Search

Tetrahymena microsomes contain cytochrome b5 and an NADH-dependent cytochrome b5 reductase, but these proteins are present at only one-tenth the levels observed in rat liver microsomes. We show that both proteins can be partially purified by techniques developed for the rat liver proteins. We can show that cyanide inhibits the rate of exhaustion of NADH, and therefore reoxidation of cytochrome b5 by microsomes, and that stearoyl CoA enhances the rate of reoxidation of the cytochrome. Also, we find that a fragment of rat liver NADH-cytochrome b5 reductase can restore NADH-dependent cytochrome b5 reduction to Tetrahymena microsomes which have been treated with N-ethylmaleimide to eliminate endogenous reductase activity. These results indicate that there is considerable resemblance between the rat and Tetrahymena systems, and that desaturation of stearoyl and oleoyl groups may occur in Tetrahymena via pathways similar to those known in liver.

The desaturation of oleoyl-CoA by a microsomal preparation from Tetrahymena has been studied. Desaturation of oleoyl-CoA required oxygen and NADH, and was inhibited by cyanide. HPLC analysis of fatty acid phenacyl esters, prepared from TLC-purified phospholipid, confirmed that radioactivity appeared in oleate, linoleate and gamma-linolenate. Both the time course of desaturation and the apparent desaturation of 1-palmitoyl-2-[14C]oleoylphosphatidylcholine suggested that phospholipid-bound oleate could be a substrate for desaturation. In the crude microsomal preparation, acylation of oleoyl-CoA to give oleoyl phospholipid was rapid. Therefore, preincubation in the absence of NADH was employed to create [14C]oleoyl phospholipids, and kinetic studies were carried out upon subsequent addition of NADH. When data were plotted in a double reciprocal form, a linear function was observed.