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Coated vesicles (CVs) have been suggested in presynaptic terminals to function as a shuttle for reutlizing synaptic vesicle (SV) membrane (recycling hypothesis of SV membrane).3~ '8> Examining the protein composition and protein kinase activities of three separate synaptic membrane fractions, the present report attempted to clarify the biochemical details of the conversion processes of the CVs to the SVs. Materials and methods. A fraction of synaptic plasma membrane (SPM) was prepared by the method of Cotman and Matthews.1 The fractions of CVs and SVs were obtained from bovine brain synaptosomes.J~ '6~ The component proteins and protein kinases of each of the three synaptic membrane fractions were analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis and auto radiagraphy.4~ The reaction mixture for a protein kinase assay contained the followings; each of the fractions (50-100 pg in protein), 50 mM Tris-HC1 buffer (pH 7.5), 5.0 mM MgC12, and [y-32P]-ATP (6 nmoles ; 3 x 107 cpm) with or without the modulators for protein kinases in a final volume of 100-200 pl. The reaction was continued for 10 min at 37°C. Modulators tested for protein kinases were cyclic AMP (100 pM) and Ca2+ plus calmodulin (CaM) (10 pM ; 20 pg). Results. A protein with a molecular weight (MW) of 52,000, termed C-52, was found to be commonly distributed throughout the three synaptic membrane fractions (Fig. 1A). The polypeptide, C-52, was phosphorylated in all the three fractions in a medium containing neither cyclic AMP nor Ca2 plus CaM (Fig. 1B: a1, b1, and c1; a horizontal bar on the right side). This kinase, common to all, seemed to agree with a casein kinase found to be present in CVs.4>

Prerequisites for conversion of coated vesicles to synaptic vesicles. A biochemical examination of synaptic-vesicle membrane recycling hypothesis.