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Partial purification of a type eta protein kinase C from murine brain: separation from other protein kinase C isoenzymes and characterization.

ABSTRACT: 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.

SUBMITTER: Zang R

PROVIDER: S-EPMC1137539 | biostudies-other | 1994 Dec

REPOSITORIES: biostudies-other

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Project description:Soluble extracts prepared from bovine thymus contain an angiotensin-I-phosphorylating activity that is activated several-fold by high concentrations of NaCl. Fractionation of this protein-tyrosine kinase activity by chromatography on DEAE-cellulose yields a major diffuse peak of activity. The enzymes responsible for this activity are found at much higher levels in extracts from bovine thymus than from bovine spleen. The peak of activity from the DEAE-cellulose column can be further separated into two major peaks by chromatography on heparin-agarose. The second peak to elute from the heparin-agarose column was previously purified through several chromatographic steps to yield a 40 kDa protein-tyrosine kinase (p40). We have now partially purified the early-eluting peak of kinase activity by chromatography on columns of butyl-agarose, protamine-agarose and Sephacryl S200. The enzyme was identified following covalent modification with 5'-fluorosulphonylbenzoyladenosine (FSBA) by reactivity with anti-FSBA antibodies. This procedure labelled a series of 52-56 kDa proteins. These proteins were also recognized by polyclonal anti-peptide antibodies raised against the C-terminal 33 amino acids of p56lck, a major T lymphocyte protein-tyrosine kinase. Peptide maps of the partially purified enzyme were identical to maps generated from p56lck obtained from LSTRA cells. These data suggest that bovine thymus p56lck is responsible for the activity found in the early-eluting peak from heparin-agarose. Antibodies raised against a peptide corresponding to amino acids 39-64 of p56lck, a sequence found near the N-terminus, recognized the slower-migrating, but not the faster-migrating, form of the enzyme, indicating that a fraction of the protein had been proteolysed near the N-terminus during purification. The partially purified bovine enzyme exhibited a restricted substrate specificity in vitro and did not readily phosphorylate human erythrocyte band 3, casein or histone, but was able to phosphorylate acid-treated enolase. The dilute enzyme present in fractions eluting from chromatography columns was unable to catalyse an autophosphorylation reaction. Autophosphorylation could be detected in more concentrated enzyme samples and was readily observed in immune-complex assays. The phosphorylation of angiotensin I by bovine thymus p56lck was weakly activated by polyionic compounds such as heparin and polylysine, and was strongly activated by high concentrations of NaCl.

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Partial purification of a type eta protein kinase C from murine brain: separation from other protein kinase C isoenzymes and characterization.

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