Purification and characterization of a polyisoprenyl phosphate phosphatase from pig brain. Possible dual specificity.
Microsomal fractions from pig and calf brain catalyze the enzymatic dephosphorylation of endogenous and exogenous dolichyl monophosphate (Dol-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 exchange chromatography, polyethylene glycol fractionation, dye-ligand chromatography, and wheat germ agglutinin affinity chromatography. Treatment of the enzyme with neuraminidase prevented binding to wheat germ agglutinin-Sepharose, indicating the presence of one or more N-acetylneuraminyl residues per molecule of enzyme. When the highly purified polyisoprenyl phosphate phosphatase was analyzed by SDS-polyacrylamide gel electrophoresis, a major 33-kDa polypeptide was observed. Enzymatic dephosphorylation of Dol-P by the purified phosphatase was 1) optimal at pH 7; 2) potently inhibited by F-, orthovanadate, and Zn2+ > Co2+ > Mn2+ but unaffected by Mg2+; 3) exhibited an approximate Km for C95-Dol-P of 45 microM; and 4) was sensitive to N-ethylmaleimide, phenylglyoxal, and diethylpyrocarbonate. The pig brain phosphatase did not dephosphorylate glucose 6-phosphate, mannose 6-phosphate, 5'-AMP, or p-nitrophenylphosphate, but it dephosphorylated dioleoyl-phosphatidic acid at initial rates similar to those determined for Dol-P. Based on the virtually identical sensitivity of Dol-P and phosphatidic acid dephosphorylation by the highly purified enzyme to N-ethylmaleimide, F-, phenylglyoxal, and diethylpyrocarbonate, both substrates appear to be hydrolyzed by a single enzyme with an apparent dual specificity. This is the first report of the purification of a neutral Dol-P phosphatase from mammalian tissues. Although the enzyme is Mg2+-independent and capable of dephosphorylating Dol-P and PA, several enzymological properties distinguish this lipid phosphomonoesterase from PAP2.
Frank
D W
DW
Department of Biochemistry, A. B. Chandler Medical Center, University of Kentucky College of Medicine, Lexington, Kentucky 40536, USA.
Waechter
C J
CJ
D000818:Animals; D001921:Brain / Q000201:enzymology; D002413:Cations, Divalent / Q000494:pharmacology; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005459:Fluorides / Q000494:pharmacology; D007526:Isoelectric Point; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D012794:Sialic Acids / Q000032:analysis; D012995:Solubility; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine; D014638:Vanadates / Q000494:pharmacology
D002413:Cations, Divalent; D003902:Detergents; D012794:Sialic Acids; D013439:Sulfhydryl Reagents; D014638:Vanadates; D010744:Phosphoric Monoester Hydrolases; C020088:dolichyl-phosphatase; D005459:Fluorides