Purification and characterization of avian liver 3-hydroxy-3-methylglutaryl coenzyme A lyase.
3-Hydroxy-3-methylglutaryl-CoA lyase has been purified to homogeneity from avian liver mitochondria. Affinity chromatography of a partially purified preparation on agarose hexane 3',5'-ADP produces enzyme of high specific activity (351 units/mg). A total purification of 1750-fold over the mitochondrial matrix fraction is achieved. The purified enzyme is stable when stored in 30% glycerol with millimolar levels of dithiothreitol. Divalent cations (e.g. Mg2+, Mn2+) and thiol-protecting agents stimulate enzyme activity under assay conditions. The enzyme binds hydroxymethylglutaryl-CoA with a Km = 8 microM. Optimal enzyme activity, measured at pH = 8.9, is 7-fold higher than activity at physiological pH. The apparent molecular weight of the native enzyme, estimated by gel filtration on Sephadex G-100, is approximately 49,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests that the enzyme is a dimer, composed of 27,000-dalton subunits. Assuming one active site per subunit, a turnover number of 158 s-1 (pH 8.2; 30 degrees C) is calculated. Antibodies have been prepared against homogeneous hydroxymethylglutaryl-CoA lyase. Ouchterlony double diffusion patterns verify the homogeneity of the preparation. Incubation of enzyme with antiserum results in virtually complete inhibition of enzyme activity.
D000214:Acyl Coenzyme A / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent; D004372:Ducks; D005779:Immunodiffusion; D007700:Kinetics; D046911:Macromolecular Substances; D015093:Meglutol / Q000031:analogs & derivatives / Q000302:isolation & purification / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification* / Q000378:metabolism
D000214:Acyl Coenzyme A; D002413:Cations, Divalent; D046911:Macromolecular Substances; D015093:Meglutol; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase