Molecular and Catalytic Properties of Mitochondrial (Keto- genie) 3-Hydroxy-3-methylglutaryl Coenzyme A Synthase of Liver*

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase has been purified to homogeneity from avian liver. The enzyme in dilute phosphate buffer, pH 7.0, has an szolu, of 5.7 S and a molecular weight of 105,000 determined by sedimentation equilibrium; the presence of 0.1 M KC1 causes dissocation to a form one-half that size, i.e. about 57,000 daltons. Since the subunit molecular weight of the synthase determined by the dodecyl sulfate acrylamide gel method is 53,000, it appears that the native enzyme is a dimer composed of weight-homogeneous subunits. A number of molecular and catalytic properties allow the mitochondrial and cytoplasmic 3-hydroxy9-methylglutarylCoA synthases to be distinguished. The p1 of the homogeneous mitochondrial enzyme is 7.2. This value, while identical to that of the single isoelectric-focusing species of broken mitochondrial preparations, differs from those of the multiple 3-hydroxyd-methylglutaryl-CoA synthases found in the cytoplasmic fraction which exhibit p1 values of 4.8 and 6.7. Rabbit antibodies against the purified mitochondrial synthase are capable of precipitating the mitochondrial, but not the cytoplasmic, synthase(s) of avian liver. Finally, the synthases differ kinetically in their responses to divalent magnesium ion, the mitochondrial enzyme being inhibited and the cytoplasmic enzyme(s) activated. It is proposed that the mitochondrial 3-hydroxy3-methylglutaryl-CoA synthase of liver functions in ketogenesis while its cytoplasmic counterpart participates in cholesterogenesis (CLINK&NBEARD, K. D., SUGIYAMA, T., REED, W. D. AND LANE, M. D. (1975) J. Biol. Chem. 250,3124-3135). Evidence presented in the preceding paper (1) and elsewhere (2-7) indicates that the enzyme system for synthesizing HMGIcoenzyme A from acetyl-CoA, i.e. acetoacetyl-Cob thiolase and HMG-CoA synthase, is present both in the mitochondrial and cytoplasmic compartments of avian and rat liver cells. This dual localization would seem to facilitate the apparent independent regulation of HMG-CoA synthesis for mitochondrial ketogenesis and cytoplasmic cholesterogenesis in liver* (1). For example, it is known that physiological states, such as fasting and diabetes which cause marked suppression of hepatic cholesterogenesis (8, 9), concomitantly stimulate hepatic ketogenesis (10, 11). Moreover, this view is supported by the finding that (-)hydroxycitrate, a potent inhibitor of cytoplasmic ATP-citrate lyase, and hence, extramitochondrial acetyl-CoA formation, blocks cholesterogenesis, but not mitochondrial ketogenesis (12). Work in this laboratory (2-6, 13, 14) has led to the isolation in homogeneous form and characterization of the enzymes from avian liver responsible for cytoplasmic HMG-CoA synthesis, i.e. cytosolic acetoacetyl-CoA thiolase and HMG-CoA synthase. These enzymes have been implicated in hepatic cholesterogenesis since their activities are subject to cholesterol feedback control (14). The present investigation describes the purification of the mitochondrial (ketogenic) HMG-CoA synthase from avian liver and its partial molecular characterization. Thus, in conjunction with the preceding paper (l), rigorous proof is provided for the existence of distinct mitochondrial and cytoplasmic forms of HMG-CoA synthase in liver.