Arachidonoyl‐Coenzyme A Synthetase and Nonspecific AcylCoenzyme A Synthetase Activities in Purified Rat Brain Microvessels

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Abstract
Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 .mu.M and 5.8 .mu.M for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol .cntdot. min-1 .cntdot. mg protein-1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 .mu.M unlabelled arachidonic acid, with a Ki of 7.1 .mu.M, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 .mu.M. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 .mu.M unlabelled palmitic acid, with a Ki of 5.0 .mu.M and to a much lesser extent by arachidonic acid (Ki, 23 .mu.M). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40.degree.C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis. These data together strongly suggest that rat brain microvessels have the capacity to catalyze specifically the formation of acyl-CoA derivatives from several polyunsaturated long-chain fatty acids, including arachidonic acid in the first place. Besides this particular arachidonoyl-CoA synthetase, palmitic acid could be activated with the aid of a second acyl-CoA synthetase.

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