PURIFICATION AND CHARACTERIZATION OF A POLYISOPRENYL PHOSPHATE PHOSPHATASE FROM PIG BRAIN - POSSIBLE DUAL-SPECIFICITY

Microsomal fractions from pig and calf brain catalyze the enzymatic de phosphorylation of endogenous and exogenous dolichyl monophosphate (Do l-P) (Sumbilla, C, A, and Waechter, C, J, (1985) Methods Enzymol. 111, 471-482), The Dol-P phosphatase (EC 3.1.3.51) has been solubilized by extracting pig brain microsomes with the nonionic detergent Nonidet P -40 and purified approximately 1,107-fold by a combination of anion ex change chromatography, polyethylene glycol fractionation, dye-ligand c hromatography, and wheat germ agglutinin affinity chromatography. Trea tment of the enzyme with neuraminidase prevented binding to wheat germ agglutinin-Sepharose, indicating the presence of one or more N-acetyl neuraminyl residues per molecule of enzyme, When the highly purified p olyisoprenyl phosphate phosphatase was analyzed by SDS-polyacrylamide gel electrophoresis, a major 33-kDa polypeptide was observed. Enzymati c dephosphorylation of Dol-P by the purified phosphatase was 1) optima l at pH 7; 2) potently inhibited by F-, orthovanadate, and Zn2+ > Co2 > Mn2+ but unaffected by Mg2+; 3) exhibited an approximate K-m for C- 95-Dol-P of 45 mu M; and 4) was sensitive to N-ethylmale-imide, phenyl glyoxal, and diethylpyrocarbonate. The pig brain phosphatase did not d ephosphorylate glucose 6-phosphate, mannose 6-phosphate, 5'-AMP, or p- nitrophenylphosphate, but it dephosphorylated dioleoylphosphatidic aci d at initial rates similar to those determined for Dol-P, Based on the virtually identical sensitivity of Dol-P and phosphatidic acid dephos phorylation by the highly purified enzyme to N-ethylmale-imide, F-, ph enylglyoxal, and diethylpyrocarbonate, both substrates appear to be hy drolyzed by a single enzyme with an apparent dual specificity. This is the first report of the purification of a neutral Dol-P phosphatase f rom mammalian tissues. Although the enzyme is Mg2+-independent and cap able of dephosphorylating Dol-P and PA, several enzymological properti es distinguish this lipid phosphomonoesterase from PAP2.