Differential regulation of three catalytic activities of platelet-activating factor (PAF)-dependent transacetylase

We have previously established that PAF-dependent transacetylase (TA) purif ied to apparent homogeneity from rat kidney membranes and cytosol contains three separate catalytic activities, namely PAF lysophospholipid transacety lase (TA(1)), PAF sphingosine transacetylase (TA(s)), and PAF acetylhydrola se (AH), In the present investigation, we studied the biochemical factors a nd mechanism(s) that differentially regulate these three TA activities of t he purified enzymes. We found that only the TA(s) activity of the TA purifi ed from the membranes was stimulated by phosphatidylserine (PS) with optima l concentration of activation occurring at 25 muM, Other acidic phospholipi ds, such as phosphatidylinositol (PI) and phosphatidylinositol I-phosphate (PIP), are partially effective, while diacylglycerol and free fatty acids h ad no effect on the TA(s) activity. PS exerted its effect on the TA(s) acti vity through the increases of both K-m and V-max. In addition, N-ethylmalim ide (NEM) and dithiobis-(2-nitro-5-thiobenzoic acid) (DTNB) strongly inhibi ted the TA(L) activity and partially decreased the TA(L) and AH activities of the purified membrane enzyme in a dose-dependent manner. The addition of PS, but not by its substrate, sphingosine, could prevented the inhibition by NEM on the basal level of TA(s). On the other hand, the inhibition of TA L by NEM and DTNB were partially protected by the substrate, lysoplasmaloge ns, Furthermore, PAF fully protects the inhibition of AH, partially protect s the inhibition of TA(L), and does not protect the inhibition of TA(s) by NEM, These results suggested that the three individual catalytic activities of TA have different dependencies on the thiol-containing residue(s) of th e enzyme, i.e., cysteine. Furthermore, the nonresponsiveness of the purifie d cytosolic TA(s) to PS activation is consistent with our previous notions that membrane and cytosolic TA are posttranslationally distinct. (C) 2001 A cademic Press.