ANALYTICAL
BIOCHEMISTRY
89, 14-21 (1978)
A Method for Rapid Microassay 3-Hydroxy-3-Methylglutaryl-CoA Reductase Activity HUGUETTE Lipid
Roux
Research Center, Lava1 Faculty of Medicine.
of
MURTHY,’ PAUL-J. LUPIEN, AND SITAL MOORJANI University Hospital, Lava1 University,
and Department Quebec, P.Q.,
of Biochemistry, Canada
Received June 9, 1977 A method is devised for the separation of mevalonolactone (MVL) from hydroxymethylglutarate (HMG) in the assay of HMG-CoA reductase activity. The main steps in the procedure consist of absorbing the reaction mixture on the bottom part of a rectangular filter paper and selectively transfering the MVL into the top part of the paper by upward elution with toluene. Under the experimental conditions described, MVL is recovered in an yield of approximately 60%, with little contamination with HMG. Among the advantages of the method are that it involves simple and very few manipulations, no internal standard is required to calculate the recovery of MVL, and simultaneous analyses of a large number of samples are possible.
HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevaionic acid (MVA), which is believed to be a limiting precursor in the synthesis of liver cholesterol (1,2). Hence the assay of this enzyme is of both fundamental and clinical importance for an understanding of abnormalities in lipid metabolism. A key step in the quantitative determination of enzyme activity is the conversion of the MVA, formed in the presence of an enzyme source, to mevalonolactone (MVL) and separation of this derivative from the more-polar HMG by an appropriate method. Thin-layer chromatography (tic) is one of the most popular techniques for this purpose because of its comparative simplicity and adaptability to microassays (3,4). However, the large amount of salt present in reaction mixtures used for the assay of HMG-CoA reductase activity, the unavailability of solvent systems that can separate HMG and MVL into discrete bands in the presence of such high concentrations of salt, and the need to use very small volumes for spotting render this technique complicated to execute and often unreliable. In order to avoid the problem of salt, some procedures resort to extraction of MVL and HMG with ether before application to tic (5,6). However, ’ All enquiries should be addressed to Huguette Roux Murthy, Lipid Research Center, C.H.U.L., 2705 Boul. Laurier, Quebec, P.Q., GIV 4G2, Canada. 0003-2697/78/0891-0014$02.00/O Copyright 6 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
14
HMG-CoA
REDUCTASE
ACTIVITY
15
there is found to be much variability in the proportion of MVL extracted in different experiments, and the use of an internal standard becomes necessary as a means of avoiding serious errors (78). Jn the following report, we describe a simple procedure for the separation of MVL from HMG which avoids several of the above difficulties. The entire reaction mixture is absorbed on the bottom part of a rectangular filter paper, and the soluble substances are carried to the top of the paper by elution with toluene. Under the conditions described in these experiments, MVL, but not HMG, migrates to the top. MVL is thus completely separated from HMG with an yield of MVL of approximately 60%. MATERIALS
AND METHODS
[3-14C]HMG (15.8 mCi/mmol) and [5-3H]MVA (DBED salt, 6.8 mCi/ mmol) were obtained from New England Nuclear Corp., Boston, Mass, NADP+, D-glucose 6-phosphate, D-glucose-6-phosphate dehydrogenase (bakers’ yeast, 300 units/mg), ATP, dithiothreitol, coenzyme ASH and N,N’-dicyclohexylcarbodiimide were purchased from Sigma Chemical Co., St. Louis, MO. Spectrofluor was from Nuclear Chicago. Plasticbacked silica gel chromogram tic sheets and 2-ml conical beakers were purchased from Eastman Kodak Co., Rochester, N. Y., and from Canadian Laboratory Supply Ltd., Montreal, respectively. Aluminum foil (0.00045 in. thick) was bought from Fisher Scientific Co., Ltd., Montreal. Whatman No. 4 filter paper was used throughout these experiments for absorption of reaction mixtures and elution of radioactive products. Preparation of [3-‘4C]HMG-CoA. [3J4C]HMG-CoA was synthesized from CoA and [3J4C]HMG anhydride by the method of Hilz et al. (9) with the improvements suggested by Louw et al. (10). The anhydride was, in turn, prepared from [3-14C]HMG according to Goldfard and Pitot (7). The radioactive HMG-CoA was purified from HMG, unreacted HMG anhydride, and CoA using the procedure of Fogelman et al. (6). The resultant product had a specific activity of 5500 dpmnmol. The aqueous solution of the purified compound, adjusted to pH 5.5, was stored at -20°C. Preparation of [.5-3H]MVL. The DBED salt of [5-3H]MVA was dissolved in 1% NaHCO, to liberate the base. The amine was removed by three successive extractions with 2 vol of diethyl ether. The residual ether in the aqueous phase was removed by a current of nitrogen, and the pH of the solution was adjusted to less than 2.0 by the addition of concentrated HCl. After 15 min of incubation at 37°C to lactonize MVA, the solution was diluted to an appropriate volume with water and stored at -20°C. Assay of HMG-CoA reductase activity. The following is the suggested procedure for the assay of HMG-CoA reductase activity. Rat liver microsomes were used as source of the enzyme in these experiments. Preparation of the microsomes, composition of the reaction mixture, and incuba-
16
MURTHY,
LUPIEN,
AND MOORJANI
tion conditions were as described by Shapiro et al. (4). The reaction was carried out in a final volume of 0.12 ml in 2-ml beakers. Following incubation, 5 JLLIof concentrated HCl was added to each beaker, and the solution was further incubated at 37°C for 30 min in order to lactonize the MVA and convert the remaining HMG-CoA to free acid. Whatman No. 4 filter paper strips (9 x 2.4 cm) trimmed at one end to fit snugly at the conical bottom of the beaker were placed inside the beakers and left at room temperature until all the solution was absorbed by the paper. The liquid generally ascended to a height of 8.0 to 8.5 cm from the bottom. The filter papers, which thus served as supports for carrying the components of the reaction mixture, were dried at room temperature and cut approximately 1.0 cm above the front to mark the migration of the solution. A narrow band of filter paper was folded in the middle, and a slit was cut in the middle of the fold to create an opening. About 2 mm of the trimmed bottom portion of the filter paper support containing the reaction mixture was inserted in the opefiing (Fig. la). The band served as a wick for transporting the solvent during the subsequent elution step. The top half of the wick as well as the entire filter paper support (except for a portion of ap4 3 2
A F
P
C
1
:
(a)
(bl
FIG. 1. Diagram of apparatus used for separation of [‘Klmevalonate from the reaction mixture. (a) Basic apparatus: (P) filter paper support attached to the (W) wick. The numbers indicate the regions of the filter support that were cut out for radioactivity determinations for the experiment of Table 1; (1) the part extending from the bottom of the support up to a distance of 2 mm below the front (F) marking the reaction mixture when it was completely absorbed; (2) from 2 mm below F up to 2 mm above F; (3) from 2 mm above F up to the end of the portion covered by aluminum foil (A); (4) the exposed portion of the support from which the solvent was permitted to evaporate. (b) Assembly for upward solvent elution of soluble substances: (W) wick, (PA) filter paper support covered by aluminum foil, (S) level of the eluting solvent in the tube; (C) pair of split corks holding,the paper.
HMG-CoA
REDUCTASE
ACTIVITY
17
proximately 2 mm at the top) were enclosed in two continuous layers of aluminum foil and pressed down on the surface and the edges to form a a compact sandwich. The sandwich was placed inside a 15ml glass centrifuge tube and held in place by means of a pair of split corks (Fig. lb). The desired eluent was then added with a Pasteur pipette almost to the top of the tube. In this manner, the paper had no access to the solvent except at the wick, and the eluting solvent evaporated at a narrow exposed surface at the top, thus ensuring continuous elution of soluble materials on the filter paper support as long as some solvent remained in the tube. After elution in this manner overnight, the top 5 mm of the sandwich was snipped off, the aluminum foil was removed, and the paper was dried at room temperature. The dried paper was placed in 10 ml of toluene-POPOP (42 ml of spectrafluor in 1 liter of distilled toluene) and agitated slowly at room temperature for 5 hr. MVL was found to be solubilized into the scintillation fluid by this treatment, whereas any small residual HMG coeluting with MVL remained on the paper. The paper was removed, and the radioactivity in the fluid was determined using a scintillation counter. Thin-layer chromatographic analysis of mevalonolactone was carried out according to Shapiro et al. (4). Protein in microsomes was determined by the biuret reaction of Goa (11). RESULTS
It is seen in Table I that, when ether was used as the eluting solvent, about a third of the MVL on the filter paper support remained within the area of absorption of the reaction mixture (regions 1 and 2, Fig. la), and two thirds of it was carried to the top portion of the filter paper (regions 3 and 4, Fig. la). Elution with toluene gave essentially the same distribution of radioactivity of MVL. In contrast, the pattern of elution of HMG appeared to depend on the solvent used for elution. Thus, with ether, HMG was distributed almost equally between filter paper regions 1 and 2 on the one hand and regions 3 and 4 on the other. With toluene, there was no migration of HMG so that all of it remained within region 1. This indicated that by eluting with toluene it is possible to separate MVL from HMG completely with an yield of MVL of approximately 60% in region 4. Figures 2 and 3 show that the formation of mevalonate as measured by the filter paper assay is proportional to concentration of microsomal protein in the reaction mixture and to incubation time, thus permitting measurement of a wide range of enzyme activities. In Table 2 are compared the results of HMG-CoA reductase activity in rat liver microsomes obtained employing the above procedure and tic. Since heat-inactivated microsomes were used in the control reaction, the ratioactivity obtained in this case represents the contamination of the MVL fraction with HMG. It is seen that the contamination was much higher in
18
MURTHY,
LUPIEN,
AND MOORJANI
TABLE DISTRIBUTION
OF [W]HMG AFTER ELUTION
1
AND [3H]MVL ON FILTER PAPER WITH TOLUENE OR ETHER”
Distribution
of radioactivity
Toluene
SUPPORTS
(%)
Region of filter paper
Ether
HMG
MVL
HMG
MVL
1 2 3 4
97.2 2.6 0.1 0.1
35.1 4.2 0.1 60.6
51.2 0.1 20.8 27.9
30.5 3.8 6.9 58.8
a Approximately 100,000 dpm of [14C]HMG or [3H]MVL was added to a solution identical in composition to the reaction medium used for HMG-CoA reductase assay (see Materials and Methods). Immediately after the addition of microsomes (1 mg/ml) concentrated HCI was added (40 $/ml), and the mixture was incubated at 37°C for 30 min. Aliquots of 0.12 ml were transfered into beakers, absorbed on filter paper supports, and eluted with toluene or ether, as described in Materials and Methods. Then the supports were cut into four portions as indicated in Fig. la, and the radioactivity in each region was determined. The figures represent averages of six to eight separate determinations.
the tic method and that it increased with increasing amount of microsomes present in the reaction mixture. The filter paper procedure gave higher MVL counts, indicating a superior recovery and, consequently, the greater sensitivity of the method. It is interesting to note that addition of HMG inhibited the activity of HMG-CoA reductase as shown in Table 2. Since this compound is known to inhibit HMG-CoA reductase activity (12,13), the experiment demonstrates that the present procedure is sufficiently sensitive to measure the action of inhibitors. DISCUSSION Most separation methods used in the assay of HMG-CoA reductase take advantage of differences in the solubility and the corresponding migration of less-polar MVL from the more-polar HMG in a variety of solvent systems. The ability of toluene to separate the bulk of MVL from HMG in our experiments appears to be due not only to the different polar characteristics of the two compounds but, at least in part, to the role of salt in their elution. For example, under the influence of toluene. MVL, unlike HMG, was transferred from a part of paper which was coated with a large quantity of salt derived from the reaction mixture (regions 1 and 2, Fig. la) to a part of paper which was free of any salt or other absorbed material (regions 3 and 4, Fig. la). On the other hand, elution with ether transferred both HMG and MVL from the region of salt to the region of no salt.
HMG-CoA
0
REDLJCTASE
0.1
0.2
03
Mg
19
ACTIVITY
0.4
0.5
0.6
protein
FIG. 2. Mevalonate formation as a function of microsome concentration. The reaction mixture contained (in a final volume of 0.12 ml): 30 nlM glucose 6-phosphate, 2.0 units/ml of glucose&phosphate dehydrogenase, 0.5 mM [%Z]HMG-CoA, 3 mM NADP*, 30 mM EDTA, 2.50 mM NaCI, 50 mM potassium phosphate, pH 7.4, 1.0 mM dithiothreitol, and the amounts of microsomal protein indicated in the figure. Incubation was at 37°C for 15 min. [‘*C]Mevalonate formed was recovered from the reaction mixture and counted as described in Materials and Methods.
This latter observation is in conformity with that of Lynen and Grass1 (5) and Shapiro and Rodwell (14) that ether treatment of HMG-CoA reductase reaction mixtures in aqueous solution or in a lyophilized state extracted varying amounts of both HMG and MVL. The manner of distribution of HMG on the filter paper support on ether elution (Table 1) indicates that spreading was due to tailing and not any
0
15 Incubation
30 time
45
60
(mins)
FIG. 3. Mevalonate formation as a function of incubation time. The reaction conditions were as in Fig. 2 except that 0.2 mg of microsomes was added to each tube.
20
MURTHY,
LUPIEN,
AND MOORJANI
TABLE
2
COMPARISON OF HMG-CoA REDUCTASE ACTIVITIES IN RAT LIVER MICROSOMES AS MEASURED BY THE FILTER PAPER TECHNIQUE AND BY THIN-LAYER CHROMATOGRAPHY” [14C]MVA formed per reaction (dpm) Additions Microsomes (100 pg) Control Complete reaction Complete reaction plus 5 mM HMG Microsomes (250 pg) Control Complete reaction Complete reaction plus 5 mM HMG
Filter paper
Thin-layer chromatography
213 k 18 6872 + 395
642 f 42 5453 2 610
3945 k 205 (44%)
2982 + 321 (51%)
245 ‘- 12 15452 k 725
928 2 112 12855 -c 1328 9050 k 1015 (32%)
9848 ‘- 465 (43%)
u Assays of HMG-CoA reductase activity were carried out as described in Fig. 1 and in Materials and Methods. Incubation was at 37°C for 30 min. Microsomes inactivated by heating at 100°C for 10 min were used for the control reaction. Figures in parentheses indicate percentage inhibition. Each value represents an average of six to eight experiments.
heterogeneity in the radioactive material, particularly in view of the lack of migration of HMG in toluene. The distribution of MVL, on the other hand, was characterized by two distinct areas of concentration of radioactivity, one in the salt region and the other in the no-salt region. This was the case whether toluene or ether was used for elution. This suggests the presence of two molecular species with different migration characteristics. Sanghvi and Parikh (15) have recently reported a partial conversion of MVL in acid medium into the less-polar AZ-3-CH,-mevalonolactone by dehydration. The method suggested in this report possesses several advantages: (I) the manipulations involved are few and simple thus minimizing sources of error; (2) the entire reaction mixture is used for analysis, rendering the method quantitatively more sensitive; (3) use of an internal standard is avoided since the recovery of MVL does not vary more than ?5-7% within replicates of the same experiment; (4) a large number of samples can be processed simultaneously, which could be of importance in clinical screening tests. REFERENCES I. Bucher, N. L. R., Overath, P.. and Lynen, F. (1960) 491-501.
Biochim.
Biophys.
Acra
40,
HMG-CoA
REDUCTASE
ACTIVITY
21
2. Siperstein, M. D., and Fagan, V. M. (1966) J. Biol. Chem. 241, 602-609. 3. Shapiro, D. J., Inblum, R. L., and Rodwell, V. W. (1969)Anal. Biochem. 31, 383-390. 4. Shapiro, D. J.. Nordstrom. J. L.. Mitschelen, J. J., Rodwell, V. W., and Schimke, R. T. (1974) Biochim. Biophys. Acfa 370, 369-377. 5. Lynen, F., and Grassl. M. (1959) Hoppe-Seyler’s Z. Physiol. Chem. 313, 291-295. 6. Fogelman, A. M.. Edmond, J., Seager, J., and Popjak, G. (1975) J. Biol. Chem. 250, 204.5-2055. 7. Goldfard, S.. and Pitot, H. C. (1971)J. Lipid Res. 12, 512-515. 8. Brown, M. S., Dana, S. E.. Dietschy. J. M.. and Siperstein, M. D. (1973)J. Biol. Chem. 248, 473 I-4738. 9. Hilz. H.. Knappe, J.. Ringelmann, E., and Lynen, F. (1958) Biochem. Z. 329, 476. 10. Louw, A. I., Bekersky, I., and Mosbach, E. H. (1969) J. Lipid Res. 10, 683-686. 11. Goa, J. (1953) Stand. J. C/in. Lab. Invest. 5, 218-222. 12. Hamprecht, B., Nussler, C., Waltinger, G., and Lynen, F. (1971)in Metabolic Effects of Nicotinic Acid and Its Derivatives (Gey, K. F.. and Carlson, L. A., eds.). Hans Huber Publishers, Bern, Stuttgart, Vienna. 13. Moorjani, S., and Lupien, P.-J. (1978) Arch. Intern. Physiol. Biochim. 85, l-10. 14. Shapiro, D. J., and Rodwell, V. W. (1971)J. Biol. Chem. 246, 3210-3216. 15. Sanghvi, A., and Parikh, B. (1976) Biochim. Biophys. Acta 444, 723-733.
BIOCHEMISTRY
89, 14-21 (1978)
A Method for Rapid Microassay 3-Hydroxy-3-Methylglutaryl-CoA Reductase Activity HUGUETTE Lipid
Roux
Research Center, Lava1 Faculty of Medicine.
of
MURTHY,’ PAUL-J. LUPIEN, AND SITAL MOORJANI University Hospital, Lava1 University,
and Department Quebec, P.Q.,
of Biochemistry, Canada
Received June 9, 1977 A method is devised for the separation of mevalonolactone (MVL) from hydroxymethylglutarate (HMG) in the assay of HMG-CoA reductase activity. The main steps in the procedure consist of absorbing the reaction mixture on the bottom part of a rectangular filter paper and selectively transfering the MVL into the top part of the paper by upward elution with toluene. Under the experimental conditions described, MVL is recovered in an yield of approximately 60%, with little contamination with HMG. Among the advantages of the method are that it involves simple and very few manipulations, no internal standard is required to calculate the recovery of MVL, and simultaneous analyses of a large number of samples are possible.
HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevaionic acid (MVA), which is believed to be a limiting precursor in the synthesis of liver cholesterol (1,2). Hence the assay of this enzyme is of both fundamental and clinical importance for an understanding of abnormalities in lipid metabolism. A key step in the quantitative determination of enzyme activity is the conversion of the MVA, formed in the presence of an enzyme source, to mevalonolactone (MVL) and separation of this derivative from the more-polar HMG by an appropriate method. Thin-layer chromatography (tic) is one of the most popular techniques for this purpose because of its comparative simplicity and adaptability to microassays (3,4). However, the large amount of salt present in reaction mixtures used for the assay of HMG-CoA reductase activity, the unavailability of solvent systems that can separate HMG and MVL into discrete bands in the presence of such high concentrations of salt, and the need to use very small volumes for spotting render this technique complicated to execute and often unreliable. In order to avoid the problem of salt, some procedures resort to extraction of MVL and HMG with ether before application to tic (5,6). However, ’ All enquiries should be addressed to Huguette Roux Murthy, Lipid Research Center, C.H.U.L., 2705 Boul. Laurier, Quebec, P.Q., GIV 4G2, Canada. 0003-2697/78/0891-0014$02.00/O Copyright 6 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
14
HMG-CoA
REDUCTASE
ACTIVITY
15
there is found to be much variability in the proportion of MVL extracted in different experiments, and the use of an internal standard becomes necessary as a means of avoiding serious errors (78). Jn the following report, we describe a simple procedure for the separation of MVL from HMG which avoids several of the above difficulties. The entire reaction mixture is absorbed on the bottom part of a rectangular filter paper, and the soluble substances are carried to the top of the paper by elution with toluene. Under the conditions described in these experiments, MVL, but not HMG, migrates to the top. MVL is thus completely separated from HMG with an yield of MVL of approximately 60%. MATERIALS
AND METHODS
[3-14C]HMG (15.8 mCi/mmol) and [5-3H]MVA (DBED salt, 6.8 mCi/ mmol) were obtained from New England Nuclear Corp., Boston, Mass, NADP+, D-glucose 6-phosphate, D-glucose-6-phosphate dehydrogenase (bakers’ yeast, 300 units/mg), ATP, dithiothreitol, coenzyme ASH and N,N’-dicyclohexylcarbodiimide were purchased from Sigma Chemical Co., St. Louis, MO. Spectrofluor was from Nuclear Chicago. Plasticbacked silica gel chromogram tic sheets and 2-ml conical beakers were purchased from Eastman Kodak Co., Rochester, N. Y., and from Canadian Laboratory Supply Ltd., Montreal, respectively. Aluminum foil (0.00045 in. thick) was bought from Fisher Scientific Co., Ltd., Montreal. Whatman No. 4 filter paper was used throughout these experiments for absorption of reaction mixtures and elution of radioactive products. Preparation of [3-‘4C]HMG-CoA. [3J4C]HMG-CoA was synthesized from CoA and [3J4C]HMG anhydride by the method of Hilz et al. (9) with the improvements suggested by Louw et al. (10). The anhydride was, in turn, prepared from [3-14C]HMG according to Goldfard and Pitot (7). The radioactive HMG-CoA was purified from HMG, unreacted HMG anhydride, and CoA using the procedure of Fogelman et al. (6). The resultant product had a specific activity of 5500 dpmnmol. The aqueous solution of the purified compound, adjusted to pH 5.5, was stored at -20°C. Preparation of [.5-3H]MVL. The DBED salt of [5-3H]MVA was dissolved in 1% NaHCO, to liberate the base. The amine was removed by three successive extractions with 2 vol of diethyl ether. The residual ether in the aqueous phase was removed by a current of nitrogen, and the pH of the solution was adjusted to less than 2.0 by the addition of concentrated HCl. After 15 min of incubation at 37°C to lactonize MVA, the solution was diluted to an appropriate volume with water and stored at -20°C. Assay of HMG-CoA reductase activity. The following is the suggested procedure for the assay of HMG-CoA reductase activity. Rat liver microsomes were used as source of the enzyme in these experiments. Preparation of the microsomes, composition of the reaction mixture, and incuba-
16
MURTHY,
LUPIEN,
AND MOORJANI
tion conditions were as described by Shapiro et al. (4). The reaction was carried out in a final volume of 0.12 ml in 2-ml beakers. Following incubation, 5 JLLIof concentrated HCl was added to each beaker, and the solution was further incubated at 37°C for 30 min in order to lactonize the MVA and convert the remaining HMG-CoA to free acid. Whatman No. 4 filter paper strips (9 x 2.4 cm) trimmed at one end to fit snugly at the conical bottom of the beaker were placed inside the beakers and left at room temperature until all the solution was absorbed by the paper. The liquid generally ascended to a height of 8.0 to 8.5 cm from the bottom. The filter papers, which thus served as supports for carrying the components of the reaction mixture, were dried at room temperature and cut approximately 1.0 cm above the front to mark the migration of the solution. A narrow band of filter paper was folded in the middle, and a slit was cut in the middle of the fold to create an opening. About 2 mm of the trimmed bottom portion of the filter paper support containing the reaction mixture was inserted in the opefiing (Fig. la). The band served as a wick for transporting the solvent during the subsequent elution step. The top half of the wick as well as the entire filter paper support (except for a portion of ap4 3 2
A F
P
C
1
:
(a)
(bl
FIG. 1. Diagram of apparatus used for separation of [‘Klmevalonate from the reaction mixture. (a) Basic apparatus: (P) filter paper support attached to the (W) wick. The numbers indicate the regions of the filter support that were cut out for radioactivity determinations for the experiment of Table 1; (1) the part extending from the bottom of the support up to a distance of 2 mm below the front (F) marking the reaction mixture when it was completely absorbed; (2) from 2 mm below F up to 2 mm above F; (3) from 2 mm above F up to the end of the portion covered by aluminum foil (A); (4) the exposed portion of the support from which the solvent was permitted to evaporate. (b) Assembly for upward solvent elution of soluble substances: (W) wick, (PA) filter paper support covered by aluminum foil, (S) level of the eluting solvent in the tube; (C) pair of split corks holding,the paper.
HMG-CoA
REDUCTASE
ACTIVITY
17
proximately 2 mm at the top) were enclosed in two continuous layers of aluminum foil and pressed down on the surface and the edges to form a a compact sandwich. The sandwich was placed inside a 15ml glass centrifuge tube and held in place by means of a pair of split corks (Fig. lb). The desired eluent was then added with a Pasteur pipette almost to the top of the tube. In this manner, the paper had no access to the solvent except at the wick, and the eluting solvent evaporated at a narrow exposed surface at the top, thus ensuring continuous elution of soluble materials on the filter paper support as long as some solvent remained in the tube. After elution in this manner overnight, the top 5 mm of the sandwich was snipped off, the aluminum foil was removed, and the paper was dried at room temperature. The dried paper was placed in 10 ml of toluene-POPOP (42 ml of spectrafluor in 1 liter of distilled toluene) and agitated slowly at room temperature for 5 hr. MVL was found to be solubilized into the scintillation fluid by this treatment, whereas any small residual HMG coeluting with MVL remained on the paper. The paper was removed, and the radioactivity in the fluid was determined using a scintillation counter. Thin-layer chromatographic analysis of mevalonolactone was carried out according to Shapiro et al. (4). Protein in microsomes was determined by the biuret reaction of Goa (11). RESULTS
It is seen in Table I that, when ether was used as the eluting solvent, about a third of the MVL on the filter paper support remained within the area of absorption of the reaction mixture (regions 1 and 2, Fig. la), and two thirds of it was carried to the top portion of the filter paper (regions 3 and 4, Fig. la). Elution with toluene gave essentially the same distribution of radioactivity of MVL. In contrast, the pattern of elution of HMG appeared to depend on the solvent used for elution. Thus, with ether, HMG was distributed almost equally between filter paper regions 1 and 2 on the one hand and regions 3 and 4 on the other. With toluene, there was no migration of HMG so that all of it remained within region 1. This indicated that by eluting with toluene it is possible to separate MVL from HMG completely with an yield of MVL of approximately 60% in region 4. Figures 2 and 3 show that the formation of mevalonate as measured by the filter paper assay is proportional to concentration of microsomal protein in the reaction mixture and to incubation time, thus permitting measurement of a wide range of enzyme activities. In Table 2 are compared the results of HMG-CoA reductase activity in rat liver microsomes obtained employing the above procedure and tic. Since heat-inactivated microsomes were used in the control reaction, the ratioactivity obtained in this case represents the contamination of the MVL fraction with HMG. It is seen that the contamination was much higher in
18
MURTHY,
LUPIEN,
AND MOORJANI
TABLE DISTRIBUTION
OF [W]HMG AFTER ELUTION
1
AND [3H]MVL ON FILTER PAPER WITH TOLUENE OR ETHER”
Distribution
of radioactivity
Toluene
SUPPORTS
(%)
Region of filter paper
Ether
HMG
MVL
HMG
MVL
1 2 3 4
97.2 2.6 0.1 0.1
35.1 4.2 0.1 60.6
51.2 0.1 20.8 27.9
30.5 3.8 6.9 58.8
a Approximately 100,000 dpm of [14C]HMG or [3H]MVL was added to a solution identical in composition to the reaction medium used for HMG-CoA reductase assay (see Materials and Methods). Immediately after the addition of microsomes (1 mg/ml) concentrated HCI was added (40 $/ml), and the mixture was incubated at 37°C for 30 min. Aliquots of 0.12 ml were transfered into beakers, absorbed on filter paper supports, and eluted with toluene or ether, as described in Materials and Methods. Then the supports were cut into four portions as indicated in Fig. la, and the radioactivity in each region was determined. The figures represent averages of six to eight separate determinations.
the tic method and that it increased with increasing amount of microsomes present in the reaction mixture. The filter paper procedure gave higher MVL counts, indicating a superior recovery and, consequently, the greater sensitivity of the method. It is interesting to note that addition of HMG inhibited the activity of HMG-CoA reductase as shown in Table 2. Since this compound is known to inhibit HMG-CoA reductase activity (12,13), the experiment demonstrates that the present procedure is sufficiently sensitive to measure the action of inhibitors. DISCUSSION Most separation methods used in the assay of HMG-CoA reductase take advantage of differences in the solubility and the corresponding migration of less-polar MVL from the more-polar HMG in a variety of solvent systems. The ability of toluene to separate the bulk of MVL from HMG in our experiments appears to be due not only to the different polar characteristics of the two compounds but, at least in part, to the role of salt in their elution. For example, under the influence of toluene. MVL, unlike HMG, was transferred from a part of paper which was coated with a large quantity of salt derived from the reaction mixture (regions 1 and 2, Fig. la) to a part of paper which was free of any salt or other absorbed material (regions 3 and 4, Fig. la). On the other hand, elution with ether transferred both HMG and MVL from the region of salt to the region of no salt.
HMG-CoA
0
REDLJCTASE
0.1
0.2
03
Mg
19
ACTIVITY
0.4
0.5
0.6
protein
FIG. 2. Mevalonate formation as a function of microsome concentration. The reaction mixture contained (in a final volume of 0.12 ml): 30 nlM glucose 6-phosphate, 2.0 units/ml of glucose&phosphate dehydrogenase, 0.5 mM [%Z]HMG-CoA, 3 mM NADP*, 30 mM EDTA, 2.50 mM NaCI, 50 mM potassium phosphate, pH 7.4, 1.0 mM dithiothreitol, and the amounts of microsomal protein indicated in the figure. Incubation was at 37°C for 15 min. [‘*C]Mevalonate formed was recovered from the reaction mixture and counted as described in Materials and Methods.
This latter observation is in conformity with that of Lynen and Grass1 (5) and Shapiro and Rodwell (14) that ether treatment of HMG-CoA reductase reaction mixtures in aqueous solution or in a lyophilized state extracted varying amounts of both HMG and MVL. The manner of distribution of HMG on the filter paper support on ether elution (Table 1) indicates that spreading was due to tailing and not any
0
15 Incubation
30 time
45
60
(mins)
FIG. 3. Mevalonate formation as a function of incubation time. The reaction conditions were as in Fig. 2 except that 0.2 mg of microsomes was added to each tube.
20
MURTHY,
LUPIEN,
AND MOORJANI
TABLE
2
COMPARISON OF HMG-CoA REDUCTASE ACTIVITIES IN RAT LIVER MICROSOMES AS MEASURED BY THE FILTER PAPER TECHNIQUE AND BY THIN-LAYER CHROMATOGRAPHY” [14C]MVA formed per reaction (dpm) Additions Microsomes (100 pg) Control Complete reaction Complete reaction plus 5 mM HMG Microsomes (250 pg) Control Complete reaction Complete reaction plus 5 mM HMG
Filter paper
Thin-layer chromatography
213 k 18 6872 + 395
642 f 42 5453 2 610
3945 k 205 (44%)
2982 + 321 (51%)
245 ‘- 12 15452 k 725
928 2 112 12855 -c 1328 9050 k 1015 (32%)
9848 ‘- 465 (43%)
u Assays of HMG-CoA reductase activity were carried out as described in Fig. 1 and in Materials and Methods. Incubation was at 37°C for 30 min. Microsomes inactivated by heating at 100°C for 10 min were used for the control reaction. Figures in parentheses indicate percentage inhibition. Each value represents an average of six to eight experiments.
heterogeneity in the radioactive material, particularly in view of the lack of migration of HMG in toluene. The distribution of MVL, on the other hand, was characterized by two distinct areas of concentration of radioactivity, one in the salt region and the other in the no-salt region. This was the case whether toluene or ether was used for elution. This suggests the presence of two molecular species with different migration characteristics. Sanghvi and Parikh (15) have recently reported a partial conversion of MVL in acid medium into the less-polar AZ-3-CH,-mevalonolactone by dehydration. The method suggested in this report possesses several advantages: (I) the manipulations involved are few and simple thus minimizing sources of error; (2) the entire reaction mixture is used for analysis, rendering the method quantitatively more sensitive; (3) use of an internal standard is avoided since the recovery of MVL does not vary more than ?5-7% within replicates of the same experiment; (4) a large number of samples can be processed simultaneously, which could be of importance in clinical screening tests. REFERENCES I. Bucher, N. L. R., Overath, P.. and Lynen, F. (1960) 491-501.
Biochim.
Biophys.
Acra
40,
HMG-CoA
REDUCTASE
ACTIVITY
21
2. Siperstein, M. D., and Fagan, V. M. (1966) J. Biol. Chem. 241, 602-609. 3. Shapiro, D. J., Inblum, R. L., and Rodwell, V. W. (1969)Anal. Biochem. 31, 383-390. 4. Shapiro, D. J.. Nordstrom. J. L.. Mitschelen, J. J., Rodwell, V. W., and Schimke, R. T. (1974) Biochim. Biophys. Acfa 370, 369-377. 5. Lynen, F., and Grassl. M. (1959) Hoppe-Seyler’s Z. Physiol. Chem. 313, 291-295. 6. Fogelman, A. M.. Edmond, J., Seager, J., and Popjak, G. (1975) J. Biol. Chem. 250, 204.5-2055. 7. Goldfard, S.. and Pitot, H. C. (1971)J. Lipid Res. 12, 512-515. 8. Brown, M. S., Dana, S. E.. Dietschy. J. M.. and Siperstein, M. D. (1973)J. Biol. Chem. 248, 473 I-4738. 9. Hilz. H.. Knappe, J.. Ringelmann, E., and Lynen, F. (1958) Biochem. Z. 329, 476. 10. Louw, A. I., Bekersky, I., and Mosbach, E. H. (1969) J. Lipid Res. 10, 683-686. 11. Goa, J. (1953) Stand. J. C/in. Lab. Invest. 5, 218-222. 12. Hamprecht, B., Nussler, C., Waltinger, G., and Lynen, F. (1971)in Metabolic Effects of Nicotinic Acid and Its Derivatives (Gey, K. F.. and Carlson, L. A., eds.). Hans Huber Publishers, Bern, Stuttgart, Vienna. 13. Moorjani, S., and Lupien, P.-J. (1978) Arch. Intern. Physiol. Biochim. 85, l-10. 14. Shapiro, D. J., and Rodwell, V. W. (1971)J. Biol. Chem. 246, 3210-3216. 15. Sanghvi, A., and Parikh, B. (1976) Biochim. Biophys. Acta 444, 723-733.
