Isolation and characterization of an ester hydrolase active on phorbol diesters from murine liver.
An esterase which cleaves the 12-ester group of phorbol-12,13-diesters has been purified to electrophoretic homogeneity from murine liver cytosol using ammonium sulfate fractionation, Sephadex G-200 gel filtration, Con A-Sepharose chromatography, and phenyl-Sepharose chromatography. The enzyme is a single chain, hydrophobic glycoprotein with a molecular weight of 60,000 and exhibits optimum activity at pH 7.5 to 8.5. The hydrolase has an isoelectric point (pI) of 5. The enzyme is heat- and acid-labile. Zinc, cobalt, and fluoride ions inhibit its activity. Phenylmethylsulfonyl fluoride is a potent inhibitor of the hydrolase. Sarkosyl also inhibits the enzyme at millimolar concentrations. The enzyme inactivates biologically active phorbol-12,13-diesters in a dose-, time-, and temperature-dependent manner. The enzyme inhibits phorbol-12,13-dibutyrate binding to its receptor in a noncompetitive manner. The inhibition constant (KI) has been found to be 6.6 X 10(-8) M.
D000818:Animals; D001921:Brain / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins; D002273:Carcinogens / Q000378:metabolism; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004950:Esterases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000378:metabolism*; D010704:Phorbols / Q000378:metabolism*; D011493:Protein Kinase C; D011955:Receptors, Drug / Q000378:metabolism; D013379:Substrate Specificity
D029742:Caenorhabditis elegans Proteins; D002273:Carcinogens; D002352:Carrier Proteins; D010703:Phorbol Esters; D010704:Phorbols; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D004950:Esterases