Chick brain synaptosomal polypeptides: phosphorylation and calcium binding studies

Abstract

Chick forebrain synaptosomes were incubated with inorganic phosphate (('32)Pi) under conditions which result in the labeling of only internal components. The synaptosomes were rapidly subfractionated into synaptic plasma membranes (SPM), crude synaptic vesicles (SV) synaptoplasm, and mitochondria. Seventeen SPM polypeptides incorporated substantial amounts of ('32)Pi with a 242,000/234,000 dalton doublet displaying greatest incorporation. This polypeptide specifically bound anti-brain spectrin IgG. These data represent an unambiguous discrimination of internally exposed SPM phosphoproteins and demonstrate that the phosphate groups on brain spectrin display rapid turnover in isolated avian nerve endings. p-Chloromercuribenzoic acid (PCMB) was shown to be a potent inhibitor of protein kinases and phosphatases. It is suggested that PCMB will be a useful tool for identifying the role of phosphorylation in neurotransmitter release. Actin, a cytoskeletal compound, is known to have one high affinity cation binding site. Five different cleavage techniques were used to investigate the identify of the calcium binding site on actin. Data suggests that the high affinity binding site is between trp 79 and glu 226. These results together with structural analysis suggest the Ca-binding site is not a classic EF-hand domain.