Effects of nucleotides on a cold labile acetyl-coenzyme A hydrolase from the supernatant fraction of rat liver

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Abstract
An acetyl-CoA hydrolyase that is labile at low temperature was purified to homogeneity from the supernatant fraction of rat liver. The monomeric molecule, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had a MW of .apprx. 63,000, while that of the purified enzyme, estimated by gel filtration, was 135,000. Thus, the enzyme consists of 2 subunits of identical MW. On addition of ATP or ADP at 25.degree. C, the dimeric form of the enzyme aggregated to tetrameric forms (MW 242,000 and MW 230,000, respectively), whereas addition of AMP had little effect on enzyme association (MW 145,000). When ATP was removed from the ATP-treated tetrameric enzyme by dialysis, the tetramer was mostly dissociation into the dimeric form. The apparent Km values for acetyl CoA of the dimeric enzyme and tetrameric enzyme, reconstituted from the former in the presence of 2 mM ATP, were 170 .mu.M and 60 .mu.M, respectively. The purified dimeric enzyme was inactivated by exposure to lower temperature, especially below 10.degree. C. The various nucleotides tested partially stabilize the dimeric enzyme at low temperature, ATP being the most effective. Sucrose density gradient centrifugation showed that loss of catalytic activity by cold treatment was accompanied by dissociation of the dimer and tetramer into protomer.