Purification and Characterization of Mouse Mevalonate Pyrophosphate Decarboxylase.

Abstract

One of the first steps in the biosynthesis of cholesterol from acetic acid is catalyzed by mevalonate pyrophosphate decarboxylase (MPD). This decarboxylase catalyzes a bimolecular reaction between mevalonate 5-pyrophosphate (MVAPP) and ATP to form isopentenyl pyrophosphate, inorganic phosphate, adenosine-5Ј-diphosphate (ADP), and CO 2 .MPD has been purifed from various sources including yeast, 1,2) latex of Hevea brasiliensis, 3) pig liver, 4,5) rat liver, [6][7][8] and chicken liver. 9) Its properties in rat and chicken have been examined in detail. Toth and Huwyler reported cDNA sequences of MPD from human liver and yeast.10) The recombinant human enzyme is a homodimer of 43-kDa subunits with 400 amino acids. We recently established a procedure for the purification of MPD from the liver of rats fed a diet containing 5% cholestyramine and 0.1% pravastatin (CP diet) using chromatography and polyclonal antiserum raised against rat MPD. 8)Epidemiological studies have indicated a negative association between the serum cholesterol level and the incidence of cerebral hemorrhage in humans.11) Spontaneously hypertensive rat stroke-prone (SHRSP) is a widely used animal model for hypertension and stroke.12) Iritani et al. reported that the serum cholesterol level in SHRSP was low when compared with that in normotensive age-matched Wistar Kyoto rats (WKY). 13) We previously reported that the low serum cholesterol level in SHRSP as compared with WKY may be attributable to the reduced activity of MPD and not 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. 14) Moreover, we found that the decline in activity was caused by a reduction in the amount of enzyme in SHRSP.15) As described above, it is important to understand the mechanism of the decrease in MPD. We are also very interested in whether MPDknockout or MPD-reduced mice experience cerebral hemorrhage. However, information on MPD in normal mice is relatively scarce.In this paper, we describe a simple procedure for the purification of MPD from mouse liver, the properties of MPD, and a comparison of this MPD with counterparts from other animals. MATERIALS AND METHODSMaterials Pharmalyte 3-10 for isoelectric focusing and Western blotting detection kits were purchased from Amersham Pharmacia Biotech. Affigel 10 and 15 were obtained from Bio-Rad. RS-[ 3 H] mevalonolactone was purchased from Du Pont-New England Nuclear. Albumin from rat fraction V was purchased from Wako Chemicals. ddy Mice (8 weeks of age) were obtained from Shimizu Experimental Animals. All other chemicals were of reagent grade and purchased from commercial sources.Radioactive Assay The activities of the crude extract as well as the purified enzyme were measured according to the method of Sawamura et al.14) Unless otherwise indicated, the reaction mixture consisted of 100 mM Tris-HCl (pH 7.2), 5 mM ATP, 5 mM MgCl 2 , 10 mM NaF, Purification of MPD in RatsPurification of rat liver MPD was carried out as described by Michihara et al. 8)Preparation of an Affinity Column with Anti-rat MPD Antibody We...

Subcellular Distribution of Mouse Mevalonate Pyrophosphate Decarboxylase

“…This decarboxylase catalyzes a bimolecular reaction between mevalonate pyrophosphate and ATP to form isopentenyl pyrophosphate, inorganic phosphate, ADP, and CO 2 . The enzyme has been purified from various sources, including yeast, 1,2) latex of Hevea brasiliensis, 3) pig liver, 4,5) rat liver, [6][7][8] mouse liver, 9) and chicken liver. 10) Toth and Huwyler reported the cDNA sequences of MPD from human liver and yeast.…”

Tissue Distribution of Mevalonate Pyrophosphate Decarboxylase in Guinea Pig

“…9,13) In the present study, we examined whether antiserum reacted with MPD in various guinea pig tissues. When crude extract of the brain of rats, mice, and guinea pigs was subjected to immunoblot analysis using MPD antiserum, this antiserum reacted with the protein of 45-kDa in rats and 46-kDa in guinea pigs as well as mice (Fig.…”

“…mouse liver, 9) and chicken liver. 10) Its properties in rats and chickens have been examined in detail.…”

Comparison of the Gene Expression Levels of Mevalonate Pyrophosphate Decarboxylase between Stroke-prone Spontaneously Hhypertensive and Wistar Kyoto Rats

“…However, there is little information on the gene expression of MPD in rats, although analyses of the MPD protein have been performed. [6][7][8][9][10][11][12][13] Thus, we examined the distribution of the mRNA levels in Wistar rat tissues by real-time PCR, to clarify the relationship between protein and mRNA levels of MPD. Next, we compared the mRNA levels of MPD in liver and brain between SHRSP and normotensive WKY.…”