Enzymes for lipolysis and fatty acid metabolism in different organelle fractions from rape seed cotyledons

Abstract

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 β-oxi-dation system of fatty acids are located in one or more membrane-bounded organelles. The organelles were isolated carefully and identified by marker-enzyme activities. Activities of neither lipase nor acylester acylhydrolase (EC 3.1.1) could be detected either in glyoxysomes or in mitochondria, even when various substrate emulsions were employed. Only after long-term incubations could the presence of a low lipolytic activity be demonstrated for different organellar fractions. This alkaline carboxylic ester hydrolase, whose activity is below the detection limit of various standard tests, cannot play a role in the lipolytic function of glyoxysomes. In addition, a complete set of enzyme activities necessary for the conversion of saturated fatty acids to acetyl CoA was found only in the glyoxysomal cell fraction. The low β-oxidation activity discovered in the mitochondrial cell fraction is evidently due to glyoxysomal contamination. Enzyme activities unique to the mitochondrial β-oxidation system such as carnitine palmitoyltransferase (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 molecular masses of 45, 63, and 70 kDa only in glyoxysomal fractions. Obviously, in the fatty rape seed neither glyoxysomes nor mitochondria are involved in triacylglycerol hydrolysis, and β-oxidation of fatty acids occurs exclusively in glyoxysomes.