Measurement of diacylglycerol acyltransferase activity in isolated hepatocytes.
An assay procedure for diacylglycerol acyltransferase that allows rapid measurement of the activity of this enzyme in isolated hepatocytes is described. The one-step procedure involves permeabilization of the plasma membrane with digitonin and simultaneous measurement of diacylglycerol acyltransferase activity. Digitonin at a concentration of 64 microg/mg of cellular protein was found to be optimal for exposing microsomal diacylglycerol acyltransferase to the components of the assay. The enzyme assay is linear with time up to 4 min and with protein concentrations in the range 0.25-2.4 mg of cellular protein/assay. It is shown that there is a good correlation of cellular enzyme activity as determined in digitonin-permeabilized hepatocytes with the rate of triacylglycerol synthesis in intact hepatocytes.
Geelen
Math J H
MJ
Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands. m.geelen@vet.uu.nl
D000217:Acyltransferases / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D051048:Diacylglycerol O-Acyltransferase; D004072:Digitonin / Q000737:chemistry / Q000494:pharmacology; D005227:Fatty Acids / Q000494:pharmacology; D015248:Gemfibrozil / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology*; D016014:Linear Models; D008297:Male; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D005227:Fatty Acids; D000217:Acyltransferases; D051048:Diacylglycerol O-Acyltransferase; D004072:Digitonin; D015248:Gemfibrozil