In situ measurement of HMG-CoA reductase activity in digitonin-permeabilized hepatocytes.
An assay procedure for HMG-CoA reductase is described which allows rapid measurement of the activity of this enzyme in isolated rat hepatocytes. In a one step procedure digitonin permeabilizes the plasma membrane and at the same time HMG-CoA reductase activity is measured. Digitonin at a concentration of 64 micrograms per mg of cell protein was found to be optimal for exposing microsomal HMG-CoA reductase to the assay components. The enzyme assay is linear with time up til 5 min and with protein concentrations in the range of 0.06-0.6 mg of cell protein per assay. It is shown that cellular enzyme activity is affected by preincubation of intact hepatocytes with a variety of short-term modulators of hepatic cholesterogenesis.
Geelen
M J
MJ
Laboratory of Veterinary Biochemistry, University of Utrecht, The Netherlands.
Papiez
J S
JS
Girgis
K
K
Gibson
D M
DM
D000818:Animals; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004072:Digitonin / Q000494:pharmacology*; D004988:Ethers, Cyclic / Q000494:pharmacology; D005934:Glucagon / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D019319:Okadaic Acid; D051381:Rats; D011919:Rats, Inbred Strains; D014667:Vasopressins / Q000494:pharmacology
D004988:Ethers, Cyclic; D007328:Insulin; D014667:Vasopressins; D019319:Okadaic Acid; D005934:Glucagon; D006903:Hydroxymethylglutaryl CoA Reductases; D004072:Digitonin