Practical Assay Method of Cytosolic Acetoacetyl-CoA Thiolase by Rapid Release of Cytosolic Enzymes from Cultured Lymphocytes Using Digitonin.

Abstract

We designed a simple approach to determine cytosolic acetoacetyl-CoA thiolase (CT) activity for differential diagnosis of ketone body catabolic 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 confirmed by immunotitration and immunoblotting using antibodies against cytosolic and mitochondrial thiolases, respectively. In the condition of best separation taken in the presence of 1 mg/ml digitonin, acetoacetyl-CoA 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 activity in the organelle fraction was doubled 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 thiolase (T2) antibody but not by anti-CT antibody. On the other hand, that in the cytosol fraction was significantly decreased by anti-CT antibody but not by anti-T2 antibody. These data suggested that T2 was collected in the organelle fraction, and that CT activity could be assessed by measurement of the thiolase activity in the cytosolic fraction. Succinyl-CoA: 3-ketoacid CoA transferase (SCOT), whose defect is the third inherited disorder of ketone body catabolism, was collected in the organelle fraction. Hence, this method will prove to be useful for accurate assessment of defects of CT as well as T2 or SCOT, all involved in ketone body catabolism.