Arachidonoyl-coenzyme A synthetase and nonspecific acyl-coenzyme A synthetase activities in purified rat brain microvessels.
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 microM and 5.8 microM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol X min-1 X mg protein-1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 microM unlabelled arachidonic acid, with a Ki of 7.1 microM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 microM. 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 microM unlabelled palmitic acid, with a Ki of 5.0 microM and to a much lesser extent by arachidonic acid (Ki, 23 microM). 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 degrees C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
D000214:Acyl Coenzyme A / Q000096:biosynthesis; D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D001667:Binding, Competitive; D001921:Brain / Q000098:blood supply*; D003066:Coenzyme A Ligases / Q000378:metabolism*; D007700:Kinetics; D008833:Microcirculation / Q000201:enzymology; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010171:Palmitoyl Coenzyme A / Q000096:biosynthesis; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D001095:Arachidonic Acids; D010169:Palmitic Acids; C026563:arachidonyl-coenzyme A; D010171:Palmitoyl Coenzyme A; D016718:Arachidonic Acid; D019308:Palmitic Acid; D003066:Coenzyme A Ligases; C033896:arachidonate - CoA ligase