Purification and properties of a pig heart thiolase with broad chain length specificity and comparison of thiolases from pig heart and Escherichia coli.
A thiolase (acetyl CoA acyltransferase, EC 2.3-1.16) which acts on substrates of various chain lengths (thiolase I) has been purified from pig heart muscle 366-fold to near homogeneity as judged by gel electrophoresis. Its molecular weight was estimated to be 200,000 in the absence and 46,000 in the presence of sodium dodecyl sulfate. Kinetic measurements with acetoacetyl coenzyme A, 3-ketohexanoyl-CoA, 3-ketooctanoyl-CoA, and 3-ketodecanoyl-CoA yielded apparent Km values of 16, 8.3, 2.4, and 1.8 micron, respectively, whereas apparent Vmax values of 65 to 69 mumol/min/mg were obtained with all substrates except for acetoacetyl-CoA, with which a value of 26.5 mumol/min/mg was observed. Antibodies prepared against this thiolase were used to demonstrate that thiolase I and acetoacetyl-CoA thilase (thiolase II) from pig heart mitochondria are immunologically unrelated. The antibodies cross-reacted, however, with thiolase I from beef heart. Kinetic constants (Km, Vmax) were also determined for thiolases I and II from Escherichia coli, as were the native and subunit molecular weights of E. coli thiolase II. Although the E. coli thiolases were found to be immunologically distinct from the pig heart enzymes, their physical and kinetic properties are strikingly similar to those of the heart thiolases. In view of this finding and in view of the known physiological functions of the E. coli thiolases, it is proposed that thiolase I from pig heart is only involved in fatty acid metabolism, whereas thiolase II functions solely in ketone body degradation.
D000102:Acetyl-CoA C-Acyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D013045:Species Specificity; D013379:Substrate Specificity; D013552:Swine
D000217:Acyltransferases; D000102:Acetyl-CoA C-Acyltransferase