Identification of protein kinase C isoenzymes in smooth muscle: partial purification and characterization of chicken gizzard PKC zeta.
The pattern of expression of protein kinase C (PKC) isoenzymes was examined in chicken gizzard smooth muscle using isoenzyme-specific antibodies: alpha, delta, epsilon, eta, and zeta isoenzymes were detected. PKC alpha associated with the particulate fraction in the presence of Ca2+ and was extracted by divalent cation chelators. PKC delta required detergent treatment for extraction from the EDTA-EGTA-washed particulate fraction. PKC epsilon, eta, and zeta were recovered in the cytosolic fraction prepared in the presence of Ca2+. PKC zeta, which has been implicated in the regulation of gene expression in smooth muscle, was partially purified from chicken gizzard. Two peaks of PKC zeta-immunoreactive protein (M(r) 76 000) were eluted from the final column; only the second peak exhibited kinase activity. The specific activity of PKC zeta with peptide epsilon (a synthetic peptide based on the pseudosubstrate domain of PKC epsilon) as substrate was 2.1 mumol P(i).min-1.(mg PKC zeta)-1 and, with peptide zeta as substrate, was 1.2 mumol P(i).min-1.(mg PKC zeta)-1. Activity in each case was independent of Ca2+, phospholipid, and diacylglycerol. Lysine-rich histone III-S was a poor substrate for PKC zeta (specific activity, 0.1-0.3 mumol P(i).min-1.mg-1). Two proteins, calponin and caldesmon, which have been implicated in the regulation of smooth muscle contraction and are phosphorylated by cPKC (a mixture of alpha, beta, and gamma isoenzymes), were also poor substrates of PKC zeta (specific activities, 0.04 and 0.02 mumol P(i).min-1.mg-1, respectively). Chicken gizzard PKC zeta was insensitive to the PKC activator phorbol 12,13-dibutyrate or the PKC inhibitor chelerythrine. The properties of PKC zeta are, therefore, quite distinct from those of other well-characterized PKC isoenzymes.
Clément-Chomienne
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Smooth Muscle Research Group, Faculty of Medicine, University of Calgary, AB, Canada.
Walsh
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MP
D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D002847:Chromatography, Agarose; D002848:Chromatography, DEAE-Cellulose; D005895:Gizzard, Avian / Q000201:enzymology; D007527:Isoenzymes / Q000096:biosynthesis* / Q000302:isolation & purification; D008840:Microfilament Proteins; D009130:Muscle, Smooth / Q000201:enzymology*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D010770:Phosphotransferases / Q000032:analysis; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011817:Rabbits; D051381:Rats; D013552:Swine; D000096985:Calponins; D000099221:Protein Kinase C zeta
D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D007527:Isoenzymes; D008840:Microfilament Proteins; D010770:Phosphotransferases; D011493:Protein Kinase C; D000096985:Calponins; D000099221:Protein Kinase C zeta