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Purification of mevalonic acid dehydrogenase from rat liver by DEAE-cellulose column chromatography
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 93, 153-156 (1961)
Purification
of Mevalonic by DEAE-Cellulose HARUO
From
Acid Dehydrogenase from Column Chromatography’
NAKAMURA2
the Department
AND
DAVID
M. GREENBERG
of Biochemistry, University of Calijornia San Francisco, California Received:
Rat Liver
School
of Medicine,
October 19, 1960
An enzyme from rat liver which catalyzes the reduction of mevaldic acid to mevaionic acid in the presence of a reduced pyridine nucleotide has been purified about 250-fold. Certain physicochemical properties of the enzyme have been determined and are reported. INTRODUCTION
Mevalonic acid dehydrogenase catalyzes the reduction of mevaldic acid to mevalonic acid by reduced diphosphopyridine nucleotide (DPNH) (in the case of liver extracts) or by reduced triphosphopyridine nucleotide (TPNH) (in the case of yeast extract,s) . The separation of this dehydrogenase from yeast was reported by Lynen (1) and from pig liver, heart, and kidney by Coon et al. (2). In this communication we are reporting a procedure for the purification of the enzyme from rat liver by chromatography on a DEAE-cellulose column, and certain physicochemical properties of the enzyme. MATERIALS TPNH and DPNH were obtained from the California Corporation for Biochemical Research, Los Angeles; nL-mevalonic acid as the dibenzylethylene diamine salt (DBED) of mevalonic acid and as the acetal of oL-mevaldic acid from the Mann Research Laboratories, Inc.; and nL-mevalonic acid2-C” as the DBED salt from Isotopes Specialties Company.
Mevaldic acid was prepared from the acetal immediately before testing by hydrolysis of the acetal at 25” with 0.1 N HCl by the method of Shunk et al. (31, and mevalonic acid was prepared from the DBED salt by ether extraction after NH8 t,reatment . ANALYTICAL
METHODS
Protein concentrations were determined at 280 and 260 rnp (4) with the Beckman model DU spectrophotometer in silica cells of l-cm. light path, and those of crude homogenate were determined by a biuret method (5), with use of human albumin as a standard. ENZYME
ASSAY
METHOD
The rate of decrease of optical density at 340 mp, which accompanies the formation of diphosphopyridine nucleotide (DPN) was determined in reaction mixtures containing 5 rmoles mevaldic acid, 200 pmoles potassium phosphate buffer, pH 6.2, 0.4 pmole DPNH, 10 pmoles ethylenediaminetetraacetic acid (EDTA), and the enzyme solution, in a final volume of 3.3 ml. Mevaldic acid was omitted from the control cell. A unit of enzyme activity is defined as a change in optical density of 0.001/min. at 340 rnp at 30” while the specific activity of the enzyme is expressed as enzyme units/mg. protein. Readings were taken every ?/z min. for 3-5 min. in the Beckman spectrophotometer at 30”. RESULTS
1 This investigation was aided by a research grant from the National Heart Institute (H-3074), National Institutes of Health and the Cancer Research Funds of the University. ? On leave from Hokkaido University School of Medicine, Sapporo, Japan, for 1959-60.
PURIFICATION
OF ENZYME
Preparation of Crude Homogenate Adult Long-Evans male rats were killed by decapitation, and the livers were re-
153
NAKAMURA AND GREEKBERG
154 2000 1.ea3 #I 1 I\ I! !!
of 0.1 M potassium phosphate buffer, pH 7.5. The resulting slightly turbid solution was dialyzed for 10 hr. against 0.005 M potassium phosphate buffer, pH 7.5, and centrifuged at 13,000 x g to remove a small amount of precipitate that had formed. The clear supernatant solution was used in the following step.
Phosphate buffer 1. 0.005 M. PH 7.5 X 002 M, pH 5.6
Chromatography
Effluent (ml ) FIG. 1. Chromatography of partially purified enzyme on DEAE-cellulose column. The bars refer to units of enzyme activity, while the curves refer to the optical density at 280 mp.
moved, washed with ice-cold water, cut into smal1 pieces, and then homogenized in a Waring blendor at half speed for 30 sec. at 4” with 10 vol. of cold 0.1 M phosphate buffer, pH 7.5, containing 5 X lop3 M EDTA. The homogenate was centrifuged for 30 min. at 13,000 x g, and the sediment was discarded without washing. Ammonium
Sulfate
Fractionation
The supernatant solution was brought to 45% saturation with ammonium sulfate by the slow additions of the required amount of solid salt [277 g./l. (6)] with constant mechanical stirring. After allowing the mixture to equilibrate for 3 hr., the precipitate was centrifuged at 13,000 X g and discarded without washing. The supernatant solution was then adjusted to 75% saturation with ammonium sulfate by the addition of solid salt [210 g./l. (6)] in the same manner. After standing for 4 hr., the precipitate was collected by centrifugation and dissolved in 5 ml.
The procedure of Peterson and Sober (7) was followed in this work. The entire operation was carried out in a cold room at 30”. The column was prepared by allowing an aqueous slurry containing 28 g. DEAE-cellulose to pack by gravity in a 45 X 2.5 cm. column. The column was washed with 0.005 M phosphate buffer, pH 7.5, before use. The enzyme solution, containing 300 mg. protein was passed down the column at the rate of 2 ml./min. The eluate was collected in lo-ml. fractions by means of an automatic fraction collector. The resin was first eluted with 0.005 M phosphate buffer, pH 7.5, and then wit,11 increasing concentration of the buffer as indicated in Fig. 1. The enzymic activity appeared in one peak (bars, Fig. 1) and accounted for nearly all the activity applied to the column. The fraction with the highest activity was used in studying the properties of the enzyme. The fractions were stored frozen. A summary of the purification procedure is given in Table I. PROPERTIES OF ENZYME
Effect of Enzyme
step Crude homogenate (NH&SOI fraction DEAE-cellulose chromatography
OF MEVALONIC
I
DEHYDROGENASE
Total activity
Protein
w&its
%.
1,040 630 195
Concentration
It was established that the rate of the reaction was linearly proportional to the enzyme concentration under the conditions of the enzyme assay. The linearity of the results is shown in Fig. 2.
TABLE PURIFICATION
on DEAE-Cellulose Column
2 x 103 3 x 102 1.5
Specificactivity 0.52 2.1 130
Yield % 100 60
19
MEVALONIC
Optimal
ACID
155
DEHYDROGENASE
pH
s x to4 4
The curve for the effect of pH on the activity of the enzyme is shown in Fig. 3. An optimum pH of 6.2 was found. Michaelis
8
12
16
8
12
16 x IO’
Cons tan ts
The effect of substrate concentration on the catalytic activity of the enzyme was determined a,t pH 6.2 at varying concentrations of mevaldic acid and with either DPNH or TPNH as the reducing agent. Plots of the results are shown in Figs. 4 and 5. From the double reciprocal plots, the K, values calculated for mevaldic acid were 1 x lo-” Ai with DPNH as the reducer and 1.25 X 1O-4 M with TPNH. g 00302 f m oom$ 00.010‘: “a
1 0
I
4
I
FIG. 4. Plots of substrate concentration ws. velocity and of their reciprocals with mevaldic acid as oxidant and DPNH as reductant.
s x IO4 , I IO I 14I , 16,
leer f
6
0.116 2
D x
/ -0.010 01
02
03
Enzyme Content (mg.1
2. Proportionality enzyme concentration. FIG.
B 7 w
-0.006
between reaction rate and
I
I 0
4
8
I2
3. 2 Fir
I
16 x IO’
FIG. 5. Plots of substrate concentration vs. velocity and of their reciprocals with mevaldic acid as oxidant and TPNH as reductant.
TABLE EFFECT Addition
II
OF SULFHYDRYL Concentration
REAGENTS “m$
Inhibition
% None PCMB PCMB Iodoacetic Iodoacetic
M 1 x
acid acid
Inhibition FIG. 3. pH-activity gcnase.
curve of mevalonic dehydro-
0.016 10-d
1 x 10-S 1 x 10-b 1 x 10-a
0
0.00
100
0.007 0.008 0.00
70 00 100
of Enzyme
Mevalonic acid dehydrogenase is a sulfhydryl enzyme, This is shown by the in-
156
NAKAMURA
AND GREENBERG
hibitory effect of the sulfhydryl reagents, p-chloromercuribenzoic acid (PCMB) and iodoacetic acid on the reaction (Table II). Identification of Reaction Product Paper Chromatography
by
The incubation medium used for the identification is as follows: 200 pmoles phosphate buffer (pH 6.2)) 21.2 pmoles mevaldic acid, 2.6 pmoles DPNH, 20 pmoles EDTA, and enzyme solut.ion (0.5 mg. enzyme) in a total volume of 3.2 ml. The incubation was continued for 2 hr. at 37”. The medium was then deproteinized by immersing the reaction vessel into boiling water for 1 min., and the protein precipitate was removed by centrifugation. The supernatant solution was then concentrated in vacua and lyophilized to dryness in a Dry Ice-acetone bath. The reaidue was dissolved in 0.1 ml. water and applied on a sheet of Whatman No. 1 filter paper. The chromatogram was developed with a mixture of ethanol-NH3-water (85: 5: 15) and sprayed with bromophenol blue.
?vIevalonic acid was the only new compound that appeared on the chromatogram of the incubation. It had an Rf value of 0.62 and gave a blue spot on a yellow background with the indicator spray. REFERENCES 1. LYNEN, F., & “Biosynthesis of Terpenes and Sterols,” p. 95. J. & A. Churchill Ltd., London, 1959. 2. COON, M. J., KUPIECKI, F. P., DEKKER, E. E., SCHLESINGER, M. J., AND DEL CAMPILLO, A., in “Biosynthesis of Terpenes and Sterols,” p. 62. J. & A. Churchill Ltd., London, 1959. 3. SHUNK, C. H., LINN, B. O., HUFF, J. W., TILFILLAN, J. L., SKEGGS, H. R., AXD FOLKERS, K., J. Am. Chem. Sot. 79, 3294 (1957). 4. WARBURG, O., AND CHRISTIAN, W., Rio&em. Z. 310, 384 (1941-2). 5. GORNALL, A. T., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem. 177,751 (1949). 6. GREEX, A. A., AND HUGHES, W. L., in “Methods in Enzymology” (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, p. 76. Academic Press, New York, 1955. 7. PETERSON,E. A., AND SOBER,H. A., J. Am. Chem. Sot. 78, 751 (1956).
Purification
of Mevalonic by DEAE-Cellulose HARUO
From
Acid Dehydrogenase from Column Chromatography’
NAKAMURA2
the Department
AND
DAVID
M. GREENBERG
of Biochemistry, University of Calijornia San Francisco, California Received:
Rat Liver
School
of Medicine,
October 19, 1960
An enzyme from rat liver which catalyzes the reduction of mevaldic acid to mevaionic acid in the presence of a reduced pyridine nucleotide has been purified about 250-fold. Certain physicochemical properties of the enzyme have been determined and are reported. INTRODUCTION
Mevalonic acid dehydrogenase catalyzes the reduction of mevaldic acid to mevalonic acid by reduced diphosphopyridine nucleotide (DPNH) (in the case of liver extracts) or by reduced triphosphopyridine nucleotide (TPNH) (in the case of yeast extract,s) . The separation of this dehydrogenase from yeast was reported by Lynen (1) and from pig liver, heart, and kidney by Coon et al. (2). In this communication we are reporting a procedure for the purification of the enzyme from rat liver by chromatography on a DEAE-cellulose column, and certain physicochemical properties of the enzyme. MATERIALS TPNH and DPNH were obtained from the California Corporation for Biochemical Research, Los Angeles; nL-mevalonic acid as the dibenzylethylene diamine salt (DBED) of mevalonic acid and as the acetal of oL-mevaldic acid from the Mann Research Laboratories, Inc.; and nL-mevalonic acid2-C” as the DBED salt from Isotopes Specialties Company.
Mevaldic acid was prepared from the acetal immediately before testing by hydrolysis of the acetal at 25” with 0.1 N HCl by the method of Shunk et al. (31, and mevalonic acid was prepared from the DBED salt by ether extraction after NH8 t,reatment . ANALYTICAL
METHODS
Protein concentrations were determined at 280 and 260 rnp (4) with the Beckman model DU spectrophotometer in silica cells of l-cm. light path, and those of crude homogenate were determined by a biuret method (5), with use of human albumin as a standard. ENZYME
ASSAY
METHOD
The rate of decrease of optical density at 340 mp, which accompanies the formation of diphosphopyridine nucleotide (DPN) was determined in reaction mixtures containing 5 rmoles mevaldic acid, 200 pmoles potassium phosphate buffer, pH 6.2, 0.4 pmole DPNH, 10 pmoles ethylenediaminetetraacetic acid (EDTA), and the enzyme solution, in a final volume of 3.3 ml. Mevaldic acid was omitted from the control cell. A unit of enzyme activity is defined as a change in optical density of 0.001/min. at 340 rnp at 30” while the specific activity of the enzyme is expressed as enzyme units/mg. protein. Readings were taken every ?/z min. for 3-5 min. in the Beckman spectrophotometer at 30”. RESULTS
1 This investigation was aided by a research grant from the National Heart Institute (H-3074), National Institutes of Health and the Cancer Research Funds of the University. ? On leave from Hokkaido University School of Medicine, Sapporo, Japan, for 1959-60.
PURIFICATION
OF ENZYME
Preparation of Crude Homogenate Adult Long-Evans male rats were killed by decapitation, and the livers were re-
153
NAKAMURA AND GREEKBERG
154 2000 1.ea3 #I 1 I\ I! !!
of 0.1 M potassium phosphate buffer, pH 7.5. The resulting slightly turbid solution was dialyzed for 10 hr. against 0.005 M potassium phosphate buffer, pH 7.5, and centrifuged at 13,000 x g to remove a small amount of precipitate that had formed. The clear supernatant solution was used in the following step.
Phosphate buffer 1. 0.005 M. PH 7.5 X 002 M, pH 5.6
Chromatography
Effluent (ml ) FIG. 1. Chromatography of partially purified enzyme on DEAE-cellulose column. The bars refer to units of enzyme activity, while the curves refer to the optical density at 280 mp.
moved, washed with ice-cold water, cut into smal1 pieces, and then homogenized in a Waring blendor at half speed for 30 sec. at 4” with 10 vol. of cold 0.1 M phosphate buffer, pH 7.5, containing 5 X lop3 M EDTA. The homogenate was centrifuged for 30 min. at 13,000 x g, and the sediment was discarded without washing. Ammonium
Sulfate
Fractionation
The supernatant solution was brought to 45% saturation with ammonium sulfate by the slow additions of the required amount of solid salt [277 g./l. (6)] with constant mechanical stirring. After allowing the mixture to equilibrate for 3 hr., the precipitate was centrifuged at 13,000 X g and discarded without washing. The supernatant solution was then adjusted to 75% saturation with ammonium sulfate by the addition of solid salt [210 g./l. (6)] in the same manner. After standing for 4 hr., the precipitate was collected by centrifugation and dissolved in 5 ml.
The procedure of Peterson and Sober (7) was followed in this work. The entire operation was carried out in a cold room at 30”. The column was prepared by allowing an aqueous slurry containing 28 g. DEAE-cellulose to pack by gravity in a 45 X 2.5 cm. column. The column was washed with 0.005 M phosphate buffer, pH 7.5, before use. The enzyme solution, containing 300 mg. protein was passed down the column at the rate of 2 ml./min. The eluate was collected in lo-ml. fractions by means of an automatic fraction collector. The resin was first eluted with 0.005 M phosphate buffer, pH 7.5, and then wit,11 increasing concentration of the buffer as indicated in Fig. 1. The enzymic activity appeared in one peak (bars, Fig. 1) and accounted for nearly all the activity applied to the column. The fraction with the highest activity was used in studying the properties of the enzyme. The fractions were stored frozen. A summary of the purification procedure is given in Table I. PROPERTIES OF ENZYME
Effect of Enzyme
step Crude homogenate (NH&SOI fraction DEAE-cellulose chromatography
OF MEVALONIC
I
DEHYDROGENASE
Total activity
Protein
w&its
%.
1,040 630 195
Concentration
It was established that the rate of the reaction was linearly proportional to the enzyme concentration under the conditions of the enzyme assay. The linearity of the results is shown in Fig. 2.
TABLE PURIFICATION
on DEAE-Cellulose Column
2 x 103 3 x 102 1.5
Specificactivity 0.52 2.1 130
Yield % 100 60
19
MEVALONIC
Optimal
ACID
155
DEHYDROGENASE
pH
s x to4 4
The curve for the effect of pH on the activity of the enzyme is shown in Fig. 3. An optimum pH of 6.2 was found. Michaelis
8
12
16
8
12
16 x IO’
Cons tan ts
The effect of substrate concentration on the catalytic activity of the enzyme was determined a,t pH 6.2 at varying concentrations of mevaldic acid and with either DPNH or TPNH as the reducing agent. Plots of the results are shown in Figs. 4 and 5. From the double reciprocal plots, the K, values calculated for mevaldic acid were 1 x lo-” Ai with DPNH as the reducer and 1.25 X 1O-4 M with TPNH. g 00302 f m oom$ 00.010‘: “a
1 0
I
4
I
FIG. 4. Plots of substrate concentration ws. velocity and of their reciprocals with mevaldic acid as oxidant and DPNH as reductant.
s x IO4 , I IO I 14I , 16,
leer f
6
0.116 2
D x
/ -0.010 01
02
03
Enzyme Content (mg.1
2. Proportionality enzyme concentration. FIG.
B 7 w
-0.006
between reaction rate and
I
I 0
4
8
I2
3. 2 Fir
I
16 x IO’
FIG. 5. Plots of substrate concentration vs. velocity and of their reciprocals with mevaldic acid as oxidant and TPNH as reductant.
TABLE EFFECT Addition
II
OF SULFHYDRYL Concentration
REAGENTS “m$
Inhibition
% None PCMB PCMB Iodoacetic Iodoacetic
M 1 x
acid acid
Inhibition FIG. 3. pH-activity gcnase.
curve of mevalonic dehydro-
0.016 10-d
1 x 10-S 1 x 10-b 1 x 10-a
0
0.00
100
0.007 0.008 0.00
70 00 100
of Enzyme
Mevalonic acid dehydrogenase is a sulfhydryl enzyme, This is shown by the in-
156
NAKAMURA
AND GREENBERG
hibitory effect of the sulfhydryl reagents, p-chloromercuribenzoic acid (PCMB) and iodoacetic acid on the reaction (Table II). Identification of Reaction Product Paper Chromatography
by
The incubation medium used for the identification is as follows: 200 pmoles phosphate buffer (pH 6.2)) 21.2 pmoles mevaldic acid, 2.6 pmoles DPNH, 20 pmoles EDTA, and enzyme solut.ion (0.5 mg. enzyme) in a total volume of 3.2 ml. The incubation was continued for 2 hr. at 37”. The medium was then deproteinized by immersing the reaction vessel into boiling water for 1 min., and the protein precipitate was removed by centrifugation. The supernatant solution was then concentrated in vacua and lyophilized to dryness in a Dry Ice-acetone bath. The reaidue was dissolved in 0.1 ml. water and applied on a sheet of Whatman No. 1 filter paper. The chromatogram was developed with a mixture of ethanol-NH3-water (85: 5: 15) and sprayed with bromophenol blue.
?vIevalonic acid was the only new compound that appeared on the chromatogram of the incubation. It had an Rf value of 0.62 and gave a blue spot on a yellow background with the indicator spray. REFERENCES 1. LYNEN, F., & “Biosynthesis of Terpenes and Sterols,” p. 95. J. & A. Churchill Ltd., London, 1959. 2. COON, M. J., KUPIECKI, F. P., DEKKER, E. E., SCHLESINGER, M. J., AND DEL CAMPILLO, A., in “Biosynthesis of Terpenes and Sterols,” p. 62. J. & A. Churchill Ltd., London, 1959. 3. SHUNK, C. H., LINN, B. O., HUFF, J. W., TILFILLAN, J. L., SKEGGS, H. R., AXD FOLKERS, K., J. Am. Chem. Sot. 79, 3294 (1957). 4. WARBURG, O., AND CHRISTIAN, W., Rio&em. Z. 310, 384 (1941-2). 5. GORNALL, A. T., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem. 177,751 (1949). 6. GREEX, A. A., AND HUGHES, W. L., in “Methods in Enzymology” (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, p. 76. Academic Press, New York, 1955. 7. PETERSON,E. A., AND SOBER,H. A., J. Am. Chem. Sot. 78, 751 (1956).