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Cross‐Reaction of Anti‐Rat B‐50: Characterization and Isolation of a “B‐50 Phosphoprotein” from Bovine Brain
Author(s) -
Oestreicher Anna Beate,
Duin Marcel,
Zwiers Henk,
Gispen Willem Hendrik
Publication year - 1984
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1984.tb12827.x
Subject(s) - phosphoprotein , isoelectric point , isoelectric focusing , proteolysis , biochemistry , biology , protein kinase a , microbiology and biotechnology , phosphorylation , endogeny , membrane protein , peptide , membrane , enzyme
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 M r 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1–24 of adrenocorticotropin (ACTH 1‐24 ) (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 M r 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 M r 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 M r of the “B‐50 protein” varies from species to species.