Purification and characterization of an isoform of protein kinase C from bovine neutrophils.
Protein kinase C (PKC) from bovine neutrophils was purified 1420-fold. Subcellular fractionation analysis of bovine neutrophil homogenate in the presence of EGTA indicated that more than 95% of the PKC activity was present in the soluble fraction. The purification procedure from cytosol involved sequential chromatographic steps on DE-52 cellulose, Mono Q, and phenyl-Sepharose. Whereas bovine brain PKC could be resolved into four isoenzymatic forms by chromatography on a hydroxylapatite column, bovine neutrophil PKC was eluted in a single peak, suggesting that it corresponded to a single isoform. The apparent molecular weight of bovine neutrophil PKC was 82,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By filtration on Sephadex G-150, a molecular weight of 85,000 was calculated, indicating that bovine neutrophil PKC in solution is monomeric. Its isoelectric point was 5.9 +/- 0.1. Bovine neutrophil PKC was autophosphorylated in the presence of [gamma-32P]ATP, provided that the medium was supplemented with Mg2+, Ca2+, phosphatidylserine, and diacylglycerol; phorbol myristate acetate could substitute for diacylglycerol. Autophosphorylated PKC could be cleaved by trypsin to generate two radiolabeled peptides of Mr 48,000 and 39,000. The labeled amino acids were serine and threonine. During the course of the purification procedure of bovine neutrophil PKC, a protein of Mr 23,000, which was abundant in the cytosolic fraction of the homogenate, was found to exhibit a strong propensity to PKC-dependent phosphorylation in the presence of [gamma-32P]ATP, Mg2+, Ca2+, phosphatidylserine, and diacylglycerol. This protein was recovered together with PKC in one of the two active peaks eluted from the Mono Q column at the second step of PKC purification.(ABSTRACT TRUNCATED AT 250 WORDS)
Dianoux
A C
AC
Départment de Recherche Fondamentale, Centre d'Etudes Nucléaires, Grenoble, France.
Stasia
M J
MJ
Vignais
P V
PV
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007527:Isoenzymes / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D011493:Protein Kinase C / Q000097:blood* / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D007527:Isoenzymes; D011493:Protein Kinase C