Properties of diacylglycerol acyltransferase from microspore-derived embryos of Brassica napus

Abstract

Particulate diacylglycerol acyltransferase (EC 2.3.1.20) from microspore-derived embryos of Brassica napus cv Reston was characterized. Diacylglycerol acyltransferase activity was mainly associated with the 10 000 and 100 000 g pellets prepared by differential centrifugation of embryo homogenate. The 100 000 g microsomal pellet was characterized in greater detail. Microsomal diacylglycerol acyltransferase activity was optimal near neutral pH and was affected by buffer composition. The microsomal enzyme was assayed optimally in a temperature range of 30–40° but appeared unstable at temperatures exceeding 40°. The enzyme activity reached a plateau at a concentration of ca 5 μM oleoyl-CoA but beyond this concentration, erucoyl-CoA was a more effective acyl donor. Considerably higher utilization rates for erucoyl-CoA were found using microsomes prepared from both high and low-erucic acid lines of Brassica napus. 2-Bromooctanoate inhibited microsomal diacylglycerol acyltransferase by 50% at a concentration of 1.5 mM. Detergents which might solubilize diacylglycerol acyltransferase were tested for enzyme inhibition. n-Octyl β- d-glucopyranoside was found to be the least inhibitory of the detergents, when tested at equivalent final concentrations.