Purification and characterization of acetoacetyl-CoA synthetase from rat liver.
Acetoacetyl-CoA synthetase (acetoacetate:CoA ligase) was purified to electrophoretic homogeneity from a rat liver supernatant by ammonium sulfate fractionation and successive chromatographies on DEAE-Sepharose, Blue-Sepharose, Red-Sepharose, CoA-Sepharose, Ultrogel AcA-44, and DEAE-Sepharose once again. The purified enzyme had a specific activity of 2.3 mumol acetoacetyl-CoA formed per min per mg protein, which constituted a 960-fold purification compared to the crude extract, with a 7.4% yield. The enzyme absolutely required ATP, CoA, a monovalent cation (K+, Rb+, Cs+ or NH4+) and a divalent cation (Mg2+, Mn2+ or Ni2+) for its activity, yielding acetoacetyl-CoA, AMP and PPi in equimolar amounts. The molecular weight of the enzyme was approximately 80 000 as determined by Ultrogel AcA-34 gel filtration, and 71 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was active only on acetoacetate and to a lesser extent on L-(+)-3-hydroxybutyrate, and the Km values for acetoacetate, L-(+)-3-hydroxybutyrate, ATP and CoA were 8 microM, 75 microM, 60 microM and 10 microM, respectively.
D000105:Acetyl Coenzyme A / Q000031:analogs & derivatives / Q000378:metabolism; D000214:Acyl Coenzyme A; D000818:Animals; D002792:Cholestyramine Resin / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D002940:Circadian Rhythm; D003066:Coenzyme A Ligases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D002792:Cholestyramine Resin; C010667:acetoacetyl CoA; D000105:Acetyl Coenzyme A; D003066:Coenzyme A Ligases; C023747:acetoacetyl-CoA synthetase