Purification and characterization of two mevalonate pyrophosphate decarboxylases from rat liver: a novel molecular species of 37 kDa.
The biosynthesis of cholesterol is regulated mainly by HMG-CoA reductase, however, recent studies indicated the pivotal role of another enzyme in cholesterol homeostasis. A previous report showed a marked decrease in the activity of mevalonate pyrophosphate decarboxylase (MPD) in stroke-prone spontaneously hypertensive rats and its possible involvement in the pathogenesis of the disorder. In this study, we purified liver MPD from rats fed a diet containing cholestyramine and pravastatin (CP diet) using conventional chromatographic techniques. We obtained two electrophoretically homogeneous enzyme preparations; 45 and 37 kDa proteins with specific activities of 8.0 and 7.4 micromol/min/mg, respectively. The enzymes showed similar molecular weights of 90 kDa, as judged on gel permeation chromatography. A kinetic study indicated apparent Km values for mevalonate pyrophosphate and ATP of 22.7 microM and 0.71 mM, respectively, for the 45 kDa MPD, and 20.0 microM and 0.80 mM, respectively, for the 37 kDa MPD. Half maximum activities were observed at 1.5 mM and 1.1 mM Mg2+ for the 45 and 37 kDa MPDs, respectively. Both enzymes required ATP as a phosphate acceptor, and in addition Mg2+, Mn2+, and Co2+ were effective as divalent cations. The optimum pH for both enzymes was 7.0. The isoelectric points for the 45 and 37 kDa MPDs were 5.6 and 5.4, respectively. Polyclonal antiserum raised against the 45 kDa enzyme detected both the 45 and 37 kDa bands on immunoblots with CP diet-induced liver crude extract as an antigen. However, non-induced liver contained the 45 kDa protein but not the 37 kDa protein. These results indicated that the CP diet induced a new species, 37 kDa, of MPD which is characteristically and immunologically very similar to the well-known 45 kDa MPD.
Michihara
A
A
Graduate School of Human and Environmental Studies, Kyoto University.
Sawamura
M
M
Nara
Y
Y
Ikeda
K
K
Yamori
Y
Y
D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D002262:Carboxy-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002784:Cholesterol / Q000096:biosynthesis; D002792:Cholestyramine Resin / Q000494:pharmacology; D004790:Enzyme Induction; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007150:Immunohistochemistry; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism; D017035:Pravastatin / Q000494:pharmacology; D051381:Rats; D011921:Rats, Inbred WKY
D000924:Anticholesteremic Agents; D007527:Isoenzymes; D009844:Oligosaccharides; D002792:Cholestyramine Resin; D002784:Cholesterol; D002262:Carboxy-Lyases; C022503:pyrophosphomevalonate decarboxylase; D017035:Pravastatin