The ability of 0.4 M KCl to extract over 80% of a short-chain .beta.-hydroxyacyl-CoA dehydrase from rat hepatic endoplasmic reticulum, while more than 80% of the long-chain .beta.-hydroxyacyl-CoA dehydrase component of the fatty acid chain elongation system remains intact, confirms the existence of > 1 hepatic microsomal dehydrase. Following extraction from the microsomal membrane, the short-chain dehydrase undergoes, at least, a 2-fold activation. Employing even-numbered trans-2-enoyl-CoA substrates ranging in carbon chain length from 4-16, the highest dehydrase specific activity of 16 .mu.mol min-1 mg protein-1 was obtained with trans-2-hexenoyl-CoA; crotonyl-CoA was the 2nd most active substrate, followed by 8 > 10 > 12 > 14 > 16. The specific activity of the short-chain dehydrase with trans-2-hexadecenoyl-CoA (C-16) was only 3% of that observed with the trans-2-hexenoyl-CoA. With crotonyl-CoA or .beta.-hydroxybutyryl-CoA as substrates, HPLC [high performance liquid chromatography] was employed to identify the products, .beta.-hydroxybutyryl-CoA, of the hydration reaction, or crotonyl-CoA, of the reverse dehydration reaction. The short-chain dehydrase catalyzed the formation of both D(-) and L(+) stereoisomers of .beta.-hydroxybutyryl-CoA. The equilibrium constant for the dehydrose-catalyzed reaction determined at pH 7.4 and 35.degree. C, was calculated to be 6.38 x 10-2 M-1, while the standard free energy change was -775 cal/mol, results similar to those obtained with crystalline crotonase. Finally, based on membrane fraction marker enzymes, substrate specificity, and heat lability of the dehydrase, the microsomal membrane contains a short-chain .beta.-hydroxyacyl-CoA dehydrase which is separate from the mitochondrial crotonase.
