PURIFICATION AND CHARACTERIZATION OF LONG-CHAIN ACYL-COA HYDROLASE FROM RAT-LIVER MITOCHONDRIA

An enzyme able to hydrolyze long‐chain acyl‐CoA esters has been purified from mitochondrial matrix of rat liver by ammonium sulfate extraction, gel chromatography, and isoelectric focusing. The specific activity of the purified enzyme represented approx. 53‐fold and 700‐fold enrichment over the initial extract in the absence and presence of serum albumin, respectively. The enzyme has been purified to homogeneity as judged by polyacrylamide‐gel electrophoresis in the absence and presence of sodium dodecyl sulfate. The enzyme has a sedimentation rate of 2.1 S in a sucrose gradient, a Stokes radius of 1.9 nm, and consists of a single polypeptide with a molecular weight of 19000 determined by gel chromatography. The isoelectric point (pI) of the enzyme is 6.0. The enzyme is specific for long‐chain (over C10) fatty acyl‐CoA esters with maximal activity for palmitoyl‐CoA. At physiological substrate concentrations the enzyme did not hydrolyze palmitoyl‐l‐carnitine, tripalmitoyl‐glycerol, dipalmitoyl‐phosphatidylcholine, or cholesterol‐oleate. The enzyme did not possess any palmitoyl‐CoA synthetase or carnitine palmitoyltransferase activity. The hydrolase activity was strongly influenced by the acyl‐CoA/protein ratio as serum albumin increased the activity. As this effect was also seen at acyl‐CoA concentrations under the critical micelle concentration it seems likely that serum albumin not only prevents the inhibition of the enzyme by binding the inhibiting micellar form of the substrate, but also stabilizes the enzyme. Similar effects were found with the nonionic detergent, Triton X‐100. Copyright © 1979, Wiley Blackwell. All rights reserved