Purification and characterization of an isoform of protein kinase C from bovine neutrophils

Journal Article · Tue Jan 24 00:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00428a003· OSTI ID:5641889

Dianoux, A C; Stasia, M J; Vignais, P V ^[1]

---

1. Centre d'Etudes Nucleaires, Grenoble (France)

Protein kinase C (PKC) from bovine neutrophils was purified 1,420-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. 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. Bovine neutrophil PKC was autophosphorylated in the presence of ({gamma}-{sup 32}P)ATP, provided that the medium was supplemented with Mg{sup 2+}, Ca{sup 2+}, phosphatidylserine, and diacylglycerol; phorbol myristate acetate could substitute for diacylglycerol. Autophosphorylated PKC could be cleaved by trypsin to generate two radiolabeled peptides of M{sub r} 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 M{sub r} 23,000 was found to exhibit a strong propensity to PKC-dependent phosphorylation in the presence of ({gamma}-{sup 32}P)ATP, Mg{sup 2+}, Ca{sup 2+}, 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. It is suggested that the M{sub r} 23,000 protein might be a natural substrate for bovine neutrophil PKC.

• OSTI ID: 5641889
• Journal Information: Biochemistry; (USA), Vol. 28:2; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
LABELLING
NEUTROPHILS
ENZYME ACTIVITY
PHOSPHOTRANSFERASES
MOLECULAR STRUCTURE
ATP
AUTORADIOGRAPHY
CATTLE
ELECTROPHORESIS
MOLECULAR WEIGHT
PHORBOL ESTERS
PHOSPHORUS 32
PHOSPHORYLATION
PURIFICATION
SERINE
THREONINE
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CARBOXYLIC ACIDS
CARCINOGENS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ENZYMES
ESTERS
HYDROXY ACIDS
ISOTOPES
LEUKOCYTES
LIGHT NUCLEI
MAMMALS
MATERIALS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
RADIOISOTOPES
RUMINANTS
TRANSFERASES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques