The oleyl-coenzyme-A desaturase of potato tubers. Enzymatic properties, intracellular localization and induction during "aging" of tuber slices.

Abstract

An active oleyl‐coenzyme‐A desaturase was associated with a microsomal fraction from “aged” slices of potato tuber; starting from [1‐14C]oleyl‐coenzyme A as a substrate, in vitro, more than 40% of the final fatty acid radioactivity was recovered in linoleic acid, the most abundant fatty acid of the intracellular membranes of potato tubers. The presence of serum albumin in the reaction mixture was essential for the desaturation reaction and for preventing the degradation of polyunsaturated fatty acids. This stabilization of the desaturase activity by serum albumin allowed a great resistance upon freezing. The microsomal oleyl‐coenzyme‐A desaturase showed a pH optimum around 7.0–7.2 and a temperature optimum near 25°C. The apparent Km of the desaturase for oleyl‐coenzyme A was 25 μM. Oxygen and pyridine nucleotide were required for oleyl‐coenzyme A desaturation (NADH or NADPH were found nearly equally effective in the desaturation). Although NADPH was more effective than NADP, NAD was a effective as NADH for desaturation, suggesting that a NADH‐regenerating system probably exists in the microsomal suspension. The oleyl‐coenzyme‐A desaturase was specific in vitro for oleyl‐coenzyme A as a substrate; no desaturation was observed with ammonium oleate, which was, however, desaturated in vivo by aged slices of potato tuber. Oleyl‐coenzyme A (and the newly labelled linoleic acid molecules) were essentially incorporated into phospholipids and particularly phosphatidylcholine. The oleyl‐coenzyme‐A desaturase was almost entirely associated with the microsomal membranes, as shown by differential centrifugation and sucrose‐gradient separations. The oleyl‐coenzyme‐A desaturase activity (absent in the freshly cut slices) was induced during the aging of slices; a linear increase with the time of aging was found in many microsomal activities, such as oleyl‐coenzyme A desaturase, NADH‐cytochrome c reductase and protein synthesis. The inhibition of all these events by cycloheximide, or the partial inhibition by actinomycin D indicated that derepression of the genetic activity and synthesis of new proteins were involved in the induction of the oleyl‐coenzyme‐A desaturase.