Immunochemical characterization of rat brain protein kinase
Polyclonal antibodies against rat brain protein kinase C (the Ca/sup 2 +//phospholipid-dependent enzyme) were raised in goat. These antibodies can neutralize completely the kinase activity in purified enzyme preparation as well as that in the crude homogenate. Immunoblot analysis of the purified and the crude protein kinase C preparations revealed a major immunoreactive band of 80 kDa. The antibodies also recognize the same enzyme from other rat tissues. Neuronal tissues (cerebral cortex, cerebellum, hypothalamus, and retina) and lymphoid organs (thymus and spleen) were found to be enriched in protein kinase C, whereas lung, kidney, liver, heart, and skeletal muscle contained relatively low amounts of this kinase. Limited proteolysis of the purified rat brain protein kinase C with trypsin results in an initial degradation of the kinase into two major fragments of 48 and 38 kDa. Both fragments are recognized by the antibodies. However, further digestion of the 48-kDa fragment to 45 kDa and the 38-kDa fragment to 33 kDa causes a loss of the immunoreactivity. Upon incubation of the cerebellar extract with Ca/sup 2 +/, the 48-kDa fragment was also identified as a major proteolytic product of protein kinase C. Proteolytic degradation of protein kinase C converts the Ca/sup 2 +//phospholipid-dependent kinase to an independent form without causing a large impairment of the binding of (/sup 3/H)phorbol 12,13-dibutyrate. The two major proteolytic fragments were separated by ion exchange chromatography and one of them (45-48 kDa) was identified as a protein kinase and the other (33-38 kDa) as a phorbol ester-binding protein. These results demonstrate that rat brain protein kinase C is composed of two functionally distinct units, namely, a protein kinase and a Ca/sup 2 +/-independent/phospholipid-dependent phorbol ester-binding protein.
- Research Organization:
- National Institutes of Health, Bethesda, MD
- OSTI ID:
- 6802813
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 261:31; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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