The pattern of expression of protein kinase C (PKC) isoenzymes was examined in chicken gizzard smooth muscle using isoenzyme-specific antibodies: α, δ, ε, η, and ζ isoenzymes were detected. PKCα associated with the particulate fraction in the presence of Ca2+ and was extracted by divalent cation chelators. PKCδ required detergent treatment for extraction from the EDTA – EGTA-washed particulate fraction. PKCε, η, and ζ were recovered in the cytosolic fraction prepared in the presence of Ca2+. PKCζ, which has been implicated in the regulation of gene expression in smooth muscle, was partially purified from chicken gizzard. Two peaks of PKCζ-immunoreactive protein (Mr 76 000) were eluted from the final column; only the second peak exhibited kinase activity. The specific activity of PKCζ with peptide ε (a synthetic peptide based on the pseudosubstrate domain of PKCε) as substrate was 2.1 μmol Pi∙min−1∙(mg PKCζ)−1 and, with peptide ζ as substrate, was 1.2 μmol Pi min−1∙(mg PKCζ)−1. Activity in each case was independent of Ca2+, phospholipid, and diacylglycerol. Lysine-rich histone III-S was a poor substrate for PKCζ (specific activity, 0.1–0.3 μmol Pi∙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 α, β, and γ isoenzymes), were also poor substrates of PKCζ (specific activities, 0.04 and 0.02 μmol Pi∙min−1∙mg−1, respectively). Chicken gizzard PKCζ was insensitive to the PKC activator phorbol 12,13-dibutyrate or the PKC inhibitor chelerythrine. The properties of PKCζ are, therefore, quite distinct from those of other well-characterized PKC isoenzymes.Key words: protein kinase C, isoenzymes, smooth muscle.
Sarcoplasmic reticulum and endothelium independently regulate venous smooth muscle [Ca2+]iand contraction
