Purification and characterization of calcium/phospholipid-dependent kinase from adult human epidermis.
Tumor-promoting phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) cause epidermal inflammation and hyperplasia similar to that observed in psoriasis. Recent evidence suggests that these effects are mediated by a calcium/phospholipid-dependent protein kinase (protein kinase C), which is quantitatively the major cellular phorbol ester receptor. This report describes the partial purification and biochemical properties of this enzyme from adult human epidermis. Protein kinase C activity was purified 30-fold from high speed supernatants prepared from homogenates of keratome biopsies obtained from healthy volunteers. The partially purified preparation had a specific activity of 1.2 nmol/min/mg protein and an apparent molecular weight of 79,400. Activity was dependent on the presence of calcium and phosphatidylserine. At low calcium concentration (less than 0.1 mM) activity was greatly stimulated by 1,2-dioleoylglycerol. TPA mimicked the effect of diglyceride on enzyme activity, and the partially purified enzyme specifically bound phorbol dibutyrate (Kd = 2 nM). Protein kinase C activity was also present in the membrane fraction from adult human epidermis, and possessed properties similar to those of the cytosolic enzyme. We conclude that protein kinase C is present in human epidermis and is activated by TPA in a manner similar to that described for this enzyme from other tissues. These data lay the foundation for studying the role of protein kinase C in the regulation of epidermal growth and maturation.
D000328:Adult; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides / Q000494:pharmacology; D004817:Epidermis / Q000201:enzymology*; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000378:metabolism; D010718:Phosphatidylserines / Q000494:pharmacology; D011493:Protein Kinase C / Q000302:isolation & purification* / Q000378:metabolism; D011955:Receptors, Drug; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004075:Diglycerides; D008565:Membrane Proteins; D010703:Phorbol Esters; D010718:Phosphatidylserines; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; C013965:diolein