Phospholipid reactivation of plasmalogen metabolism

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Abstract
This report is concerned mainly with the properties of an enzyme from rat liver microsomes which hydrolyzes the alkenyl ether bond of 1-(1′-alk-1′-enyl)-glycero-3-phosphoryl-choline (alkenyl-GPC hydrolase). Destruction of the normal environment of the microsomes by treatment with phospholipases A or C caused inactivation of the alkenyl-GPC hydrolase, which was then partially reactivated by the addition of exogenous phospholipids. Both sphingomyelin and diacyl-GPC were efficient in restoring activity; diacyl-GPE was less effective; and monoacyl-GPC and monoacyl-GPE were ineffective. The presence of two long hydrocarbon chains in the lipid activator is apparently required for reactivation, suggesting that interaction of hydrophobic areas of the enzyme with the phospholipid is necessary for maximal activity. High concentrations of sucrose mimicked the effect of phospholipids, and because the sucrose and diacyl-GPC did not show an additive effect, they may reactivate the enzyme in a similar manner. Disrupting the enzyme's environment by freezing and thawing the preparation also resulted in a loss of enzymatic activity, which was restored by added exogenous phospholipids. The alkenyl-GPC hydrolase was inhibited by imidazole and some of its derivatives. Histidine and N-acetyl histidine did not inhibit the enzyme, presumably due to the presence of a negative charge on the carboxyl group rather than the steric bulk of that group, since histidine methyl ester did inhibit the enzyme. Kinetic evidence showed imidazole to be a competitive inhibitor. The enzymatic activity of imidazole-treated microsomes also increased following addition of exogenous phospholipids. Imidazole inhibition differed from the phospholipase A-inactivation in that it was partially reversed by KCl, but not by sucrose. Imidazole did not inhibit other microsomal enzymes tested, indicating that it is not a general inhibitor of membrane-associated enzymes.