Purification and characterization of mouse mevalonate pyrophosphate decarboxylase

Article abstract of DOI:10.1248/bpb.25.302

Mevalonate pyrophosphate decarboxylase (MPD) in mouse liver was purified by affinity chromatography. The purified enzyme was a homodimer of 46-kDa subunits and had an isoelectric point of 5.0. Kinetic analysis revealed an apparent K_m value of 10 μM for mevalonate pyrophosphate. The enzyme required ATP as a phosphate acceptor and Mg as a divalent cation, which could be substituted with Mn or Co. Its optimum pH was 4.0-7.0. A comparison with MPD from various other sources revealed the mouse MPD to have essentially the same properties as rat MPD, expect for the optimum pH range. An excess of rabbit anti-rat MPD antibody deleted approximately 80% of the MPD activity in the crude extract of mouse liver. These results suggested that the homodimer of 46-kDa subunits represents the major active form of MPD in mice.

Full text of DOI:10.1248/bpb.25.302

3
02
Biol. Pharm. Bull. 25(3) 302—306 (2002)
Vol. 25, No. 3
Purification and Characterization of Mouse Mevalonate Pyrophosphate
Decarboxylase
,
a
a
b
a
Akihiro MICHIHARA,* Kenji AKASAKI, Yukio YAMORI, and Hiroshi TSUJI
a
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Gakuencho, Fukuyama, Hiroshima 729–0292,
b
Japan and WHO Collaborating Center, Kyoto 606–8413, Japan.
Received September 10, 2001; accepted November 7, 2001
Mevalonate pyrophosphate decarboxylase (MPD) in mouse liver was purified by affinity chromatography.
The purified enzyme was a homodimer of 46-kDa subunits and had an isoelectric point of 5.0. Kinetic analysis
revealed an apparent Km value of 10 mM for mevalonate pyrophosphate. The enzyme required ATP as a phos-
phate acceptor and Mg as a divalent cation, which could be substituted with Mn or Co. Its optimum pH was
4
.0—7.0. A comparison with MPD from various other sources revealed the mouse MPD to have essentially the
same properties as rat MPD, expect for the optimum pH range. An excess of rabbit anti-rat MPD antibody
deleted approximately 80% of the MPD activity in the crude extract of mouse liver. These results suggested that
the homodimer of 46-kDa subunits represents the major active form of MPD in mice.
Key words mevalonate pyrophosphate decarboxylase; purification; characterization; mouse; liver
One of the first steps in the biosynthesis of cholesterol MATERIALS AND METHODS
from acetic acid is catalyzed by mevalonate pyrophos-
phate decarboxylase (MPD). This decarboxylase catalyzes
a bimolecular reaction between mevalonate 5-pyrophosphate Western blotting detection kits were purchased from Amer-
MVAPP) and ATP to form isopentenyl pyrophosphate, inor- sham Pharmacia Biotech. Affigel 10 and 15 were obtained
Materials Pharmalyte 3—10 for isoelectric focusing and
(
3
ganic phosphate, adenosine-5Ј-diphosphate (ADP), and CO2. from Bio-Rad. RS-[ H] mevalonolactone was purchased
MPD has been purifed from various sources including from Du Pont-New England Nuclear. Albumin from rat frac-
1
,2)
3)
4,5)
6—8)
yeast, latex of Hevea brasiliensis, pig liver, rat liver,
tion V was purchased from Wako Chemicals. ddy Mice (8
9
)
and chicken liver. Its properties in rat and chicken have weeks of age) were obtained from Shimizu Experimental An-
been examined in detail. Toth and Huwyler reported cDNA imals. All other chemicals were of reagent grade and pur-
sequences of MPD from human liver and yeast. The re- chased from commercial sources.
1
0)
combinant human enzyme is a homodimer of 43-kDa sub-
Radioactive Assay The activities of the crude extract as
units with 400 amino acids. We recently established a proce- well as the purified enzyme were measured according to the
1
4)
dure for the purification of MPD from the liver of rats fed a method of Sawamura et al. Unless otherwise indicated, the
diet containing 5% cholestyramine and 0.1% pravastatin (CP reaction mixture consisted of 100 mM Tris–HCl (pH 7.2),
diet) using chromatography and polyclonal antiserum raised 5 mM ATP, 5 mM MgCl , 10 mM NaF, 30 mM nicotinamide,
2
8
)
3
against rat MPD.
250 nmol [ H]MVAPP, and crude extract, cytosol fraction,
Epidemiological studies have indicated a negative associa- or purified enzyme. Preparation of [ H]MVAPP from [ H]-
tion between the serum cholesterol level and the incidence of mevalonic acid was carried out based on the method de-
cerebral hemorrhage in humans. Spontaneously hyperten- scribed by Cardemil and Jabalquinto.
sive rat stroke-prone (SHRSP) is a widely used animal model Purification of MPD in Rats Purification of rat liver
for hypertension and stroke. Iritani et al. reported that the MPD was carried out as described by Michihara et al.
serum cholesterol level in SHRSP was low when compared Preparation of an Affinity Column with Anti-rat MPD
with that in normotensive age-matched Wistar Kyoto rats Antibody We previously reported that anti-rat MPD anti-
3
3
1
1)
16)
1
2)
8)
1
3)
8)
(
WKY). We previously reported that the low serum choles- serum contained albumin antibody. To remove the antibody,
terol level in SHRSP as compared with WKY may be attrib- we carried out the following procedure. The anti-rat MPD
utable to the reduced activity of MPD and not 3-hydroxy-3- antiserum was applied to a column of Albumin-Affigel 15
1
4)
methylglutaryl coenzyme A (HMG-CoA) reductase. More- (2 mg of albumin/ml of packed gel) equilibrated with phos-
over, we found that the decline in activity was caused by a re- phate-buffered saline (PBS) containing 0.15 M NaCl (buffer
1
5)
duction in the amount of enzyme in SHRSP. As described A). The Albumin-Affigel 15 was washed with three column
above, it is important to understand the mechanism of the de- volumes of buffer A. The effluents were applied to a column
crease in MPD. We are also very interested in whether MPD- of MPD-Affigel 15 (0.5 mg of MPD/ml of packed gel) equili-
knockout or MPD-reduced mice experience cerebral hemor- brated with buffer A. The column was washed with buffer A
rhage. However, information on MPD in normal mice is rela- until the absorbance at 280 nm dropped to the baseline level
tively scarce.
(0.005—0.01). The anti-rat MPD antibody bound to the col-
In this paper, we describe a simple procedure for the pu- umn was eluted with glycine–HCl (pH 3.0) containing 0.15 M
rification of MPD from mouse liver, the properties of MPD, NaCl. The elutant was adjusted to neutral pH by adding 1 M
and a comparison of this MPD with counterparts from other Tris solution and then concentrated with the Amicon Diaflo
animals.
apparatus to obtain a purified anti-rat MPD antibody. A
column of anti-rat MPD antibody-Affigel 10 (0.5 mg of anti-
∗
To whom correspondence should be addressed. e-mail: mitihara@fupharm.fukuyama-u.ac.jp
© 2002 Pharmaceutical Society of Japan

March 2002
303
Table 1. Purification of MPD from Mouse Liver (8 g)
Total protein
Total activity
(nmol/min)
Specific activity
(nmol/min/mg)
Purification
(fold)
Recovery
(%)
Purification step
(
mg)
Cytosol fraction
Antibody column
526.7
0.123
181.4
28.6
0.34
232.7
100
15.7
682
rat MPD antibody/ml of packed gel) was prepared using the
purified antibody.
Purification of MPD in Mice Mice livers (8 g wet
weight) were washed with buffer B (100 mM sodium phos-
phate [pH 7.5] containing 10 mM 2-mercaptoethanol, 1 mM
EDTA, 1 mM leupeptin, 1 mM pepstatin A, 1 mM antipain, and
1
mM chymostatin), and then homogenized in 3 volumes of
ice-cold homogenate buffer (buffer B containing 0.5 mM
phenylmethylsulfonylfluolide) using a homogenizer. The ho-
mogenate was centrifuged at 100000ϫg for 1 h to obtain a
cytosol fraction. The resultant supernatant (cytosol fraction)
was applied to the column of anti-rat MPD antibody-Affigel
1
2
0 equilibrated with PBS containing 0.15 M NaCl and 10 mM
-mercaptoethanol (buffer C). The column was washed with
buffer C until the absorbance at 280 nm dropped to the base- Fig. 1(a). SDS-PAGE of Purified MPD
line level (0.005—0.01). Proteins suspected of being MPD
were eluted with 0.5 M NaCl and then concentrated with the
Amicon Diaflo apparatus.
The relative positions of the molecular weight standards are shown in the left margin.
Lane 1, cytosol fraction from mouse liver (10 mg); lane 2, purified mouse MPD (2 mg);
lane 3, purified rat MPD (2 mg).
Molecular Weight Determination The molecular weight
of the purified MPD was determined at room temperature on
Sephacryl 300 (1.5ϫ90 cm) equilibrated with 50 mM sodium
phosphate (pH 7.0), 0.1 mM EDTA, 10 mM 2-mercaptothanol,
and 0.15 M NaCl. The molecular weight markers used were
aldolase (158 kDa), bovine serum albumin (BSA; 68 kDa),
and ovalbumin (45 kDa).
Isoelectric Focusing Isoelectric focusing was done for
2
4 h at 4 °C in the presence of Pharmalyte (pH 3.0—10.0).
Immunoblotting Western blot analysis was carried out
using a detection kit (Amersham Pharmacia Biotech).
Gel Electrophoresis Sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) was carried out on
1
7)
1
0% acrylamide gel as described by Laemmli. Proteins
were stained with Coomassie Blue R-250.
Protein Determination Proteins were determined by the
1
8)
method of Lowry et al. with BSA as the standard.
RESULTS
Fig. 1(b). Native Molecular Weight of MPD
Purified mouse MPD (50 mg) was loaded onto a Sephacryl 300 column. The activity
in each fraction was measured. The MW of the purified enzyme was determined using
the molecular weight standard.
Purification of MPD When the cytosol fraction from
mouse liver was subjected to immunoblot analysis using the
purified anti-rat MPD antibody, a 46-kDa protein was de-
tected (data not shown). To test the possibility that the 46- purified 46-kDa protein on immunoblotting (data not shown).
kDa protein was MPD or a subunit thereof, we purified it These results show that the 46-kDa protein purified from the
using an affinity column. The protein was obtained from the cytosol fraction of mouse liver was immunologically similar
cytosol fraction of mouse liver using the column of anti-rat to rat MPD.
MPD antibody-Affigel 10. Table 1 shows the results of the
Characterization of Mouse MPD As shown in Fig.
purification. As compared with the MPD in the cytosol 1(b), the molecular weight of the native enzyme as deter-
fraction, the protein was purified approximately 682-fold mined on Sephacryl 300 was approximately 100 kDa, indi-
with a recovery of 15.7% and a specific activity of 232.7 cating a homodimer of 46-kDa subunits. The apparent Km
nmol/min/mg protein. The protein purified from the cytosol and Vmax were calculated to be 10 mM and 77 nmol/min/mg,
fraction exhibited a MW of 46 kDa on SDS-PAGE (Fig. respectively, from Lineweaver–Burk plots (Fig. 1(c)). The
1
(a)). We found that the MPD in rats was 1 kDa smaller than isoelectric point was 5.0, as determined on slab gel (Fig.
the protein in mice. The anti-rat MPD antibody detected the 1(d)). The mouse MPD showed a broad pH (4.0—7.0) de-

3
04
Vol. 25, No. 3
Table 2. Effect of Nucleotides and Cations on the Activity of the Liver
MPD
Nucleotide and cation (5 mM)
Relative activity (%)
ATPϩMgCl2
ATPϩMnCl2
ATPϩCoCl2
ATPϩCaCl2
ATPϩCdCl2
ATPϩKCl
ATPϩBaCL2
GTPϩMgCl2
CTPϩMgCl2
UTPϩMgCl2
None
100
105
85
50
28
26
21
25
12
13
15
Activity in the presence of Mg is 100%. Data are the means of four identical experi-
ments and each value varies within 10%.
Fig. 1(c). Kinetics of MPD
Table 3. Effect of Sulfhydryl Reagent on the Activity of Liver MPD
Purified mouse MPD (2 mg) was incubated with various amounts of MVAPP, after
which the initial velocities were estimated. The values are the means of three identical
experiments and each varies within 5%.
SH reagent (10 mM)
MPD activity (%)
IAA
NEM
DTNB
PCMBS
85
60
40
50
Activity in the presence of 5 mM ATP and 5 mM MgCl is 100%. Data are the means
2
of four identical experiments and each value varies within 10%.
pendency, as assayed in 0.1 M glycine–HCl (pH 3—5) or
0
.1 M Tris–HCl (pH 5—10) (Fig. 1(e)). As shown in Table 2,
Mg could be substituted with Mn or Co with little change in
activity. ATP was the only nucleotide required for the activ-
ity. Alvear et al. reported that chicken MPD was sensitive
9
)
to sulfhydryl-directed reagents. Our purified mouse MPD,
however, showed 85% activity in the presence of 10 mM
iodoacetamide (IAA) (Table 3). However, when MPD activ-
ity in mice was measured in the presence of 10 mM N-
ethylmaleimide (NEM), 10 mM 5,5-dithiobis (2-nitrobenzoic
acid) (DTNB), and 10 mM p-mercuribenzenesulfonic acid
(PCMBS), a decrease of 60%, 40%, and 50%, respectively,
Fig. 1(d). Isoelectric Points of MPD
was observed.
The relative positions of the isoelectric focusing standards are shown in the left mar-
gin. Purified mouse MPD (0.5 mg) was electrofocused, and the proteins were visualized
by silver staining.
Immunoprecipitation of MPD MPD in mouse liver ex-
tract was immunoprecipitated with the column of anti-rat
MPD antibody-Affigel 10 in a dose-dependent manner. More
than 80% of the activity was depleted from the extract with
an excess of the antibody (Fig. 2A). When the same amount
of crude extract was immunoprecipitated with anti-rat MPD
antibody-protein A, more than 80% of the MPD activity was
lost with 0.5 mg as well as with 1 mg of the antibody (Fig.
2
B). These results indicate that the purified MPD represents
the major active form in crude extract of mouse liver.
DISCUSSION
In studies on the regulation of serum cholesterol, attention
has focused on the biosynthesis of cholesterol, in paticular,
the effect of reduced MPD activity. We are very interested in
whether MPD-knockout mice or MPD-reduced mice experi-
ence cerebral hemorrhage. However, information on MPD in
normal mice is scarce. MPD has now been purified to homo-
geneity from mouse liver and characterized. As shown in
Fig. 1(e). Effect of pH on Activities of MPD
Purified mouse MPD (100 ng) was assayed in 0.1 M glycine–HCl (᭺) or 0.1 M
Tris–HCl (᭝). Each point represents the average of triplicate determinations.
Table 4, the apparent K value for MVAPP of mouse MPD
m

March 2002
305
Table 4. Comparison of the Properties of Animal MPD
Cofactor requirement
Metal ion
Molecular weight
Specific
activity MVAPP
Km for
Optimum
pH
Enzyme source
pI
Nucleotide
GTP UTP CTP
Ref. no.
Native
Subunit
(kDa)
(
unit/mg)
(mM)
(
kDa)
Mg
Mn
Co
Ca
Human
Pig
Chicken
Rat
2.4
3.5
6.3
0.056
2
13
14
10
86
88
85.4
126
100
90
90
100
43
46
44
35
45
45
37
46
10
5
9
6
7
8
8
5.0—8.0 100
4.0—6.5 100
37
97
82
13
90
82
44
29
42
10
9
29
No
No
No
No
No
No
4.8
5.2
100
4
8
7
.6
.0
.4
20
22.7
20.0
10
5.6
5.4
5.0
7.0
100
126
149
105
125
91
85
55
38
50
15
15
25
10
11
13
8
11
12
6.0—8.0 100
4.0—7.0 100
Mouse
0.23
This study
center of the enzyme. Biochemical analysis using the crude
extract from mouse liver revealed the K value and optimum
m
pH to be similar to the values obtained for the purified MPD.
These results indicate that MPD in mice has essentially the
8
)
same properties as MPD in rats, expect for a difference in
the optimum pH range.
After crude extract had been immunoprecipitated with
anti-rat MPD antibody-Affigel 10 (0.5 mg/ml) or anti-rat
MPD antibody-protein A (1 mg/0.2 ml), the 46-kDa MPD
subunit band of the resultant supernatant in mice was not de-
tected by immunoblot analysis (data not shown). Therefore
we consider that the protein with MPD activity (20%) in the
supernatant was immunologically different from the MPD of
mice. Two types of MPD have been purified from normal rat
tissue. Shama Bhat and Ramasarma reported that rat liver
MPD was composed of four subunits of 35 kDa each and was
6
)
unstable when stored at 4 °C. On the other hand, Toth et
7
)
al. 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. However, no data
to support this possibility were obtained. Further study is
needed to understand the physiological role of MPD in mice.
In conclusion, we reported the purification and properties
of mouse MPD. The findings of the present study will be
Fig. 2. Immunoprecipitation of MPD
The liver crude extracts (10 nmol/min MPD activity) dissolved in homogenate buffer useful for analyses of MPD-knockout or MPD-reduced mice.
were (A) added to increasing amounts of anti-rat MPD antibody-Affigel 10 (0.1, 0.3,
0
.5, or 1.0 ml of 0.5 mg of antibody/1 ml of packed gel) equilibrated with homogenate
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buffer and rotated at 4 °C for 2 h prior to centrifugation at 3000ϫg for 5 min. MPD ac-
tivity in the supernatant was measured.
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(