Discontinuous sodium dodecyl sulfate slab gel electrophoresis of Gl globulin from several strains of Phaseolus vulgaris L. seed permitted dear resolution of the constituent polypeptides. Three strains (Tendergreen, Canadian Wonder, and BBL 240) had subunits of molecular weight 53,000, 47,000 and 43,000 while two strains (Seafarer and PI 229,815) had 50,500, 47,000 and 43,000 molecular weight subunits. F1 seed from the cross BBL 240 x PI 229,815 showed four polypeptides on dissociation of the Gl protein; however, the amount of each of the 53,000 and 50,500 subunits was hbaf that of the 47,000 subunit. This is interpreted as evidence that both the maternal and paternal loci for these polypeptides are transcribed and translated with similar efficiency. All of the polypeptides were found to have associated sugar residues.We have shown previously that Gl globulin, the major storage protein of French bean (Phaseolus vulgaris L.) seeds, has three polypeptide subunits (4, 10). These subunits are synthesized by classical ribosomal systems, and each appears to be an individual gene product, there being no evidence for posttranslational cleavage of a large precursor molecule (9). One of the strains studied, PI 229,8152 was found to have a modified subunit composition in which the largest subunit was nearly the same size as the middle subunit (5). Thus, after electrophoretic separation on continuous SDS acrylamide gels, the polypeptide pattern appeared two-banded, while that of the cultivar Tendergreen was clearly three-banded.Using different conditions for electrophoresis we now show unequivocally the three-banded nature of the modified strain and provide a model for the phenotypic expression of the large subunit, which is inherited as expected for a polypeptide controlled by a single Mendelian gene (5). We also confirm that each of the polypeptide subunits has associated sugar residues. MATERIALS AND METHODSPlant Material. Bean Gl Globulin Extraction. Dry mature seeds were covered with a small amount of glass wool and ground with a pestle and mortar to a flour using a freshly broken Pasteur pipette as a grinding aid. Protein was extracted at room temperature by addition of 0.5 M NaCl in 0.025 N HCI (20 ml/g flour). The suspension was centrifuged (J-20 rotor on a Beckman J-2 1 centrifuge) at 20,000 rpm for 15 min and the pellet was discarded. Five volumes of distilled H20 (0 C) were added to the clear supernatant. The precipitated globulin was sedimented at 20,000 rpm for 10 min and finally dissolved in 0.5 M NaCl. The protein concentration was determined by UV absorption, assuming A°7l6 protein = 1. Prior to electrophoresis in the Gi protein was dissociated by heating for 2 min at 100 C with an equal volume of cracking buffer (3), 100 ml of which contained:10 ml 625 mm tris-HCI (pH 6.8), 2 g SDS, 2 ml ,8-mercaptoethanol, 40 g sucrose, 10 ,ug bromophenol blue, and 1 ml 0.2 M EDTA.Discontinuous SDS Slab Gel Electrophoresis. Slab gels (0.75 mm thick) were prepared according to Laemmli (3) as modified by Knowland (2...

Electrophoretic analysis of the major seed protein, GI globulin, from four strains of Phaseolus vulgaris L. revealed a three-banded pattern for two strains having a high methionine content (BBL 240 and PI 302,542). The other two strains (PI 207,227 and P1 229,815) known to have a lower seed methionine content, had a two-banded subunit pattern for the GI globulin. Analytical ultracentrifugation confirmed that globulin from the two-banded strains underwent pHdependent reversible dissociation similar to that previously found for a three-banded cultivar; additionally, the protomer molecular weight showed that three subunits of about 50,000 molecular weight each were present in the GI globulin of the two-banded strain. Gel patterns of Gl globulin from the two strains used as parents, BBL 240 and PI 229,815, showed differences in the largest subunit, which appeared as either a 53,000 molecular weight polypeptide known to be present in the three-banded strain, or While soybeans have the highest protein content and economic value, the oil present in these seeds is a hindrance to protein studies, and seeds of Phaseolus vulgaris L. were selected as the preferred material. Dry seeds of P. vulgaris contain 20 to 30% protein by weight, depending on the cultivar (5). Of this protein, 50 to 75 % is globulin (requiring appreciable salt for solubility) which can be separated into two fractions. In Osborne's studies on legumes he adopted the term legumin (first used by Braconnot in 1827) for the fraction requiring the highest level of salt for solubility, and coined the term vicilin for the fraction soluble in lower salt concentrations (9). Through the years, misuse of these terms has occurred; they have been applied to proteins from many genera despite the differing properties of protein in a given fraction from species to species. To avoid the resulting confusion, we designated the globulin fraction which first precipitates on addition of water to a saline solution of P. vulgaris protein as the Gl fraction, and the second fraction (obtained by extensive dialysis) as the G2 fraction (7).Despite the apparently simple separation of the two globulin fractions on the basis of solubility, great difficulty has been experienced in obtaining them free from traces of cross-contamination. We developed an acidic extraction procedure (7) which achieves this goal, permitting characterization of the Gl and G2 globulin fractions (15). Recent articles (12, 18) have described separation of globulins using zone precipitation, which performs the same function as our isoelectric separation, but is lengthier and limits the amount of material that can be handled.Although the G2 fraction gives a single boundary sedimenting at 6.6S in the analytical ultracentrifuge (16), it is not homogeneous, and contains several growth-inhibiting substances. This fraction has a high content of sugar-containing proteins, and a major component, glycoprotein I (10), possesses hemagglutinating properties, i.e., it is a lectin. Purification of lectin from Phas...

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