An improved assay of 3-hydroxy-3-methylglutaryl-CoA reductase activity in Reuber H35 hepatoma cells without microsomes isolation.
The optimal conditions for measuring 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity in Reuber H35 hepatoma cells are described in this paper. Cells in the exponential phase of growth were lysed by incubation with Brij 97 detergent for 30 min. We used imidazole buffer supplemented with EDTA and leupeptine, two inhibitors of proteases. Disrupted cells were then centrifuged at 12,000 g. Although microsomes are usually reported as enzyme preparations for measuring HMG-CoA reductase, our data showed that hepatoma cells may be used without previous isolation of microsomes. The 12,000 g supernatant showed similar levels of total and specific activities to those found in the microsomal fraction obtained after 105,000 g centrifugation. The soluble fraction showed less than 10% of reductase activity. Reductase activity from Reuber H35 hepatoma cells increased proportionally to the reaction time from 30 to 90 min and to the amount of protein added in a range of 50-500 micrograms. Our modified method was very sensitive and reproducible, because very low specific activity (about 15-100 pmol min-1 per mg protein) could be quantified in different assay conditions obtaining similar values.
García-Pelayo
M C
MC
Department of Biochemistry and Molecular Biology, University of Granada, Spain. garciap@goliat.ugr.es
García-Peregrín
E
E
Martínez-Cayuela
M
M
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D051381:Rats; D014407:Tumor Cells, Cultured
D000214:Acyl Coenzyme A; C008047:3-hydroxy-3-methylglutaryl-coenzyme A