HPLC measurement of testicular long chain acyl-CoA synthetases with different substrate specificities

Abstract

Acyl-CoA synthetase activity with various long chain fatty acid substrates was measured in microsomes from rat testes, isolated spermatids and testes of hypophysectomized adult rats, using reversed-phase high performance liquid chromatography (HPLC). The spectrophotometric HPLC method produced results comparable to those of parallel radiometric assays and was highly specific for acyl-CoA products. At optimal pH and cofactor concentrations, specific activity from whole testis was similar for 18:1, 20:4 and 22:5 but somewhat lower for 16:0 over the substrate range 0.01-3.2 mM. Activity from spermatids or from testes of hypophysectomized rats was much lower with 22:5 than with 18:1 or 20:4, whereas activities with 18:1 and 20:4 were similar at all substrate concentrations. All substrates exhibited Michaelis-Menten type saturation kinetics and linear Lineweaver-Burke plots at lower substrate concentrations but inhibited activity at higher concentrations. Apparent values of KM for 16:0, 18:1 and 20:4 were more than twice that of 22:5, whereas both observed and calculated maximum velocities were similar for the four fatty acids. Differences in pseudokinetic parameters and differential expression of the testicular acyl-CoA synthetase activities with different fatty acids suggest the presence of multiple enzymes, at least one of which may be hormonally regulated.