Purification and characterization of mitochondrial thioesterase from rabbit myocardium and its inhibition by palmitoyl carnitine.
Mitochondrial thioesterase from rabbit myocardium was purified to homogeneity by sequential ion exchange, hydroxylapatite, chromatofocusing and gel filtration chromatographies. The purified protein had an apparent molecular mass of 170 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining revealed a single band (Mr 43 000) demonstrating that the protein is a tetramer. The specific activity of the purified thioesterase for palmitoyl-CoA hydrolysis was 1.8 mumol/mg per min. Thioesterase activity was maximal at pH 8 and was activated by Mg2+ but inhibited by Ca2+. Pathophysiological concentrations of L-palmitoyl carnitine (20-400 microM) competitively inhibited enzymic activity. The purified enzyme was also inhibited by high concentrations of substrate (over 20 microM palmitoyl-CoA).
D000818:Animals; D002122:Calcium Chloride / Q000378:metabolism; D002331:Carnitine / Q000031:analogs & derivatives*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D015636:Magnesium Chloride; D008929:Mitochondria, Heart / Q000201:enzymology*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010172:Palmitoylcarnitine / Q000494:pharmacology*; D011817:Rabbits; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
D015636:Magnesium Chloride; D009243:NAD; D010172:Palmitoylcarnitine; D013869:Thiolester Hydrolases; D008274:Magnesium; D002122:Calcium Chloride; D002331:Carnitine