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Title: A 23-kDa protein as a substrate for protein kinase C in bovine neutrophils. Purification and partial characterization

In {sup 32}P{sub i}-loaded bovine neutrophils stimulated with phorbol myristate acetate (PMA), radioactivity was preferentially incorporated into a protein of low molecular mass, suggesting a PKC-dependent phosphorylation. This protein, termed 23-kDa protein, was predominantly localized in the cytosol. The apparent molecular mass of the purified protein range between 20 and 23 kDa. In the absence of mercaptoethanol, a dimer accumulated. Homogeneity of the 23-kDa protein was verified by 2D-PAGE analysis. Gel isoelectric focusing (IEF) of the purified 23-kDa protein followed by Coomassie blue staining allowed the visualization of our discrete protein bands with isoelectric points ranging between pH 6.3 and 6.7. Phosphorylation of the 23-kDa protein by ({gamma}-{sup 32}P)ATP in the presence of bovine neutrophil PKC supplemented with Ca{sup 2+}, phosphatidylserine, and diacylglycerol or with PMA occurred on serine and required the presence of mercaptoethanol. IEF of the {sup 32}P-labeled 23-kDa protein followed by autoradiography revealed for discrete bands with distinct isoelectric points similar to those of the bands stained by Coomassie blue after IEF on nonlabeled 23-kDa protein. The bands of the 23-kDa protein resolved by IEF and transfered to nitrocellulose showed ability to bind ({sup 35}S)GTP-{gamma}-S. The immunoreactivity of antibodies raised in rabbits against the bovine neutrophil 23-kDamore » protein was demonstrated on immunoblots after SDS-PAGE. The 23-kDa protein differed also from several other proteins of similar molecular mass that have been identified in neutrophils, namely, calmodulin, the small subunit of the low-potential cytochrome b, and a low molecular weight protein which is ADP-ribosylated by the botulinum toxin.« less
Authors:
; ;  [1]
  1. (Centre d'Etudes Nucleaires (France))
Publication Date:
OSTI Identifier:
7138153
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (USA); Journal Volume: 28:25
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PHORBOL ESTERS; BIOLOGICAL EFFECTS; PHOSPHOTRANSFERASES; BIOCHEMISTRY; PROTEINS; MOLECULAR STRUCTURE; ATP; CATTLE; ELECTROPHORESIS; MOLECULAR WEIGHT; NEUTROPHILS; PHOSPHORUS 32; PHOSPHORYLATION; PURIFICATION; SUBSTRATES; SULFUR 35; ANIMALS; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY FLUIDS; CARCINOGENS; CHEMICAL REACTIONS; CHEMISTRY; DAYS LIVING RADIOISOTOPES; DOMESTIC ANIMALS; ENZYMES; ESTERS; EVEN-ODD NUCLEI; ISOTOPES; LEUKOCYTES; LIGHT NUCLEI; MAMMALS; MATERIALS; NUCLEI; NUCLEOTIDES; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; PHOSPHORUS ISOTOPES; PHOSPHORUS-GROUP TRANSFERASES; RADIOISOTOPES; RUMINANTS; SULFUR ISOTOPES; TRANSFERASES; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques