Purification of a beta 35 form of the beta gamma complex common to G-proteins from human placental membranes.
The significance of the 36,000-35,000-dalton doublet of proteins referred to as the beta-subunit of the G-proteins remains unresolved. An immunological distinction between the 36,000 (beta 36)- and 35,000 (beta 35)-dalton proteins has been reported (Roof, D. J., Applebury, M. L., and Sternweis, P. C. (1985) J. Biol. Chem. 260, 16242-16249; Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 265-269). The availability of homogeneous preparations of beta 36 and beta 35 will facilitate studies designed to address the significance of the doublet structure. This manuscript presents the preparative purification of the beta 35 protein from a highly enriched source, human placenta. Unlike the beta gamma complex associated with G-proteins from placenta, the beta 35 preparations consist predominantly of the 35,000-dalton protein. The gamma-peptide associated with beta 35 is indistinguishable electrophoretically and immunologically from that associated with the placental G-protein oligomers. The beta 35 preparation and beta-subunit doublet exhibit similar specific activities in inhibiting human platelet adenylate cyclase activity. The preparations have proven useful for the generation of a panel of rabbit polyclonal antisera that recognize beta 35, Gt beta 36, and both beta 36 and beta 35 in the doublet associated with Gs, Gi, Go, and Gp. One antiserum generated to beta 35 recognizes the gamma-peptide associated with these preparations and human placental G-protein oligomers. The antiserum does not recognize Gt gamma. Protease digestion of Gt beta 36 and human placental beta 35 with Staphylococcus aureus V8 protease identified a unique peptide generated from beta 35 which is absent in beta 36 digests.
D000918:Antibody Specificity; D002462:Cell Membrane / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D005260:Female; D019204:GTP-Binding Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007159:Immunologic Tests; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy; D012697:Serine Endopeptidases
D000939:Epitopes; D007106:Immune Sera; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D019204:GTP-Binding Proteins