Synthesis of arachidonoyl coenzyme A and docosahexaenoyl coenzyme A in retina.
The synthesis of 14C-labeled arachidonoyl 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 docosahexaenoate and arachidonate esterified to phospholipids. 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- lowered the Km and Vmax values to 7.64 +/- 0.11 and 3.03 +/- 0.12, respectively. The Km and Vmax values for arachidonate 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 arachidonate were 24 degrees C and 28 degrees C, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
D000214:Acyl Coenzyme A / Q000096:biosynthesis*; D000818:Animals; D002417:Cattle; D003066:Coenzyme A Ligases / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology; D011894:Rana pipiens; D051381:Rats; D011919:Rats, Inbred Strains; D012160:Retina / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000201:enzymology; D013045:Species Specificity; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature
D000214:Acyl Coenzyme A; C026563:arachidonyl-coenzyme A; D003066:Coenzyme A Ligases