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.