of basic polypeptides (e.g. Bia, Bib, B2, B4) that migrate together at Mr = 20,000 (4). ... in electrophoretic mobility of glycinin and ,B-conglycinin sub- units that are ...
Plant Physiol. (1984) 76, 840-842 0032-0889/84/76/0840/03/$0 1.00/0
Short Communication
Urea-Elicited Changes in Relative Electrophoretic Mobility of Certain Glycinin and B-Conglycinin Subunits' Received for publication July 11, 1984
ELIZABETH PACHECO BATISTA FONTES, MAURILIO AVES MOREIRA, CORINNE S. DAVIES, AND NIELS C. NIELSEN* Department of Chemistry, Federal University of Vicosa, Vicosa, MG, Brazil (E. P. B. F., M. A. M.); and United States Department ofAgriculture (N. C. N.) and Agronomy Department, Purdue University, West Lafayette, Indiana 47907 (C. S. D., N. C. N.) amide gels which contain SDS. This method is rapid but not entirely satisfactory, because it does not resolve the group of acidic polypeptide components (e.g. AIa, A,lb, A2, A4) that migrate together in the gel at Mr = 37,000, nor does it separate the group of basic polypeptides (e.g. Bia, Bib, B2, B4) that migrate together at Mr = 20,000 (4). Two dimensional electrophoresis permits better resolution, but it is time-consuming. Recently, we observed that the relative positions of several major electrophoretic components changed when 6 M urea was included in SDSpolyacrylamide gradient gels. This report describes the changes in electrophoretic mobility of glycinin and ,B-conglycinin subunits that are elicited by urea, as well as the features of several genetic variants whose glycinin and ,B-conglycinin subunits are differentially affected by urea in SDS-polyacrylamide gels.
ABSTRACT Six molar urea in sodium dodecyl sulfate-polyacrylamide gels altered the relative electrophoretic mobility of several soybean protein subunits. Glycinin acidic polypeptide components A3 and A4 could be resolved from the other acidic polypeptides. A variant of the 6' subunit of B-conglycinin was identified.
Cultivated soybean varieties contain about 40% protein on a dry weight basis, of which about 70% is either glycinin or ,8conglycinin (1). Both of these proteins are complexes made up of nonidentical subunits. Glycinin can be purified in a 350,000 mol wt form that consists of six subunits. Each subunit is composed of two parts: an acidic polypeptide and a basic polypeptide (7) which are linked via a single disulfide bond (9). Thus far, five major glycinin subunits have been identified on the basis oftheir NH2-terminal sequence and are designated AiaB2, A,bB,b, A2Bia, A3B4, and A5A4B3 (4, 5, 7). Small amounts of another acidic polypeptide, A6, have been detected with a NH2-terminal sequence homologous to A4 (10). This polypeptide is probably part of another subunit, although other polypeptide components associated with it are unknown. ,3-Conglycinin can be isolated as a 150,000 mol wt complex that consists of three nonidentical subunits. At least four different subunits, designated a', a, fl, and y, have been identified (1 1) and characterized with respect to apparent mol wt and NH2terminal sequence (3). Clear evidence exists for a polymorphism with respect to subunit composition for (-conglycinin (1 1). It is likely that all of the 7S subunits are structurally equivalent to one another and associate randomly to form the 150,000 mol wt complex. The various subunits associated with glycinin and ,3-conglycinin are commonly visualized by electrophoresis in polyacryl'Supported by National Science Foundation grant no. INT-8212594 to N.C.N. and CNPq grant to M.A.M. as part of the United States Brazil Bilateral Scientific Exchange Program, as well as grants from the Amer-
MATERIALS AND METHODS Plant Material. Seeds of the varieties Raiden and Century were obtained from plants grown at the Purdue Agronomy Farm (West Lafayette, IN) during the summer of 1982. PI 54608-1, which exhibits the 'a-shifted' and 'a'-shifted' phenotypes, PI 88.302-1 which exhibits the 'double fl' phenotype, and Kura which specifically lacks A3, were obtained from the Northern USDA Soybean Germplasm Collection maintained at Urbana, IL by Dr. R. Bernard. The varieties Jackson, Hood, and Uniao, which exhibit the a'-shifted phenotype were obtained from a Brazilian Germplasm Collection maintained by Dr. R. Khil at Empresa Basileira de Pesquisa Agropecuaria (EMPRAPA) in Londrina, Parana, Brazil. The varieties Jackson and Hood were also obtained from the Southern USDA Soybean Germplasm Collection maintained by Dr. E. Hartwig at Stoneville, MS, and they gave results which were identical to those obtained with seed from Brazil. Electrophoretic Methods. Seed samples were ground to a fine powder and then a 15-mg sample was suspended in 2 ml of sample buffer (50 mM Tris-HCl [pH 8.0], 0.2% SDS, 10 mm 2mercaptoethanol, and 5 M urea). Samples were extracted 30 min at room temperature and then centrifuged to remove seed debris. Proteins in the seed extracts were separated in SDS-polyacrylamide gradient gels which either contained or did not contain 6 M urea. A 7 to 13% acrylamide gradient gel (2) was used for samples separated in gels devoid of urea. A 10 to 18% acrylamide gradient was used for gels which contained urea, because it gave superior resolution of bands. These gels were prepared as follows: solution A contained 29.2 g acrylamide, 0.8 g bis-acrylamide, and 36 g urea in a final volume of 100 ml. Solution B contained 18.15 g Tris, 0.2 g SDS, 36 g urea, 24 ml 1 M HCI in a final
ican Soybean Association Research Foundation and United Nations Educational, Scientific, and Cultural Organization. Cooperative research between United States Department of Agriculture-Agricultural Research Service and the Purdue Agricultural Experiment Station. Agricultural Experiment Station Journal No. 9830. 840
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FIG. 1. Effect of urea on the relative electrophoretic mobilities of glycinin and fl-conglycinin subunit bands. Seed meal was extracted with SDSsample buffer. The proteins were separated either in 7 to 13% SDS-polyacrylamide gradient gels (lA) or in 10 to 18% SDS-polyacrylamide gradient gels which contained 6 M urea (I B). Lane 1, Century; lane 2, PI 88.302-1; lane 3, PI 54608-1; lane 4, Hood; lane 5, Kura; lane 6, A3 protein; lane 7, A2 protein; lane 8, Aja protein; lane 9, 11 S protein from Raiden; lane 10, Riaden; lane 11, 1 IS protein from Century; lane 12, Century.
FRO"'NTES volume of 100 ml. The 10% acrylamide solution consisted of 8 ml of solution A, 7 ml of solution B, 1.2 ml of 1.4% ammonium sulfate, and 20,1 of TEMED (N,N,N',N'-tetramethylethylenediamine) in a final volume of 24 ml. The 18% acrylamide solution consisted of 14.8 ml of solution A, 7 ml of solution B, 1.2 ml of 1.4% ammonium sulfate, 20,l of TEMED in a final volume of 24 ml. A gradient gel was formed which measured 18 cm x 8.5 cm x 3 mm, and then a stacking gel 18 cm x 4.5 cm x 3 mm was cast above it. The stacking gel consisted of 4 ml of ,ul solution A, 7 ml of solution B, 2 ml of 1.4% (NH4)2SO4, 20 Il) were of TEMED in a final volume of 24 ml. Samples (10-20 applied to the gels, and they were run at a constant voltage of 100 v for1 hr and then at 125 v for 5 h.
842
RESULTS AND DISCUSSION In the standard SDS-polyacrylamide gradient gels (7-13%), the a', a, andjB-subunits in whole-seed extracts migrated with Mr = 72,000, 68,000, and 52,000, respectively (Fig.1A). While not normally resolved, the -y-subunit occasionally migrated slightly faster than the p3-subunit. These results are consistent with earlier reports (3, 11). In the case of the glycinin polypeptides, A3 migrated slightly slower (Mr = 42,000) than the major group of acidic ones at Mr = 37,000 (e.g. Ala, Aib, A2, A4). Occasionally, the resolution of the polypeptides in this Mr = 37,000 region was sufficient to detect several components. Polypeptide A5 had Mr =10,000 and was located near the bottom of the gel. The polypeptide B3 migrated in the gel at a position which is slightly above the other basic components (e.g. B,, B2, B4). When urea was included in SDS-polyacrylamide gradient gels (10-18%), changes were observed in the region of the electro,-conglycinin phoretic pattern located between the #-subunit of and the Mr = 37,000 region where the majority of acidic polypeptides from glycinin migrate (Fig. 1 B). A new electrophoretic band appeared just below the,-subunit band and another was located at a position similar to, but not identical with, the position of the A3 band in gels which lacked urea. Experiments were undertaken to determine the identity of the new bands in gels with urea. Glycinin was purified from seeds of the varieties Century and Raiden. The former contains a full complement of glycinin andjB-conglycinin subunits, whereas the latter is homozygous for a recessive allele which causes glycinin subunit A5A4B3 to be eliminated. Both whole-seed extracts and purified glycinin from Raiden seeds lacked the lower of the two new bands observed in gradient gels with urea (Fig. 1B). In addition, bands that corresponded to A5 and B3 were not detected on either SDS gels without urea, as reported previously (10), or on SDS gels with urea. Two observations led to identification ofthe position occupied by the A3 polypeptide in gels with urea. The A3-polypeptide purified from the variety Raiden had the same mobility as the upper, new, heavily stained electrophoretic component. Further, this band was missing in Kura, a variety in which A3 is either absent or drastically reduced in gradient gels without urea (Davies and Nielsen, unpublished observation). Thus, the relative electrophoretic mobilities of both A3 and A4 are reduced in gels that contain urea compared to the mobilities of the other acidic polypeptide components of glycinin. The reasons underlying the changes in mobility of A3 and A4 are not clear, although two characteristics of the glycinin polypeptides seem relevant. First, determination of the complete amino acid sequence of A2 revealed that it had a calculated mol wt of 29,500 ± 100 compared to Mr = 37,000 determined in SDS-gels which lack urea (8). In contrast, the mol wt of Bia
Plant Physiol.Vol. 76, 1984 calculated from its amino sequence matched almost exactly that determined (8). The acidic polypeptides must,
ET AL.
electrophoretically
therefore, possess structural features that cause them to migrate nonideally in SDS-electrophoretic systems. Second, glycinin subunits can be separated into two groups based on sequence homologies (6). A3B4 and A5A4B3 comprise the group II subunits and exhibit about 90% homology between their NH2-terminal the acidic sequences. In contrast, the NH2-terminal sequences ofonly 50 to and basic polypeptides of the group-Il subunits are 60% homologous with those of the group I subunits but are likewise more homologous with each other. Therefore, it is the nonideal probable that whatever structural changes lead to those features components, electrophoretic behavior ofthe acidic in the group II subunits respond in a more extreme fashion than those in groupI. A change in relative electrophoretic mobility in response to urea was not limited to the glycinin polypeptides. In the course of screening entries from the EMBRAPA germplasm collection Jackson) were
with the urea gels, several varieties (Uniao, Hood, found in which the a'-subunit band shifted to a higher position in the gel (as in Fig. 1, lane 4). Interestingly, when extracts of Hood seeds were separated in non-urea gels, the mobilities of the were indistinguishable in all other varieties tested. a'-subunits in mobility Thus, the inclusion of urea in the gel elicited the shiftthe original of a' in Hood extracts. Progeny generated from a'-shifted the which exhibited seed all of sample Uniao produced Studies with this and other varieties which also phenotype. exhibit the a'-shifted characteristic reveal that the trait is genetcontrolled (Fontes and Davies, unpublished observations). ically An electrophoretic variant (PI 54608-1) in which the a-subunit undergoes an analogous shift in electrophoretic mobility has been found in the Northern USDA germplasm collection. However, in this instance, the shift in mobility is ojbserved inPI gradient which do not contain urea (Fig. 1). Interestingly, 54608gels 1 also contained an a'-subunit which, like Uniao, had a shifted in urea. Preliminary data indicate that the shifted amobility subunit in this line is conditioned by a codominant allele of the a-structural gene (Davies, unpublished observations). Genetic tests are underway to confirm this and to determine the linkage relationship between loci that condition the a-shifted and the a'shifted subunits.
LITERATURE CITD 1. DERBYSHIRE E, DJ WRIGHT, D BOULTER 1976 Legumin and vicilin, storage proteins of legume seeds. Phytochemistry 15: 3-24 2. KITAMURA K, CS DAVIES, NC NIELSEN 1984 Inheritance of null-alleles for the a' subunit of -wconglycinin and the A"A4B3 subunit of glycinin in soybean. Theor Appl Genet 68: 253-257
3. MEDEIROS JS 1982 Characterization of the subunits of -conglycinin and the determination of the contents of,-conglycinin application of ELISA to the and glycinin in soybean. PhD thesis. Purdue University, West Lafayette, IN 4. MOREIRA MA, MA HERMODSON, BA LARKINS, NC NIELSEN 1979 Partial characterization of the acidic and basic polypeptides ofglycinin. J Biol Chem 254: 9921-9926 5. MOREIRA MA, MA HERMODSON, BA LARKINS, NC NIELSEN 1981 Comparison ofthe primary structure of the acidic polypeptides ofglycinin. Arch Biochem Biophys 210: 633-642 6. NIELSEN NC 1983 The chemistry of legume storage proteins. Phil Trans R Soc Lond B304: 287-296
7. STASWICK PE, MA HERMODSON, NC NIELSEN 1981 Identification of the acidic and basic subunit complexes of glycinin. J Biol Chem 256: 8752-8755 8. STASWICK PE, MA HERMODSON, NC NIELSEN 1984 The amino acid sequence of the A2BA. subunit of glycinin. J Biol Chem. In press of the 9. STASWICK PE, MA HERMODSON, NC NIELSEN 1984 Identificationsubunits. which link the acidic and basic components of the glycinin cystines J Biol Chem. In press 10. STASWICK PE, NC NIELSEN 1983 Characterization of a soybean cultivar lacking certain glycinin subunits. Arch Biochem Biophys 223: 1-8 11. THANH VH, K OKUBO, K SHIBASAKI 1975 Isolation and characterization of the multiple 7S globulins of soybean proteins. Plant Physiol 56: 19-22
