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J Biol Chem, Vol. 273, Issue 19, 11791-11798, May 8, 1998
From the Department of Biochemistry, A. B. Chandler Medical
Center, University of Kentucky College of Medicine,
Lexington, Kentucky 40536
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 µM; 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.
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