PRACTICAL ASSAY-METHOD OF CYTOSOLIC ACETOACETYL-COA THIOLASE BY RAPIDRELEASE OF CYTOSOLIC ENZYMES FROM CULTURED LYMPHOCYTES USING DIGITONIN

We designed a simple approach to determine cytosolic acetoacetyl-Cob t hiolase (CT) activity for differential diagnosis of ketone body catabo lic defects, using rapid cell-subfractionation of cultured lymphocytes with digitonin. Efficiency of cell subfractionation was determined by measurement of lactate dehydrogenase and citrate synthetase as marker enzymes for cytosol and organelle fractions, respectively, and confir med by immunotitration and immunoblotting using antibodies against cyt osolic and mitochondrial thiolases, respectively. In the condition of best separation taken in the presence of 1 mg/ml digitonin, acetoacety l-Cos thiolase activities in the presence of K+ ion in the cytosol and organelle fractions were 138.3 +/- 39.2 and 84.0 +/- 16.2 nmol/min/ml , respectively. The thiolase acitivity in the organelle fraction was d oubled by the presence of K+ ion, whereas that in the cytosol fraction was not affected. The thiolase activity in the organelle fraction was reduced by the treatment of anti-mitochondrial acetoacetyl-Coa thiola se (T2) antibody but not by anti-CT antibody. On the other hand, that in the cytosol fraction was significantly decreased by anti-CT antibod y but not by anti-T2 antibody. These data suggested that T2 was collec ted in the organelle fraction, and that CT activity could be assessed by measurement of the thiolase activity in the cytosolic fraction. Suc cinyl-CoA: 3-ketoacid CoA transferase (SCOT), whose defect is the thir d inherited disorder of ketone body catabolism, was collected in the o rganelle fraction. Hence, this method will prove to be useful for accu rate assessment of defects of CT as well as T2 or SCOT, all involved i n ketone body catabolism. (C) 1998 Tohoku University Medical Press.