This report describes the partial characterization of the enzymatic activity responsible for the hydrolysis of
acetate from 1-alkyl-2-acetyl-sn-glycerol, the immediate precursor in the de novo synthesis of PAF (platelet-activating factor or 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by Ehrlich
ascites cells. The highest acetylhydrolase activity for this neutral
lipid was associated with the membrane fractions from Ehrlich
ascites cells (greater than 90% of total activity); only a minimal level of activity (less than 10%) was observed in the cytosol which contrasts with the cytosolic site of
PAF acetylhydrolase in normal cells. Hydrolysis of 1-[3H]hexadecyl-2-acetyl-sn-
glycerol by the membrane fraction at pH 7.5 and 37 degrees C gave apparent values for Km and Vmax of 45 microM and 179 nmol/min per mg
protein, respectively. Hydrolysis of
acetate from 1-[3H]hexadecyl-2-acetyl-sn-
glycerol by the membrane fraction was not affected by 5 mM concentrations of Ca+2, Mg+2 or
EDTA, but was significantly inhibited (80% reduction) by 10 mM NaF. Based on differences in both the subcellular distribution and response to inhibition by NaF, the neutral
lipid acetylhydrolase does not appear to be the same
enzyme that hydrolyzes
acetate from
platelet-activating factor. In contrast to inhibition of
diacylglycerol lipase by p-chloromercuribenzoate and
N-ethylmaleimide, we found no significant inhibition of
acetate hydrolysis from 1-[3H]hexadecyl-2-acetyl-sn-
glycerol by either of these compounds. Also,
p-nitrophenyl acetate (a
nonspecific esterase substrate) failed to inhibit
acetate hydrolysis of 1-[3H]hexadecyl-2-acetyl-sn-
glycerol. Our studies of this
enzyme would indicate that it may play an important role in regulating the levels of
platelet-activating factor synthesized by the de novo pathway via hydrolysis of the immediate precursor of PAF.