Euphaseolin, the Predominant Reserve Globulin of Phaseolus vulgaris Cotyledons

Botany School, University of Melbourne, Parkville Vic. 3052, Australia

Euphaseolin, the Predominant Reserve Globulin of Phaseolus vulgaris Cotyledons DAVID R. MURRAY and JANET A. CRUMP With 6 figures Received February 19, 1979 . Accepted March 22, 1979

Summary A single globulin accounts for 72 % of the total salt-extractable protein from the cotyledons of kidney bean, Phaseolus vulgaris L. cv. Hawkesbury Wonder. This globulin is mainly (but not entirely) insoluble at pH 4.7 and comprises three subunits of MW 54,000, 49,000 and 46,000, which associate non-covalently in the ratio 1 : 1 : 1. Heterogeneity of the polysaccharide moieties associated with the polypeptide subunits was indicated by the small proportion of the total globulin capable of interacting with the lectin concanavalin A. The name «euphaseolin» is preferred for this globulin, since its properties clearly distinguish it from both legumin and vicilin. Key words: Cotyledon, euphaseolin, glycoprotein, globulin, Phaseolus vulgaris.

Introduction The major globulins from pea (Pisum sativum 1.) and broad bean (Vicia faba 1.) cotyledons have been studied extensively and are called legumin and vicilin (OSBORNE and HARRIS, 1907; MILLERD, 1975; DERBYSHIRE et a!., 1976; THOMSON et a!., 1978). Legumin is insoluble at its isoelectric point at about pH 4.7, while vicilin remains soluble at this pH. Legumin is larger than vicilin, having an aggregate molecular weight (MW) of approximately 330,000 daltons, compared to 180,000 for vicilin. Their relative sizes are frequently given as 11 to 13 S compared to 7 S respectively. Globulins from many species of legumes have been equated with either legumin or vicilin solely on the basis of this solubility difference or by comparison of sedimentation coefficients (DANIELSON, 1949; DERBYSHIRE et a!., 1976). There is at present considerable confusion in the literature concerning the major reserve globulin from the cotyledons of kidney bean, Phaseolus vulgaris L. OSBORNE gave this fraction the name «phaseolin» (OSBORNE and CLAPP, 1907), but currently different authors are claiming that this globulin is analogous both to legumin (SUN and HALL, 1975; SUN et a!., 1975) and to vicilin (BOLLINI and CHRISPEELS, 1978; ERICSON and DELMER, 1978). This globulin has also been referred to by several other names (Table 1). It has been reported to be insoluble at pH 4.7 (SUN and HALL,

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1975) and at pH 5.0 (PUSZTAI and WATT, 1970). However, BARKER et a1. (1976) and DERBYSHIRE and BOULTER (1976) reported that the major reserve globulin from P. vulgaris was soluble at pH 4.7. Table 1: Terms used to refer to the major globulin from seeds or cotyledons of P. vulgaris L. Name

Authors

Phaseolin

OSBORNE and CLAPP (1907) KLOZ et ai. (1966) KLOZ (1971)

Glycoprotein II

PUSZTAI and WATT (1970) RACUSEN and FOOTE (1971) BARKER et ai. (1976)

GI globulin

McLEESTER et ai. (1973) ROMERO et ai. (1975) SUN and HALL (1975) SUN et ai. (1975) HALL et ai. (1977)

Euphaseolin

KLOZ and KLOzovA (1974)

In view of these apparent contradictions, the properties of the major globulin fraction from the cotyledons of P. vulgaris cv. Hawkesbury Wonder, a variety of red kidney bean, have been studied and compared with the reported properties of globulin fractions from other bean cultivars, and with legumin and vicilin from pea cotyledons. Materials and Methods Bean seeds, Phaseolus vulgaris L. cv. Hawkesbury Wonder, were from the same batch used in our enzyme studies (CRUMP and MURRAY, 1978). The experiments reported here were completed within 12 months of the previous work. Pea seeds were imbibed under the same conditions as before (COLLIER and MURRAY, 1977).

Preparation of Cotyledon Extracts Samples of 6-9 cotyledons from the same number of imbibing seeds were weighed and thoroughly ground in a cold mortar using acid-washed sand and chilled extraction medium, at a ratio of 1 : 5 (w/v). The extraction media used were 25 mM Na phosphate, pH 7.25, 0.1 M Tris-CI, pH 7.8, or 0.1 M Na borate, pH 8.0, each containing 0.5 mM 2-mercaptoethanol (ME). The homogenates were stirred continuously at room temperature for a further 40 min, squeezed through 2 layers of cheesecloth and centrifuged at 20,000 X g for 20 min at 5 dc. Samples of the clear extracts were withdrawn from beneath the surface lipid layer. Preparation of Seed Meal Extracts The yields of total protein extracted by various media were compared using seed meal prepared using an electric coffee grinder. In addition to those media listed above, 0.5 M NaCI in 50 mM Na phosphate, pH 7.5 (BARKER et aI., 1976) and 0.5 M NaCI, 0.25 M ascorbic acid, pH 2.4 (ROMERO et aI., 1975) were used. Equal portions of meal were extracted with

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each medium at a ratio of 1 : 10 (w/v) by continuous stirring at 25°C for periods up to 3 h. Homogenates were clarified as above.

Fractionation of Proteins Extracted from Cotyledons Small volumes (5-10 ml) of phosphate (pH 7.25) extracts from bean cotyledons were measured into dialysis tubing (9.5 mm wide) and dialysed for 18 h at 5°C against several changes of 0.1 M Na acetate buffer, pH 4.7, containing 0.5 mM ME. Following dialysis, the contents of the dialysis sac were carefully resuspended before being squeezed into a centrifuge tube. Samples (0.5 or 1.0 ml) were removed and centrifuged separately using a bench centrifuge with swing-out head. The pH 4.7 insoluble precipitates were washed once by resuspension with 0.1 M Na acetate and recentrifuged. Recovery of protein following dialysis was 95 Ofo or better. Preparation of Euphaseolin and «Gl Globulin» The major bean globulin was isolated after passage of cotyledon extracts (pH 7.25) through DEAE-Sephadex A50 (COLLIER and MURRAY, 1977) and Sephadex G-200. The «GI» fraction was prepared according to ROMERO et al. (1975) by diluting 0.5 ml samples of pH 2.4 extract with 2.5 ml of cold distilled water and collecting the resultant precipitates by centrifugation. The precipitates were redissolved in 0.5 M NaCI and reprecipitated by addition of water in the same ratio as before (5 : 1, v/v). Preparation of Legumin and Vicilin After initial separation of pea cotyledon extracts into albumin and globulin fractions (MURRAY et aI., 1978), the globulins were redissolved and separated into legumin and vicilin fractions by isoelectric precipitation of legumin against 0.1 M Na acetate buffer, pH 4.7. Protein Determination Samples of the various soluble fractions were treated with ethanol and the precipitates washed once with 80 Ofo ethanol (MURRAY, 1979). All precipitates were dissolved in 0.25 M NaOH and protein was measured by the biuret reaction using lipid-extracted bovine serum albumin (BSA) as standard (COLLIER and MURRAY, 1977). Polyacrylamide Gel Electrophoresis Disc gel electrophoresis in the presence of SDS and in the presence and absence of ME was performed according to WEBER and OSBORN (1969). Gels were set from 10 Ofo acrylamide unless specified otherwise. The molecular weights of polypeptide bands were estimated by comparison of mobilities with the same standards as before (MURRAY et aI., 1978). Double Diffusion Tests These were carried out in 1 % agarose gels set on microscope slides as described by MURRAY and KNOX (1977). Separation of bean cotyledon fractions by electrophoresis prior to double diffusion against concanavalin A (Con A) was performed in 1 Ofo agarose gels set in Tris-barbitone buffer (pH 8.8), which was also the running buffer (MURRAY and KNOX, 1977).

Results Extraction of Proteins from Bean Cotyledons

Although borate buffer at alkaline pH has been favoured by a number of workers and WATT, 1970; BOLLINI and CHRISPEELS, 1978), comparison of borate medium with phosphate medium revealed that the borate medium extracted only (PUSZTAI

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DAVID R. MURRAY and JANET A. CRUMP 100

~

_

.....c.

I~

75

f!

50

II

II II /1

25 1

a

15 30 TIME

60

120

(min)

Fig. 1: Time course of extraction of protein from seed meal by 25 mM Na phosphate, pH 7.25 (6) and by 0.1 M Na borate, pH 8.0 (0). Each buffer contained 0.5 mM ME.

90010 (± 2010) of the total protein extractable from seed meal (Fig. 1) or from imbibed cotyledons. The addition of 0.5 M NaCI to phosphate buffer (BARKER et al., 1976) did not significantly increase the amount of protein extracted by phosphate medium alone. Tris-CI, pH 7.8, also extracted as much protein as the phosphate medium. Borate is clearly unsuitable for the quantitative extraction of protein from bean cotyledons. Polypeptide Composition of the Major Fractions from Bean Cotyledons

The major globulin isolated from bean cotyledons accounted for 72 010 of the total phosphate-extractable protein and showed a polypeptide composition dominated by three components of MW 54,000, 49,000 and 46,000 (Fig. 2). Traces of polypeptides of MW 23,000, 21,000 and 18,500 were also present in preparations of this major globulin, which we shall refer to as «euphaseolin» following the suggestion of KLOZ and KLOZOVA. (1974). Additional polypeptides of various sizes were present in «GI globulin» fractions (Fig. 3) prepared exactly as described by ROMERO et al. (1975). One band of approximate MW 92,000 was dissociated by ME (Fig. 3) but this polypeptide is too large to correspond to the <
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-

Fig. 2: Polypeptide composition of euphaseolin (e) from P. vulgaris cv. Hawkesbury Wonder. (MW X 10-3). b = BSA, CI> C 2 = cytochrome c.

BOLLINI and CHRISP EELS (1978), but must be 1 : 1 : 1, a formula which is consistent with the protomeric MW of 160,000 determined by ROMERO et al. (1975) and with the mobility of the protomer in slab gels of varied pore size (MURRAY et aI., 1978). Fig. 4 shows scans of SDS-polyacrylamide gels following electrophoresis of pH 4.7 soluble, pH 4.7 insoluble and total protein fractions from 2-day imbibed cotyledons. Euphaseolin is distributed as the main constituent of the pH 4.7 insoluble fraction, and a very minor component of the pH 4.7 soluble fraction. The latter showed major components of MW 30,000 and > 100,000, corresponding to the monomeric and tetrameric forms of phytohaemagglutinin (PHA) (ALLAN and CRUMPTON, 1971; PUSZTAI and WATT, 1974; LIENER, 1976).

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DAVID R. MURRAY and JANET A. CRUMP

+ME

-

5~

+ME -.~ -~.....

10%

Fig. 3: Polypeptide composition of «Gl globulin» from seed meal of P. vulgaris cv. Hawkesbury Wonder. Electrophoresis was conducted in the presence or absence of ME using gels set from 5 % and 10 Ofo acrylamide (as indicated).

Interaction of Bean Glycoproteins with Concanavalin A Con A binds to glycoproteins or polysaccharides possessing terminal a-mannosyl or a-glucosyl residues or internal 2-0-linked mannosyl units (GOLDSTEIN et a!., 1973). PHA from both P. vulgaris and P. lunatus are known to be glycoproteins which bind fully to Con A (BESSLER and GOLDSTEIN, 1973; JAFFE et aI., 1974). It was

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54

49

>100 30

46

pH 4.1 Soluble

Total

pH4.7 Insoluble

Fig:- 4; D~llsitometric scans- of polyacrylamide gels following electrophoresis of fractions from 2-day imbibed bean cotyledons. Numbers indicate molecular weight (X 10-3).

Ph.v. \

A

L

Fig. 5: Double diffusion tests for interaction of the jack bean lectin Con A (centre well) with euphaseolin (E) from P. vulgaris cotyledons, and with components of albumin (A), legumin (L) and vicilin (V) fractions from cotyledons of Pisum sativum L. T = total extractable protein from cotyledons in each case. Z. PJlanzenphysiol. Bd. 94. S. 339-350. 1979.

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noticed by GLEESON and JERMYN (1977) and by JERMYN (1975) that although the total Con A-interacting fraction from bean meal possessed haem agglutinating activity, its electrophoretic analysis revealed the presence of protein components which were not characteristic of bean lectins: additional polypeptides of MW 56,000 and 50,000 were prominent. These observations suggested to us that the major bean globulin might interact with Con A, since it was already known that D-mannose and D-glucosamine are the chief constituents of the polysaccharide moiety, which represents 5.5 % (w/w) of the total glycoprotein (PUSZTAI and WATT, 1970; RACUSEN and FOOTE, 1971). This prediction was confirmed (Fig. 5). However, we are able to conclude that only a minor proportion of euphaseolin exists in forms able to interact with Con A.

s

T

s

Fig. 6: Double diffusion of Con A (centre channel) against components of cotyledon extracts separated by electrophoresis: S, pH 4.7 soluble; I, pH 4.7 insoluble and T, total (pH 7.25 soluble) fractions from bean cotyledons imbibed for 1 day.

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Double diffusion tests conducted following electrophoresis confirmed that the pH 4.7 insoluble fraction contained only anodal Con A-interacting glycoprotein components, corresponding to the position of similar components from purified euphaseolin, whereas the pH 4.7 soluble fraction contained both immobile and anodal components (Fig. 6). The immobile components correspond to forms of PHA (or «protein II» of KLOZOVA and TURKOVA, 1978). Double diffusion tests with serially diluted fractions showed that the concentration of Con A-interacting glycoproteins in the pH 4.7 soluble fraction was lower than the concentration of such compounds in the pH 4.7 insoluble fraction (redissolved to original extract volume) by only a factor of 2. This is much less than expected if all or most of the major globulin in the pH 4.7 insoluble fraction were interacting with Con A. Furthermore, our calculations show that for this variety of bean, euphaseolin accounts for ca. 16 Ofo of the dry matter of the cotyledons, but the total Con A-interacting material represents only 4 Ofo of dry matter (JERMYN, 1975; GLEESON and JERMYN, 1977). Discussion

According to OSBORNE: «phaseolin is a globulin which forms nearly all of the protein substance of the white or kidney bean (Phaseolus vulgaris)>> (OSBORNE and CLAPP, 1907). Although OSBORNE'S preparation was not completely pure (KLOZ and KLOZOVA, 1974), there can be no doubt that subsequent investigators have been studying essentially the same globulin, despite the unnecessary proliferation of terms used to describe it (Table 1). Examination of our own data for cv. Hawkesbury Wonder, together with that published for other varieties, establishes beyond reasonable doubt that the major bean globulin is neither legumin nor vicilin. This globulin is not legumin because: 1. The size of the polymeric form of the major bean globulin is ca. 19 S (PUSZTAI and WATT, 1970), much larger than legumin (11-13 S). 2. The bean globulin is not invariably insoluble at pH 4.7 (see Table 2). 3. The subunit composition of legumin is entirely different, and the association of the two main legumin subunits involves the formation of disulphide bridges (THOMSON et al., 1978). 4. Legumin has a much lower polysaccharide content (BASHA and BEEVERS, 1976) and does not interact significantly with Con A (Fig. 5; DAVEY and DUDMAN, 1978).

Neither can this bean globulin be vicilin, since: 1. Vicilin requires a lower salt concentration for complete solution (OSBORNE and CLAPP, 1907; OSBORNE and HARRIS, 1907; SUN and HALL, 1975). 2. Vicilin is composed of a larger number of subunits ranging in size from 12,000 to 75,000 daltons (THOMSON et al., 1978). 3. Vicilin has an extremely low polysaccharide content (BASHA and BEEVERS, 1976). Z. Pjlanzenphysiol. Bd. 94. S. 339-350. 1979.

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4. Not all of the vicilin subunits possess glycosyl mOletles (DAVEY and DUDMAN, 1978) whereas all three subunits of euphaseolin are glycosylated (HALL et aI., 1977). 5. Euphaseolin is completely soluble at pH 4.7 only if a mutation has occurred leading to deletion of portion of the largest subunit (Table 2). Table 2: Some reported properties of the major seed globulin from different cultivars of

P. vulgaris.

Subunit sizes (X 10-3 dalton)

Cultivar

Solubility at pH 4.7

Iinsoluble I

Tendergreen

53,

47,

43

(A, B)

Canadian Wonder Canadian Wonder Greensleeves Hawkesbury Wonder

53, 53, 52, 54,

47, 49, 49, 49,

43 45 46 46

(A) (D) (E) (F)

Seafarer Seafarer BBL 240 PI 229, 815 BBL 240 X } Fl PI 229, 815 A B C D

(ref.)

53,

50,

47

(D)

47, 47, 47,

43 43 43

(A) (A, G) (A)

50.5,

47,

43

(A)

E F G

(C)

not stated not stated not stated

50.5, 53, 50.5,

HALL et al. 1977 McLEESTER et al. 1973 SUN and HALL 1975 BARKER et al. 1976

(ref.)

Iinsoluble I Isoluble I

(F)

(D)

not stated not stated not stated

not stated BOLLINI and CHRISPEELS 1978 MURRAY and CRUMP 1979 (this paper) ROMERO et al. 1975

Finally, it must be pointed out that DUD MAN and MILLERD (1975) failed to detect proteins from P. vulgaris cotyledons bearing antigenic determinants identical to those of legumin and vicilin. While we disagree with BOLLINI and CHRISPEELS over their super-ficial use of the name vicilin, we agree with these authors that the terms «Glycoprotein II» and «Gl Globulin» should be abandoned. We would adhere to the name «phaseolin» devised by OSBORNE except for the assumption of that term by CRUICKSHANK and PERRIN (1963) to refer to a particular type of phytoalexin produced by P. vulgaris tissues in response to fungal invasion. Instead, we endorse the use of the term «euphaseolin» introduced by KLOZ and KLOZOVA. (1974). Acknowledgements We thank Mr. I. PORTER for excellent technical assistance, Dr. R. DAVEY and Dr. S. L. DurGAN for helpful discussion, and the Australian Research Grants Committee for financial support.

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KLOZ, J. and E. KLOZovA: The protein euphaseolin In Phaseolinae - a chemotaxonomical study. BioI. Plantar., 16,290-300 (1974). KLOzovA, E. and V. TURKovA: Variability of some seed proteins of the species Phaseolus vulgaris and their relationship to phytohaemagglutinating activity. BioI. Plantar., 20, 129-134 (1978). LIENER, I. E.: Phytohemagglutinins (phytolectins). Annu. Rev. Plant PhysioI., 27, 291-319 ( 1976). McLEESTER, R. c., T. C. HALL, S. M. SUN, and F. A. Buss: Comparison of globulin proteins from Phaseolus vulgaris with those from Vicia faba. Phytochemistry, 12, 85-93 (1973). MILLERD, A.: Biochemistry of legume seed proteins. Annu. Rev. Plant PhysioI., 26, 53-72 (1975). MURRAY, D. R.: The seed proteins of Kowhai, Sophora microphylla AlT. Z. PflanzenphysioI., 93, 423-428 (1979). MURRAY, D. R. and R. B. KNOX: Immunofluorescent localization of urease in the cotyledons of jack bean, Canavalia ensiformis. J. Cell Sci., 26, 9-18 (1977). MURRAY, D. L., W. J. ASHCROFT, R. D. SEPPELT, and F. G. LENNOX: Comparative biochemical and morphological studies of Acacia sophorae (LABILL.) R. BR. and A. longifolia (ANDREWS) WILLD. Aust. J. Botany, 26, 755-771 (1978). OSBORNE, T. B. and S. H. CLAPP: Hydrolysis of phaseolin. Amer. J. PhysioI., 18, 295-308 (1907). OSBORNE, T. B. and I. F. HARRIS: The proteins of the pea (Pisum Sativum). J. BioI. Chern. 3,213-217 (1907). PUSZTAI, A. and W. B. WATT: Glycoprotein II. The isolation and characterization of a major antigenic and nonhemagglutinating glycoprotein from Phaseolus vulgaris. Biochim. Biophys. Acta, 217, 413-431 (1970). - - Isolectins of Phaseolus vulgaris. A comprehensive study of fractionation. Biochim. Biophys. Acta, 365, 54-71 (1974). RACUSEN, D. and M. FOOTE: The major glycoprotein in germinating bean seeds. Can. J. Bot., 49, 2107-2111 (1971). ROMERO, J., S. M. SUN, R. C. McLEESTER, F. A. Buss, and T. C. HALL: Heritable variation in a polypeptide subunit of the major storage protein of the bean, Phaseolus vulgaris L. Plant Physiol., 56, 776-779 (1975). SUN, S. M. and T. C. HALL: Solubility characteristics of globulins from Phaseolus seeds in regard to their isolation and characterization. J. Agr. Food Chern., 23,184-189 (1975). SUN, S. M., B. U. BUCHBINDER, and T. C. HALL: Cell-free synthesis of the major storage protein of the bean, Phaseolus vulgaris L. Plant PhysioI., 56, 780-785 (1975). THOMSON, J. A., H. E. SCHROEDER, and W. F. DUDMAN: Cotyledonary storage proteins in Pisum sativum. I. Molecular heterogeneity. Aust. J. Plant PhysioI., 5, 263-279 (1978). WEBER, K. and M. OSBORN: The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis. J. BioI. Chern., 244, 4406-4412 (1969). D. R. MURRAY, Botany School, University of Melbourne, Parkville Vic. 3052, Australia.

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