Cross-reaction of anti-rat B-50: characterization and isolation of a "B-50 phosphoprotein" from bovine brain.
Antibodies to the phosphoprotein B-50 of rat brain were used to trace cross-reacting brain proteins of vertebrates. With the SDS-gel-immunoperoxidase method, a cross-reacting protein (CP) of apparent Mr 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1-24 of adrenocorticotropin (ACTH1-24) (10(-5) M and 10(-4) M) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B-50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two-dimensional gel analysis of bovine and rat ASP showed that the cross-reacting protein had an isoelectric point less acidic than B-50. Limited proteolysis by Staphylococcus aureus protease yielded a "peptide map" analogous to B-50. Two major fragments of Mr 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B-50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross-reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the Mr of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross-reacting bovine protein is a "B-50 protein," and (2) the Mr of the "B-50 protein" varies from species to species.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001923:Brain Chemistry; D002417:Cattle; D011232:Chemical Precipitation; D003366:Cosyntropin / Q000494:pharmacology; D019922:GAP-43 Protein; D006801:Humans; D007124:Immunoenzyme Techniques; D007525:Isoelectric Focusing; D010446:Peptide Fragments; D010750:Phosphoproteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity; D013570:Synaptic Membranes / Q000032:analysis
D000906:Antibodies; D019922:GAP-43 Protein; D010446:Peptide Fragments; D010750:Phosphoproteins; D003366:Cosyntropin; D011494:Protein Kinases; D011493:Protein Kinase C