Development and Optimization of an Assay of Scp-2/Thiolase Activity in Rat Liver, and a Study of the Substrate Stereospecificity of Permfe-2
The peroxisome is the site of side-chain shortening of the bile acid precursor 3a, 7a, 12a-trihydroxy-5ß-cholestanoyl-CoA (THC-CoA) by a ß-oxidative mechanism. Sterol carrier protein 2 (SCP-2/Thiolase) has recently been shown to harbor the peroxisomal thiolase activity responsible for cleavage of 24-oxo-THCA-CoA to C₃-CoA and cholyl-CoA. To characterize this enzyme further, an assay that is sensitive and specific is needed. An HPLC method has been developed which allows the use of a גּ-fraction from rat liver for an assay of thiolase activity toward 24-oxo-THCA-CoA, as measured by C₃-CoA production over incubation time. The HPLC method used for this assay is a modification of Demoz et al.'s method for measuring short-chain fatty acyl-CoA derivatives in rat liver homogenates. It is demonstrated that the assay provides linear, reproducible results, and that the method can be extended to allow studies of other enzymatic activites as well. An investigation of the stereospecificity of another peroxisomal enzyme from rat liver, perMFE-2, is also described. The conclusions from this study indicate that perMFE-2 harbors both the hydratase and dehydrogenase activities responsible for normal production of 24-oxo-THCA-CoA from (E) -24-ene-THCA-CoA in rat livers.
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