Properties of the lipid body lipase of Pinus eduUs and electrophoretic purification of its 64 kDa subunit

Abstract

Lipase (triacyiglycerol acylhydrolase, EC 3.1.1.3) in oilseeds can be associated with either the lipid body or glyoxysomal membrane and can have various pH optima and substrate specificities. There is conflicting evidence for the subcellular location of lipase in gymnosperms, and little information exists on its activity characteristics. In this report, Pinus edulis (pinyon) was found to have an acid lipase, which was associated with the lipid body fraction and the activity of which increased during germination. Active lipase from the solubilized lipid body membrane was determined by gel permeation chromatography to have a molecular weight of 260 000. Further attempts to purify the active enzyme were unsuccessful. A lipid body membrane protein of 64 kDa which increased in parallel with lipase activity during germination was isolated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. excised, and polyclonal antibodies were made against it. Using these antibodies, active lipase was immunoprecipitated from solution, thus indicating that the 64 kDa protein is a subunit of the lipase. Pinus edulis lipase had a pH optimum of ca 4.5. and it exhibited little specificity for triacyiglycerol substrates in vitro. The lipase was specific in activity against fluorometric substrates, with the highest activity against methyl‐umbelliferyl laurale. Lipase activity was inhibited by high concentrations of non‐ionic detergent. This lipid body acid lipase appears to be primarily responsible for lipid hydrolysis during pinyon germination.