TROUT LIVER PHOSPHOLIPASE A2 AND ITS ROLE IN MEMBRANE ADAPTATIONS TO TEMPERATURE

The deacylating enzyme, phospholipase A(,2) (PLA(,2); E. C. 3.1.1.4) from liver microsomes of 5(DEGREES)- and 20(DEGREES)-acclimated rainbow trout (Salmo gairdneri) was studied using fixed time assays in the presence of an exogenous substrate of 1-acyl,2-{('3)H}-oleoyl phosphatidylcholine and measuring the accumulation of radiolabel in free fatty acid. The enzyme occurred in conjunction with phospholipase A(,1) in microsomes, but unlike the mammalian phopholipases, PLA(,2) accounted for the preponderance of deacylating activity in that subcellular fraction. Incubation of microsomes in the presence of Triton X-100 resulted in extracting the PLA(,2) protein from the native membrane. A 30-fold purification and the removal of all detectable traces of phospholipid were achieved by precipitation of the solubilized enzyme with 35-65% ammonium sulfate and gel filtration chromatography on Sephadex G-200. The molecular weight was estimated at 73,000 daltons.;To determine the substrate preference of PLA(,2), ratios of kinetic constants, V/K(,m), were measured for 1-palmitoyl phosphatidylcholines with three different 18-carbon fatty acids esterified to the sn-two carbon. PLA(,2) from cold-acclimated fish showed an order of preference for the acyl moieties of 18:1 > 18:2 > 18:0. Warm-acclimated trout generally preferred 2-18:0 phosphatidylcholine, but the actual order of preference depended on the temperature of the assay and the presence of endogenous lipids. The preference in warm-acclimated fish for disaturated species may be responsible for their absence in trout cell membranes at high temperatures. The preference for monoenoic species and the compensation of catalytic rate in cold-acclimated fish provide a biochemical basis for the previously reported decrease in monoenes at the sn-two position of phosphatides following cold acclimation in trout.;In light of its intracellular distribution, perfect compensation and temperature-dependent substrate preference, trout liver PLA(,2) is implicated as an important component and possible regulatory step in thermally induced remodeling of cell membranes via a deacylation-reacylation cycle.;The enzyme exhibited perfect compensation for temperature at non-saturating substrate concentrations regardless of whether assays were performed using microsomal (particulate) preparations or lipid-free preparations indicating that temperature compensation was an attribute of the protein and not its interaction with the lipid microenvironment. Compensation was due primarily to a decrease in apparent K(,m) following cold acclimation. Positive thermal modulation with acute temperature change in warm-acclimated fish, i.e., the lowering of apparent K(,m) in 5(DEGREES) assays versus 20(DEGREES) assays, was evident only in particulate enzyme, hence was an adaptive phenomenon attributable to the presence of endogenous lipids.