Isolation and characterization of protein kinase C from rat brain synaptosome cytoskeleton.
Synaptosomes prepared from brain tissues are known to retain morphological and functional characteristics of the nerve ending. Little information is available, however, as to the biochemical events underlying synaptogenesis and transmitter release. Increasing body of evidence suggests that protein kinase C (PKC) plays crucially important roles through phosphorylation of membrane proteins such as GAP-43 (for 43-kDa growth-associated protein) and 87-kDa MARCKS (for myristoylated, alanine-rich C kinase substrate) in many aspects of the neuronal function. Among them, arrangement of membrane cytoskeletal protein is proposed to be one of the primary sites of PKC action. The present study is an attempt to isolate and characterize PKC associated with synaptosomal membrane cytoskeleton. Rat brain synaptosomal Triton X-100 insoluble elements (cytoskeleton) contains specific [3H]phorbol dibutyrate binding activity and 78-kDa protein which reacts with an antibody against beta II-PKC subspecies. Although 78-kDa protein could not be solublized by the treatment with various ionic and non-ionic detergents and/or high concentrations of salts such as NaCl and LiBr, the fragment of 78-kDa protein was produced and solublized from cytoskeleton by limited proteolysis with calpain II, which cleaves PKC at one or two specific sites of the enzyme to produce catalytic and regulatory fragments. The solubilized 46-kDa fragment was identical with the catalytic fragment of beta II-PKC. The results indicate that this PKC subspecies is tightly associated with the cytoskeletal network of synaptic membranes.
Tanaka
S
S
Department of Biochemistry, Kobe University School of Medicine.
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D003599:Cytoskeleton / Q000201:enzymology*; D015151:Immunoblotting; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011493:Protein Kinase C / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013574:Synaptosomes / Q000201:enzymology*
D010446:Peptide Fragments; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C