Characterization of Bovine Aortic Protein Kinase C with Histone and Platelet Protein P47 As Substrates

Overview

Journal Biochem J

Specialty Biochemistry

Date 1988 Sep 1

PMID 3178768

Citations 6

Authors

K R Dell

M P Walsh

D L Severson

Affiliations

Soon will be listed here.

Abstract

A Ca2+- and phospholipid-dependent protein kinase (protein kinase C) was partially purified from the media of bovine aortas by chromatography on DEAE-Sephacel and phenyl-Sepharose. Enzyme activity was characterized with both histone and a 47 kDa platelet protein (P47) as substrates, because the properties of protein kinase C can be modified by the choice of substrate. Both phosphatidylserine and Ca2+ were required for kinase activity. With P47 as substrate, protein kinase C had a Ka for Ca2+ of 5 microM. Addition of diolein to the enzyme assay caused a marked stimulation of activity, especially at low Ca2+ concentrations, but the Ka for Ca2+ was shifted only slightly, to 2.5 microM. With histone as substrate, the enzyme had a very high Ka (greater than 50 microM) for Ca2+, which was substantially decreased to 3 microM-Ca2+ by diolein. A Triton X-100 mixed-micelle preparation of lipids was also utilized to assay protein kinase C with histone as the substrate. Under these conditions kinase activity was almost totally dependent on the presence of diolein; again, diolein caused a large decrease in the Ka for Ca2+, from greater than 100 microM to 2.5 microM. The increased sensitivity of protein kinase C to Ca2+ with P47 rather than histone, and the ability of diacylglycerol to activate protein kinase C without shifting the Ka for Ca2+, when P47 is the substrate, illustrate that the mechanism of protein kinase C activation is influenced by the exogenous substrate used to assay the enzyme.

Citing Articles

Effect of cis-unsaturated fatty acids on aortic protein kinase C activity.

Dell K, Severson D Biochem J. 1989; 258(1):171-5.

PMID: 2930504 PMC: 1138337. DOI: 10.1042/bj2580171.

Protein kinase C zeta subspecies from rat brain: its structure, expression, and properties.

Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, NISHIZUKA Y Proc Natl Acad Sci U S A. 1989; 86(9):3099-103.

PMID: 2470089 PMC: 287072. DOI: 10.1073/pnas.86.9.3099.

Protein kinase C does not regulate diacylglycerol metabolism in aortic smooth muscle cells.

Chuang M, Dell K, Severson D Mol Cell Biochem. 1990; 96(1):69-77.

PMID: 2233706 DOI: 10.1007/BF00228454.

Protein phosphorylation associated with the stimulation of neutrophils. Modulation of superoxide production by protein kinase C and calcium.

Heyworth P, Badwey J J Bioenerg Biomembr. 1990; 22(1):1-26.

PMID: 2160451 DOI: 10.1007/BF00762842.

Calmodulin inhibits the protein kinase C-catalysed phosphorylation of an endogenous protein in A10 smooth-muscle cells.

Zhao D, Hollenberg M, Severson D Biochem J. 1991; 277 ( Pt 2):445-50.

PMID: 1859372 PMC: 1151254. DOI: 10.1042/bj2770445.

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