PRELIMINARY CHARACTERIZATION OF PHOSPHOLIPASE-A(2) IN LAGENIDIUM-GIGANTEUM

Phospholipase A2 (PLA2) hydrolyses the fatty acyl ester bond at the sn -2 position in glycerophospholipids. To better understand its regulato ry roles, factors affecting PLA2 activity in Lagenidium giganteum were investigated: divalent ions; chelators; inhibitors; pH; and substrate concentration. PLA2 activity of L. giganteum whole cell homogenates w as determined using 1-stearoyl-2-[C-14]arachidonoyl phosphatidylcholin e as substrate. The divalent cations Ca2+, Mg2+, and Mn2+ all enhanced PLA2 activity, while Co2+, Fe2+, and Zn2+ were either slightly inhibi tory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while h igh concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca2+ and Mn2+ than Mg2+, reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both ac id (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors , nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenac ylbromide, reduced PLA2 activity by about 80% at 5 mM concentration, 5 0% with 1 mM inhibitor, and had no effect at 100 muM. The relatively h igh levels of these compounds needed to inhibit PLA2 hydrolysis may ha ve been due to the presence of a cocktail of enzymes, some of which we re not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without a dverse effects to the mycelia. PLA2 activity was highest in the late o ospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA2 activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes , but there was significant activity in cytosolic fractions. Separatio n of cell homogenates by column chromatography revealed that there wer e at least nine enzymes capable of cleaving fatty acids in the sn-2 po sition of phospholipids. (C) 1994 Academic Press, Inc.