Characterization of the enzymatic hydrolysis of acetate from alkylacetylglycerols in the de novo pathway of PAF biosynthesis.

Abstract 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 (> 90% of total activity); only a minimal level of activity ( 3 H]hexadecyl-2-acetyl- sn -glycerol by the membrane fraction at pH 7.5 and 37°C gave apparent values for K m and V max of 45 μM and 179 nmol/min per mg protein, respectively. Hydrolysis of acetate from 1-[ 3 H]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-[ 3 H]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-[ 3 H])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.