Characterization of rat brain microsomal acyl-coenzyme A ligases: different enzymes for the synthesis of palmitoyl-coenzyme A and lignoceroyl-coenzyme A.
Palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by microsomal membranes but lignoceric acid solubilized with Triton WR-1339 was not an effective substrate even though the detergent dispersed the same amount of these fatty acids and was also not inhibitory to the enzyme [I. Singh, R. P. Singh, A. Bhushan, and A. K. Singh (1985) Arch. Biochem. Biophys. 236, 418-426]. This observation suggested that palmitoyl-CoA and lignoceroyl-CoA may be synthesized by two different enzymes. We have solubilized the acyl-CoA ligase activities for palmitic and lignoceric acid of rat brain microsomal membranes with Triton X-100 and resolved them into three separate peaks (fractions) by hydroxylapatite chromatography. Fraction A (palmitoyl-CoA ligase) had high specific activity for palmitic acid and Fraction C (lignoceroyl-CoA ligase) for lignoceric acid. Specific activity of palmitoyl-CoA ligase for palmitic acid was six times higher than in Fraction C and specific activity of lignoceroyl-CoA ligase for lignoceric acid was four times higher than in Fraction A. At higher concentrations of Triton X-100 (0.5%), lignoceroyl-CoA ligase loses activity whereas palmitoyl-CoA ligase does not. Lignoceroyl-CoA ligase lost 60% of activity at 0.6% Triton X-100. Palmitoyl-CoA ligase (T1/2 of 4.5 min) is more stable at 40 degrees C than lignoceroyl-CoA ligase (T1/2 of 1.5 min). The pH optimum of palmitoyl-CoA ligase was 7.7 and that of lignoceroyl-CoA ligase was 8.4. Similar to our results with intact membranes, palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by palmitoyl-CoA ligase whereas lignoceric acid when solubilized with Triton WR-1339 was not able to act as substrate for lignoceroyl-CoA ligase. Since solubilized enzyme activities for synthesis of palmitoyl-CoA and lignoceroyl-CoA from microsomal membranes can be resolved into different fractions by column chromatography and demonstrate different properties, we suggest that in microsomal membranes palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes.
D000214:Acyl Coenzyme A / Q000096:biosynthesis*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002846:Chromatography, Affinity; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005260:Female; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D017830:Octoxynol; D010171:Palmitoyl Coenzyme A / Q000096:biosynthesis*; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012995:Solubility; D013379:Substrate Specificity; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D000214:Acyl Coenzyme A; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D010171:Palmitoyl Coenzyme A; C018582:lignoceroyl-coenzyme A; D011092:Polyethylene Glycols; D017830:Octoxynol; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase; C016811:tyloxapol