Molecular and Catalytic Properties of Cytosolic Acetoacetyl Coenzyme A Thiolase from Avian Liver

A cytosolic thiolase, presumably involved in hepatic cholesterogenesis, has been purified to homogeneity from avian liver. This enzyme is the predominant acetoacetyl-CoA thiolase of chicken liver comprising 70% of the total thiolase activity in laying hens. In addition to the cytosolic enzyme, two mitochondrial species were also detected by isoelectric focusing; the cytosolic species exhibits a pI of 7.1, which is identical with that of the purified enzyme, whereas the mitochondrial species have pI values of 5.9 and 8.3. The cytosolic thiolase has a sedimentation coefficient (s20,w) of 7.94 S and a molecular weight, determined by sedimentation equilibrium, of 169,000. At pH 8.2 and 30°, the native enzyme catalyzes the thiolytic cleavage of 54,000 moles of acetoacetyl-CoA per min per mole of enzyme; under comparable conditions, but with 50 mm Mg2+ added, the rate in the reverse (condensation) direction is 1,770 moles per min per mole of enzyme. Dissociation of the thiolase and electrophoresis on sodium dodecyl sulfate polyacrylamide gels gives rise to a single stained protein band whose mobility corresponds to a subunit weight of 41,000 daltons. Thus it appears that the native enzyme is composed of four identical or similar polypeptide chains. The combined actions of purified cytosolic thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase rapidly converts acetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA in a thermodynamically favorable manner; equilibrium (at pH 8.0 and 30°) is attained when approximately 68% of the acetyl-CoA added has been converted to 3-hydroxy-3-methylglutaryl-CoA. Thus, regulation of one or both of these enzymes would appear necessary to prevent depletion of the cytoplasmic acetyl-CoA pool when cholesterogenesis is shut off. Although feeding 2% cholesterol to chickens for 2 to 7 days depresses liver cytosolic thiolase activity by 30 to 54%, this reduction is insufficient to account for the nearly complete cessation of hepatic cholesterogenesis that has been reported under identical conditions of cholesterol feeding (Sakakida, H., Shediac, C. C., and Siperstein, M. D. (1963) J. Clin. Invest. 42, 1521).