Purification of Rat Liver Mevalonate Pyrophosphate Decarboxylase

Toth, Matthew J.; Huwyler, Leslie; Park, John

doi:10.1080/10826069608000049

Cited by 19 publications

(18 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Shama Bhat and Ramasarma reported that rat liver MPD was composed of four subunits of 35 kDa each and was unstable when stored at 4°C. 6) On the other hand, Toth et al 7) reported a homodimer of 45-kDa MPD subunits in rats that was stable at 4°C. It is likely that the activity remaining in the supernatant after immunoprecipitation with anti-rat MPD antibody-Affigel 10 or anti-rat MPD antibody-protein A is from an unstable form of the enzyme like the rat MPD reported by Shama Bhat and Ramasarma.…”

Section: Fig 1(d) Isoelectric Points Of Mpdmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Purification and Characterization of Mouse Mevalonate Pyrophosphate Decarboxylase.

Michihara

Akasaki

Yamori

et al. 2002

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

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...

show abstract

Section: Fig 1(d) Isoelectric Points Of Mpdmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Purification and Characterization of Mouse Mevalonate Pyrophosphate Decarboxylase.

Michihara

Akasaki

Yamori

et al. 2002

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

show abstract

“…This decarboxylase catalyzes a bimolecular reaction between mevalonate pyrophosphate and adenosine 5Ј-triphosphate (ATP) to form isopentenyl pyrophosphate, inorganic phosphate, ATP, 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] and chicken liver. 9) Toth and Huwyler reported the cDNA sequences of MPD from human liver and yeast.…”

mentioning

confidence: 99%

“…[6][7][8] The cDNA sequences of 45-kDa MPD from rats were described by gene bank. We have purified and characterized 45-kDa MPD from rat liver and polyclonal antiserum raised against 45-kDa MPD.…”

mentioning

confidence: 99%

Tissue Distribution of a Major Mevalonate Pyrophosphate Decarboxylase in Rats.

Michihara

Akasaki

Yamori

et al. 2001

Biological & Pharmaceutical Bulletin

View full text Add to dashboard Cite

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 pyrophosphate and adenosine 5Ј-triphosphate (ATP) to form isopentenyl pyrophosphate, inorganic phosphate, ATP, 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] and chicken liver. 9) Toth and Huwyler reported the cDNA sequences of MPD from human liver and yeast.10) The recombinant human enzyme is a homodimer of a 43-kDa subunit with 400 amino acids.The 45-and 35-kDa MPD subunits have been purified from normal rat liver. [6][7][8] The cDNA sequences of 45-kDa MPD from rats were described by gene bank. We have purified and characterized 45-kDa MPD from rat liver and polyclonal antiserum raised against 45-kDa MPD.8) The native 45-kDa MPD had a molecular weight of 90 kDa, consisted of two identical subunits of 45 kDa, and was stable when stored at 4°C. Shama Bhat and Ramasarma reported that rat liver MPD was composed of four subunits with a molecular weight of 35 kDa and highly unstable when stored at 4°C. 6)The relationship between 45-and 35-kDa MPD remains unclear.In this study, we first examined the relationship between 45-and 35-kDa MPD and quantitatively measured the tissue distribution of a major MPD in rat liver. MATERIALS AND METHODS MaterialsMale Wistar rats weighing 200 g were obtained from Shimizu Experimental Animals (Kyoto). [35 S]-Lmethionine was purchased from HAS (Hungary). Protein ASepharose CL-4B was obtained from Amersham Pharmacia. Affigel 15 or 10 was obtained from Bio-Rad. All other chemicals were of reagent grade and purchased from various commercial sources. Radioactive AssayThe enzyme activities of the crude extract were measured according to the method of Sawamura et al. 11)Purification of MPD in Rat Liver Purification of MPD in rat liver was carried out as described by Michihara et al. 8)Purification of Anti-rat 45-kDa MPD Antibody and Preparation of Anti-MPD Antibody-Affigel 10 We previously reported that anti-rat 45-kDa MPD antiserum contained albumin antibody. 8) Therefore, to remove the albumin antibody from anti-rat 45-kDa antiserum, we carried out the following procedure. The anti-rat 45-kDa MPD antiserum prepared by Michihara et al. 8) was applied to a column of Albumin-Affigel 15 (2 mg of albumin/ml of packed gel) equilibrated with phosphate buffered saline (PBS) containing 0.15 M NaCl (buffer A). The Albumin-Affigel 15 was washed with three column volumes of buffer A. The flow-through solution was applied to a column of purified 45-kDa MPD-Affigel 15 (0.5 mg of MPD/ml of packed gel) equilibrated with buffer A. The column of purified 45-kDa MPD-Affigel 15 was washed with buffer A until the absorbance at 280 nm dropped to the base line level (0.005-0.01). The anti-rat 45-kDa MPD antibody bound in column was eluted with glycine-HCl (pH 3.0) containing 0.15 M NaCl. The elutant ...

show abstract

The Saccharomyces cerevisiae Mevalonate Diphosphate Decarboxylase (Erg19p) Forms Homodimers In Vivo, and a Single Substitution in a Structurally Conserved Region Impairs Dimerization

et al. 1999

View full text Add to dashboard Cite

The wild-type ERG19 gene of the yeast Saccharomyces cerevisiae encoding mevalonate diphosphate decarboxylase (MVD) and the mutated recessive erg19-34 allele leading to a decrease of sterol production and to a thermosensitive phenotype have been characterized [2]. The mutated erg19-34 allele bears a single amino acid leucine 79-to-proline (L79P) substitution. It was shown that this mutation does not affect the level of production of the enzyme. We performed a two-hybrid assay to show that the yeast Saccharomyces cerevisiae MVD forms homodimers in vivo and that the single point mutation drastically impairs the oligomerization of the protein, thereby explaining the deficiency of MVD activity observed in the temperature-sensitive strain.

show abstract

Purification of Rat Liver Mevalonate Pyrophosphate Decarboxylase

Cited by 19 publications

References 4 publications

Purification and Characterization of Mouse Mevalonate Pyrophosphate Decarboxylase.

Purification and Characterization of Mouse Mevalonate Pyrophosphate Decarboxylase.

Tissue Distribution of a Major Mevalonate Pyrophosphate Decarboxylase in Rats.

The Saccharomyces cerevisiae Mevalonate Diphosphate Decarboxylase (Erg19p) Forms Homodimers In Vivo, and a Single Substitution in a Structurally Conserved Region Impairs Dimerization

Contact Info

Product

Resources

About