Subcellular localization of enzyme activities involved in the metabolism of platelet-activating factor in rainbow trout leukocytes

Abstract

The subcellular distribution of an alkyllyso-GPC: acetyl-CoA acetyltransferase (EC 2.3.1.67) and transacylase, two important enzyme activities involved in the remodeling pathway for the biosynthesis of platelet-activating factor (1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine, PAF) have been examined in leukocytes isolated from the pronephros of the rainbow trout, Oncorhynchus mykiss. Contrary to mammalian systems, in which the acetyltransferase is localized to intracellular membranes, the subcellular distribution of an acetyltransferase activity in rainbow trout leukocytes was localized to the plasma membrane. Analysis of the acetyltransferase products by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) confirmed synthesis of two subclasses of PAF, 1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine and 1-acyl-2-acetyl- sn-glycero-3-phosphocholine. The transacylase activity in this study was detected in membrane fractions in two domains of the intermediate density region which also contained the NADH dehydrogenase activity, a marker enzyme for the endoplasmic reticulum. Acylation of lysoPAF (1- O-alkyl-2-lyso- sn-glycero-3-phosphocholine) exhibited approximately 95% specificity for ω-3 fatty acids. Acylation patterns were not significantly different in either domain of the endoplasmic reticulum. A model is proposed herein for the metabolism of PAF in rainbow trout leukocytes.