-

135

160- 190

230-

245

300440500-

Fig. 1: Coomassie brilliant blue-stained proteins following gradient polyacrylamide gel electrophoresis of water-insoluble (G, 47 ~g) and water-soluble (A, 49 ~g) protein fractions from Abrus precatorius embryos. MW x 10- 3 daltons.

550

distinguished by their violet colour after staining with Coomassie brilliant blue, in contrast to the more usual blue colour displayed by all the other bands. The most prominent albumins were those of MW 65,000, 88,000 and 190,000 daltons. In addition, a prominent broad band of MW 135,000 daltons and a well defined band of 245,000 daltons were distributed in both fractions. Disc Gel Electrophoresis

For the water-soluble fraction, reduction with ME allowed the dissociation of a major polypeptide of MW 70,000 into component subunits of MW 36,000 and 34,000 daltons (Fig. 2). The same polypeptides are present in the globulin fraction (Fig. 3), but here they represent a smaller proportion of the total protein in the fraction. The main component of the globulin fraction is the polypeptide of MW 60,000 that does not dissociate on reduction with ME (Fig. 3). Two other non-dissociating polypeptides of MW 65,000 and 55,000 daltons in the water-soluble fraction (Fig. 2) were violet after staining and correspond to the proteins of MW 65,000 and 53,000 observed after gradient electrophoresis (Fig. 1). Other polypeptides associating via disulphide-linkages are those of MW 29,000 (Figs. 2, 3) and 21,000 (Fig. 3). The latter corresponds in size to the small subunit of legumin, but is apparently derived from the polypeptide of MW 42,000 confined to the globulin fraction (Fig. 3 a).

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DAVID R. MURRAY and FRANKLIN VAIRINHOS

474

a

70

b

c

>100 7560-

- 65 - 55

b

c

60

42-

-- ~6 4

-H

29

- 17

a

29

-

Fig. 2

21

--

-

Fig. 3

Fig. 2: SDS-polyacrylamide disc gels of the water-soluble fraction dissociated in the absence (a) and presence of ME (b, c). The acrylamide content of the gel is 10% (a, b) or 14% (c). MW x 10- 3 daltons. Fig. 3: SDS-polyacrylamide disc gels of the globulin fraction dissociated in the absence (a) and presence of ME (b, c). The acrylamide content of the gel is 10 % (a, b) or 14 % (c). MW X 10- 3 daltons.

Double Diffusion vs. Concanavalin A As shown in Fig. 4, a component of the globulin fraction migrating towards the anode is a glycoprotein capable of interacting with the lectin Con A. A second almost immobile interacting species is a component of the water-soluble fraction, which did not show any of the anodal component.

t

I~I~~I"IIII~IIIIIII~I

Fig. 4: Double diffusion of Con A (centre channel) against components of globulin (g) and total (t) protein fractions from embryo extracts following electrophoresis in agarose. The dye marker ran 2.8 cm towards the anode (right).

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Discussion

The Distribution ofAbrin and Agglutinin The most prominent of the polypeptides linked by disulphide bonds are those of MW 36,000 and 34,000, occurring in both the water-soluble (Fig. 2 b, c) and globulin fractions (Fig. 3 b, c). It is clear from the studies of Olsnes et al. (1974 b) that these two polypeptides comprise the main lectin (agglutinin) of A. precatorius. Olsnes et al. (1974 b) concluded that the purified agglutinin of this species consisted of non-covalent associations of disulphide-linked polypeptide pairs of MW 36,000 + 33,000 = 69,000 and 35,000 + 33,000 = 68,000 daltons. By comparison, our seed sample did not display this variation in the size of the larger subunit. This would have been detected using 14 % gels (Figs. 2 c, 3 c). The fully associated lectin accounts for the region of MW 135,000 shared by both fractions prior to dissociation with SDS (Fig. 1). The shared band of MW 245,000 (Fig. 1) could represent a higher aggregate of the same lectin subunits. The relatively minor polypeptide of MW 29,000 (Figs. 2, 3) represents the A chain of abrin, which consists of this subunit linked covalently to one of the larger chains of the type found in the agglutinin (Olsnes et al., 1974 a, b). In its possession of high seed lectin content, A. precatorius resembles Canavalia ensi· formis (jack bean) and allied species, which possess from 23 % to 32 % of total seed protein in the form of the lectin Con A (Hague, 1975).

Relationships ofAbrus Seed Proteins to Legumin and Vicilin Dudman and Millerd (1975) reported that proteins interacting with antisera directed against authentic legumin and vicilin from Vicia faba were detectable in extracts from seeds of A. precatorius. Within the globulin fraction (Fig. 1), a protein corresponding to vicilin in size (MW 160,000) is more prominent than another corresponding approximately to that expected for legumin (MW 300,000). The anodal Con A-interacting glycoprotein which is confined to the globulin fraction (Fig. 4) is probably vicilin. It cannot be the agglutinin or abrin, since these are distributed in both fractions. Nor can it be legumin, since legumin from Pisum sativum does not interact with Con A, whereas vicilin does (Murray and Crump, 1979). The vicilin from Abrus must consist mainly of polypeptide(s) of MW 60,000, the predominant polypeptide of the globulin fraction in both the absence and presence of ME (Fig. 3). It is thus simpler than the vicilin of Cicer, which includes polypeptides of MW 73,000,37,000 and 27,000 daltons, and indeed simpler than the vicilin of any member of the Vicieae (Table 2 ofVairinhos and Murray, 1982 b). The major components of legumin from representatives of Vicieae and Cicereae are disulphide-linked pairs of polypeptides of estimated MW 36,000 to 46,000 and 20,000 to 22,000 daltons respectively (Vairinhos and Murray, 1982 b). Amongst the disulphide-linked polypeptides of the globulin fraction of A. precatorius there is none that obviously corresponds to the large subunit of legumin. The polypeptides of MW 36,000 and 34,000 are the paired subunits of the agglutinin (see above). Their fairly

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equal staining intensities preclude the coincidence of a legumin large subunit with either (Fig. 3). We must conclude that the legumin of Abrus consists primarily of disulphide-linked subunits of equal size, 21,000 + 21,000 = 42,000 (Fig. 3). This major component of legumin in A. precatorius has already been detected as a minor constituent of the globulins of Cicer arietinum, and is similar to other minor disulphidelinked polypeptide pairs occurring in members of Vicieae (Table 1 of Vairinhos and Murray, 1982 b). Until this recent evidence, we had not considered that these were related to legumin. Acknowledgements We thank Dr. R. J. Whelan for providing the seeds of Abrus precatorius and Mrs. M. Cordova-Edwards for assistance.

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