ENZYMES FOR LIPOLYSIS AND FATTY-ACID METABOLISM IN DIFFERENT ORGANELLE FRACTIONS FROM RAPE SEED COTYLEDONS

This study addresses the still open question of whether or not in oily storage tissues, e.g. cotyledons of germinating rape (Brassica napus L.) seedlings' lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) and the beta-oxidation system of fatty acids are located in one or more me mbrane-bounded organelles. The organelles were isolated carefully and identified by marker-enzyme activities. Activities of neither lipase n or acylester acylhydrolase (EC 3.1.1) could be detected either in glyo xysomes or in mitochondria, even when various substrate emulsions were employed. Only after longterm incubations could the presence of a low lipolytic activity be demonstrated for different organellar fractions . This alkaline carboxylic ester hydrolase, whose activity is below th e detection limit of various standard tests, cannot play a role in the lipolytic function of glyoxysomes. In addition, a complete set of enz yme activities necessary for the conversion of saturated fatty acids t o acetyl CoA was found only in the glyoxysomal cell fraction. The low beta-oxidation activity discovered in the mitochondrial cell fraction is evidently due to glyoxysomal contamination. Enzyme activities uniqu e to the mitochondrial beta-oxidation system such as carnitine palmito yltransferase (EC 2.3.1.21), carnitine acetyltransferase (EC 2.3.1.7), and acyl-CoA dehydrogenase (EC 1.3.99.3) were absent, indicating that mitochondria are not involved in fatty acid metabolism. In addition, on Western blots, antibodies raised against malate synthase (EC 4.1.3. 2) and acyl-CoA oxidase (EC 1.1.3)recognized three polypeptides with m olecular masses of 45, 63, and 70 kDa only in glyoxysomal fractions. O bviously, in the fatty rape seed neither glyoxysomes nor mitochondria are involved in triacylglycerol hydrolysis, and beta-oxidation of fatt y acids occurs exclusively in glyoxysomes.