Synthesis of arachidonoyl coenzyme A and docosahexaenoyl coenzyme A in retina

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Abstract
The synthesis of 14c-labeled arachidonoy1 coenzyme A (CoA) and docosahexaenoyl CoA was studied in the human bovine, rat and frog retina. The synthesis of arachidonoyl CoA was two- to fourfold higher than that of docosahexaenoyl CoA in the retinal membranes examined. The enzyme involved in the synthesis of these polyenoyl CoAs, long-chain acyl-CoA synthetase, had a species variation and was most active in microsomal membranes from frog retina. In the retina, 60% of the enzyme's total activity was in the microsomal fraction, whereas only 4-7% of the long-chain acyl-CoA synthetase activity was in the rod outer segments, which contain large amounts of docosa-hexaenoate and arachidonate esterified to phospho-lipids. This fact implies that despite the high concentration of docosahexaenoate and arachidonate in rod outer segments, there is little turnover of these acyl chains, at least through the formation of thioesters. The apparent Km (uM) and Vmax (nmol/min/mg protein) values for the labeled docosahexaenoate were 9.84 ± 0.86 and 5.26 ± 0.46, respectively. The presence of Triton X-100 lower-ed the Km and Vmax values to 7.64 ± 0.11 and 3.03 ± 0.12, respectively. The Km and Vmax values for araehidonate were 40 and 13.3, respectively. The apparent Km value for ATP was 270 ± 33 uM and for CoA, 3.70 ± 0.23 uM. The transition temperatures obtained from Arrhenius plots for docosahexaenoate and araohidonate were 24°C and 28°C, respectively. The calculated apparent activation energies were 75-94 KJ/mol below and 10-13 KJ/mol above the transition temperature. The high affinity that the activating enzyme has for the polyenoic acids, especially docosahexaenoic acid, may be responsible, at least in part, for the active retention of these fatty acids in the retina.