In situ measurement of HMG-CoA reductase activity in digitonin-permeabilized hepatocytes

Biochem Biophys Res Commun. 1991 Oct 31;180(2):525-30. doi: 10.1016/s0006-291x(05)81096-4.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Membrane Permeability*
  • Cells, Cultured
  • Digitonin / pharmacology*
  • Ethers, Cyclic / pharmacology
  • Glucagon / pharmacology
  • Hydroxymethylglutaryl CoA Reductases / metabolism*
  • Insulin / pharmacology
  • Kinetics
  • Liver / drug effects
  • Liver / enzymology*
  • Male
  • Okadaic Acid
  • Rats
  • Rats, Inbred Strains
  • Vasopressins / pharmacology

Substances

  • Ethers, Cyclic
  • Insulin
  • Vasopressins
  • Okadaic Acid
  • Glucagon
  • Hydroxymethylglutaryl CoA Reductases
  • Digitonin