Cross-Reaction of Anti-Rat B-50: Characterization and Isolation of a "B-50 Phosphoprotein" from Bovine Brain |
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Authors: | Anna Beate Oestreicher Marcel van Duin Henk Zwiers Willem Hendrik Gispen |
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Institution: | Division of Neurobiology, Institute of Molecular Biology and Rudolf Magnus Institute for Pharmacology, State University of Utrecht, Utrecht, The Netherlands |
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Abstract: | Abstract: 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 M r of the "B-50 protein" varies from species to species. |
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Keywords: | ACTH Antibodies Bovine brain Phosphoproteins Rat B-50 Synaptic plasma membranes |
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