Factors Involved in Fatty Acyl CoA Desaturation by Fungal Microsomes. The Relative Roles of Acyl CoA and Phospholipids as Substrates

1 Microsomes were prepared from strain 74a Neurospora crassa mycelium. Gas-liquid chromatographic analysis indicated that approximately 80 % of the microsomal fatty acids contained 18 carbon atoms; 90% of the 18-carbon fatty acids were unsaturated, and of these linoleic acid was the major component. 2 Microsomes suspended in 0.1 M phosphate buffer pH 7.1 could desaturate both [1-14C]-stearyl CoA and [1-14C]oleyl CoA; however, the latter Δ 12 desaturation was always an order of magnitude slower than the Δ 9 desaturation as assayed by radiogaschromatography. Attempts to solubilize the desaturase activity were unsuccessful. 3 Oxygen and a nucleotide were required for both Δ 9 and Δ 12 desaturation. The rate of desaturation was higher with NADH than with NADPH, and NADP was almost as effective as NADPH. Δ 9 desaturation in the presence of NAD was slow, and no detectable desaturation at Δ 12 occurred with this oxidized nucleotide. 4 Mycelial microsomes were used to test the hypothesis that phospholipids are intermediates in Δ 9 and Δ 12 desaturation. Microsomes were incubated at 30°C with 14C-labeled acyl CoA substrates in the absence of reduced nucleotides, then washed to remove unused acyl CoA, and, finally, incubated at 30° C with NADH. No Δ 9 desaturation of stearyl CoA occurred under these conditions; however, the [14C]oleyl-labeled microsomes were converted to linoleyl phospholipid at a normal rate. 5 Stearyl CoA was rapidly converted to oleyl phospholipid in the presence of NADH and to free stearic acid in the absence of NADH. 6 Oleyl CoA was much more rapidly incorporated into phospholipid than hydrolyzed to the free fatty acid, even in the absence of a reduced nucleotide. 7 Stearyl CoA, free stearic acid, and oleyl CoA were all tightly, but not covalently, bound to the microsomes. 8 The data rule out the possibility of stearyl phospholipid being an intermediate in the Δ 9 desaturation. However, these experiments are consistent with oleyl phospholipid's being an intermediate in the conversion of oleyl CoA to linoleyl phospholipid. 9 The data do not establish whether or not phospholipids are the direct substrates for Δ 12 desaturases in fungal microsomes.