Differentiation of microsomal from lysosomal triacylglycerol lipase activities in rat liver

Abstract

Non-Iysosomal triacylglycerol lipase (EC 3.1.1.3) and cholesteryl esterase (EC 3.1.1.13) activities from rat liver were distinguished from lysosomal activities using assays based on the release of oleic acid from glycerol-[ 3H]oleate or cholesteryl-[1- 14C]oleate. Dispersion of the lipid substrates in acetone and the addition of a 1:1 (w/w) mixture of sonicated phosphatidylcholine/phosphatidylserine to the incubation mixture permitted the development of sensitive assays that allowed comparison under identical conditions. MnCl 2 stimulated the non-lysosomal triacylglycerol lipase and cholesteryl esterase activities 2-fold but inhibited the lysosomal activities 85%. The lysosomal activities could be solubilized by osmotic disruption with water, whereas the non-lysosomal activities repelleted after centrifugation at 100 000 × g for 1 h. pH optima of the lysosomal and non-lysosomal activities were 5.5 and 6.0, respectively, under the conditions employed. Cobalt acetate, CaCl 2 and MgCl 2 inhibited the lysosomal triacylglycerol lipase and cholesteryl esterase activities greater than 60% but had no effect on the non-lysosomal activities. Upon subcellular fractionation, the Mn 2+-stimulated triacylglycerol lipase and cholesteryl esterase activities partitioned with microsomal marker enzymes. Pronase inactivated the Mn 2+-stimulated activities 75–82% under conditions in which mannose-6-phosphatase latency and activity remained unchanged, suggesting that the Mn 2+-stimulated activities contain protease-sensitive sites exposed on the cytoplasmic surface of microsomal vesicles. The triacylglycerol lipase and cholesteryl esterase activities in fibroblasts from patients with Wolman's disease and cholesteryl ester storage disease were stimulated by MnCl 2, had pH optima of 6.0–6.5, and were not solubilized by osmotic disruption, suggesting that human fibroblasts may also contain both the microsomal and the lysosomal activities. Microsomal triacylglycerol lipase and cholesteryl esterase activities may function to hydrolyze endogenously synthesized triacylglycerol and cholesteryl esters.