Modification of the radiochemical assay of rat liver mevalonate-5-diphosphate decarboxylase and induction and stabilization of the activity.
Problems encountered in attempts to purify mevalonate-5-diphosphate decarboxylase from rat liver are addressed. These are the quantitative, facile separation of [14C]isopentenol in the radiochemical assay (2) the instability of the enzyme activity and (3) the very low activity in rat liver. The assay was modified by using Sep Pac C18 filters to bind and release [14C]isopentenol. Authentic isopentenol was quantitated by absorbance at 210 nm wavelength and the extinction coefficient estimated to be epsilon m = 3.26 X 10(3). Recovery of authentic isopentenol from aqueous solution after binding and elution into methanol was quantitative from 10-100 nmols. Recovery of [14C]ispentenol from assay mixtures using 2-[14C]mevalonate-5-diphosphate and alkaline phosphatase to hydrolyze phosphate was quantitative using Sep Pac filter but not using petroleum ether extraction. Enzyme activity was stabilized by phenylmethylsulfonyl fluoride, aprotinin and leupeptin and was stable at -73 degrees C for 3 months. Activity of the decarboxylase was increased by 5-fold after feeding young rats 2.5% cholestyramine for ten days to four weeks.
Hulcher
F H
FH
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157-1016.
D000818:Animals; D002250:Carbon Radioisotopes; D002262:Carboxy-Lyases / Q000032:analysis* / Q000096:biosynthesis / Q000378:metabolism*; D002792:Cholestyramine Resin / Q000494:pharmacology; D004790:Enzyme Induction; D004795:Enzyme Stability; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D000439:Pentanols / Q000032:analysis; D011865:Radioisotope Dilution Technique; D051381:Rats; D011919:Rats, Inbred Strains
D002250:Carbon Radioisotopes; D000439:Pentanols; D002792:Cholestyramine Resin; C056744:isopentenol; D002262:Carboxy-Lyases; C022503:pyrophosphomevalonate decarboxylase