Partial purification of a type eta protein kinase C from murine brain: separation from other protein kinase C isoenzymes and characterization.
Various murine tissues were tested, by using a protein kinase C-eta-specific antiserum, for the expression of type eta protein kinase C. Brain was found to be the richest source of a type eta isoenzyme. Native protein kinase C-eta was partially purified from the cytosol of murine brain by chromatography on DEAE-Sepharose, hydroxyapatite and protamine-agarose. This procedure resulted in a separation of protein kinase C-eta from the other phorbol 12-myristate 13-acetate (PMA)-responsive isoenzymes (alpha, beta, gamma, delta, epsilon) and allowed, for the first time, characterization of the native enzyme. The protein kinase C of type eta from mouse brain is a phospholipid-dependent Ca(2+)-unresponsive protein kinase. Both PMA and bryostatin activate the kinase for phosphorylation of a substrate as well as for autophosphorylation. Various pseudo-substrate-related peptides are suitable as substrates for the eta-type kinase, peptide delta being the best and peptides eta and epsilon the poorest substrates. The enzyme is inhibited by staurosporine and staurosporine-related compounds, such as K252a and Gö 6976. However, protein kinase C-eta, like protein kinase C-delta, is around two orders of magnitude less sensitive towards Gö 6976 than are the Ca(2+)-responsive isoenzymes (alpha, beta, gamma). The eta-type protein kinase C exhibits an extreme tendency to lose its PMA-responsiveness. Consequently, purification of the enzyme to homogeneity has not yet been successful.
Zang
R
R
German Cancer Research Center, Heidelberg, Federal Republic of Germany.
Müller
H J
HJ
Kielbassa
K
K
Marks
F
F
Gschwendt
M
M
D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001921:Brain / Q000201:enzymology*; D054713:Bryostatins; D002460:Cell Line; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D018014:Gene Transfer Techniques; D007163:Immunosorbent Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D054713:Bryostatins; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; D011994:Recombinant Proteins; C046785:bryostatin 1; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate