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A mevalonate kinase assay
iNALYTICAL
BIOCHEMISTRY
38, 130-138
(1970)
A Mevalonate
Kinase
T. R. GREEN Department
Received
D. J. BAISTED
AND
of Biochemistry and Cowallis,
Assay
Biophysics, Oregon Oregon 97331
February
State
University,
26, 1970
Mevalonic acid (MVA) is the precursor of numerous terpenoids in animals, plants, and microorganisms (1). Implicated in the biosynthesis of these compounds are several phosphorylated intermediates arising from MVA: 5-phosphomevalonate (MVAP) (2, 3), 5pyrophosphomevalonate (MVAPP) (3)) A3-isopentenyl pyrophosphate (IPP) (3)) y,ydimethylallyl pyrophosphate (DMAPP) (4, 5), geranyl pyrophosphate (GPP) (4), farnesyl pyrophosphate (FPP) (4), and geranylgeranyl pyrophosphate (GGPP) (6)) in the order that they are formed from MVA. Further isopentenylation can lead to higher acyclic terpenoids while further condensations and cyclizations of these intermediates can lead to a great variety of terpenoids and .also sterols (7). The initial phosphorylation of mevalonic acid is catalyzed by an enzyme, mevalonate kinase (EC 2.7.1.36), which has been isolated from yeast (2), liver (8,9) and several higher plants (10). The kinase has been assayed in three ways: (1) radiochromatographically using Dowex-formate columns (2) for the measurement of the rate of disappearance of mevalonic .acid-2-W; (2) by paper chromatography (11, 12) and thin-layer chromatography (13) for the measurement of the appearance of phosphorylated products; (3) spectrophotometrically (11) as a coupled reaction with pyruvate kinase and lactate dehydrogenase to measure NADH consumption. This consumption ultimately depends upon the formation of ADP during the reaction. There are certain disadvantages with all of the assay procedures. The spectrophotometric assay may only be used with relatively pure preparations of the kinase as it is ultimately dependent upon the measurement of ADP formed during conversion of MVA to MVAP by ATP. However, reactions such as the further conversion of MVAP to MVAPP or of MVAPP to IPP utilize ATP and also produce ADP in the process. Furthermore, preparations of mevalonic kinase are frequently contaminated by enzymes catalyzing these two reactions and as a consequence MVA 130
MEVALONATE
KINASE
ASSAY
131
kinase activities of such preparations measured by this procedure must be high. Other difficulties encountered using this assay for crude preparations have also been discussed (13). The assay involving paper and thin-layer chromatography of the extract for quantitation of the products of MVA metabolism must be carried out on small volumes to avoid overloading of the paper or plate. Although they have the advantage of requiring very small amounts of substrate, the paper chromatographic assay, at least, suffers from a relatively large inaccuracy (11). The Dowex-formate column chromatography of the assay mixture is the most rigorous method for the measurement of disappearance of substrate but it is also the most laborious. In this paper we describe a procedure for the assay of MVA kinase in a 40,OOOg supernatant from homogenates of germinated pea seeds. The assay is based upon measurement of the rate of disappearance of MVA2-14C under conditions of first-order kinetics with respect to the substrate. The residual MVA in the extracts is removed by a modification of a procedure of Porter and Guchhait for the isolation of MVA lactone (14). That the residual radioactivity is associated only with MVA lactone is confirmed by paper and gas chromatography. In addition, all radioactive metabolites of MVA in the extract were identified by ion-exchange and paper chromatography and confirmed the fact that disappearance of MVA must have occurred only through the kinase. Phosphatase activity in the extract was established as having no influence on the assay. MATERIALS
RS-MVA-~-I~C was obtained from New England Nuclear Corporation as the N,N’-dibenzylethylenediamine salt1 The salt was converted to the sodium salt in aqueous solution and diluted to appropriate activity with water. Solution activities normally ranged between 0.707 and 2.095 X lo6 dpm/lO ,ul (specific activity 5.9 pCi/pmole). Adenosine 5’triphosphate was obtained from P-L Biochemicals, Inc. Chromosorb W (60-80 mesh) and butanediol succinate polyester were both purchased from Perco Supplies. Pea seeds (Pisum sativum) were the Blue Bantam strain of the W. Atlee Burpee Seed Company. An Fii ammonium sulfate fraction of pork liver prepared according to the method of Popjak (13) was used for the synthesis of R-MVAP-~-~C from RS-MVA-~-~~C. The product was isolated and identified by elution from a Dowex-formate ionexchange resin (3). The identity of the product and its purity were confirmed by its paper chromatographic behavior in an n-propanol/ammonia/ ‘Symbols
R
and s according to Cahn-Ingold-Prelog nomenclature.
132
GREEN
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BASTED
water (60: 20: 20) system (15). Radiochromatographic scanning of the paper indicated better than 99% of the radioactivity applied to the paper was associated with a peak, the Rf of which coincided with that of MVAP. METHODS
Protein Determinations. All protein measurements were made by the biuret method at 540 nm according to the method of Gornall et al. (16). Enzyme Preparation. Batches of 100 seeds were germinated in the dark for 9 hr with distilled water in glass Petri dishes. After washing them thoroughly, they were transferred to a Waring blender and mixed with 75 ml ice-cold 0.1 M phosphate buffer, pH 6.8, which was 0.01 M in MgS04, 0.01 M in glutathione, and 0.45 M in sucrose. After 30 set homogenization, the homogenate was strained through four layers of cheese cloth, and then centrifuged at 40,OOOg for 10 min to give a clear cell-free supernatant. This supernatant was used as the source of mevalonate kinase. Protein concentrations normally ranged between 40 and 45 mg/ml. Where necessary, the protein concentrations were decreased by dilution of the supernatant with phosphate buffer. Enzyme preparations were carried out at cold room temperatures. Mevalonate Kinase Assay. A series of tubes, each containing 3.0 ml of the crude 40,OOOg supernatant containing 0.18 pmole ATP, were thermally equilibrated in a water bath to 24” and 10 ~1 MVA-2-14C solution was added to each tube. After incubating the tubes for various intervals of time, 2.0 ml hot 20% KOH was added to each tube to terminate the reaction and then 2 mg cold MVA lactone was added to each sample as carrier. Each ,sample was transferred quantitatively to a continuous ether extractor; the solutions were acidified to pH 1 with 12 N H,SO, and allowed to stand at 37” for 15-30 min to lactonize nonmetabolized MVA. The incubations were terminated with 20% KOH rather than acid because it was found that protein precipitated in the presence of acid and made the quantitative transfer of the solution to the extractor difficult. The alkali treatment maintained a clear solution. Following lactonization, the samples were continuously extracted with diethyl ether for 12-14 hr under reflux. The ether extracts were concentrated to a small volume on a steam bath under a hood, transferred to graduated test tubes in tetrahydrofuran (5-10 ml), and aliquots (l/100) removed for scintillation counting on a Packard Tri-Carb scintillation counter. Bray scintillation fluor (17) was used and each sample aliquot counted and internally spiked with a toluene-14C standard in order to detect quenching. The decrease in radioactivity of the ether extract with time of reaction provided the basis of the mevalonate kinase assay. For routine measurements of kinase activity in pea seed homogenates an incubation time of 2.0 min was used.
MEVALONATE
KINASE
ASSAY
133
RESULTS AND DISCUSSION For the assay to be valid it was necessary to establish the following: (1) that under the conditions of the assay the 14C content of the ether extract was truly mevalonolactone, (2) that the enzyme assayed was mevalonate kinase, (3) that the assay was not influenced by phosphatase, and (4) that the recovery of mevalonolactone was not impaired by binding to protein. To substantiate that the 14C content of the ether extract represented MVA-2-l% lactone, aliquots from incubations allowed to proceed for different lengths of time were subjected to gas and paper chromatography. Gas chromatography was conducted on a 6’ X 1/4#’ column of Chromosorb W coated with 20% butanediol succinate polyester according to the procedure of VandenHeuvel (18). The column was run at 195” and a helium flow rate of 40-50 ml/min in a Beckman GC 4 instrument equipped with a flame ionization detector. The effluent was split 10: 1 in favor of passage through maNuclear-Chicago gas ionization chamber, so that carrier MVA lactone and radioactivity were measured simultaneously on a two-channel recorder equipped with Disc integrators for peak area measurement. (If no gas ionization chamber is available, the lactone can be trapped and counted in a scintillation vial as described by Porter and Guchhait.) In our experiments no labeled compounds other than mevalonolactone were detected in the diethyl ether extract until more than 90% of the R-MVA had been metabolized. From Figure 1 it can be seen that this represents an incubation time greater than 6 min. Where other labeled compounds are found in the ether extract, internal normalization of the radiochromatographic tracings with respect to mevalonolactone can be used to determine the proportion of 14C associated with mevalonolactone. Sensitivity of our counting equipment permitted the detection of less than 2% of the emerging radioactivity in a single peak. In cases in which internal normalization cannot be used due to selective adsorption of labeled compounds to the column, the acidified samples can be extracted with petroleum ether prior to diethyl ether extraction (13, 14). Ascending paper chromatography of the extract in a n-propanol/ammonia/water (60:20: 20) system followed by radiochromatographic scanning on a Packard model 7201 scanner also demonstrated no other contaminating component to be present until 90% of the R-MVA had been metabolized. The measure of the 14C content of the diethyl ether extract under the conditions of the assay was then concluded to represent the nonmetabolized MVA. It was necessary to correlate the disappearance of MVA with the appearance of products resulting from metabolism through the kinase. An incubate in which 32% of the RS-MVA had metabolized was subjected to the following procedures:
134
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TIME (min) FIG. 1. Stereospecificity of MVA kinase for one enantiomer of RS-MVA-2-%. 3.0 ml aliquots of the 40,OOOg supernatant were incubated with 159 mamoles of the racemic substrate as described in the text. Disappearance was followed versus incubation period.
(a) DEAE-cellulose chromatography under conditions that separated MVA from the other radioactive components (19). These metabolites were subjected to paper chromatography in three systems: n-propanol/ ammonia/water (60: 20: 20) (15) ; tert-butanol/formic acid/water (40 : 10: 16) (3)) and n-butanol/formic acid/water (77: 10: 13) (2,20). (5) Dowex-formate chromatography of the acid-stable intermediates (3, 21). (c) Acid hydrolysis and evaporation of volatile compounds for determination of ally1 derivatives (11). By these methods the distribution of radioactivity in the incubate was found to be: MVA (68%), MVAP (6-7%), MVAPP (9-10%/o), IPP (4-5%), DMAPP (11%) and IP (~1%). No higher sally1 phosphates or pyrophosphates beyond dimethylallyl pyrophosphate were observed. By these procedures we were able to account for all of the radioactivity in the incubate as products resulting from metabolism of the MVA through MVA kinase. If phosphatase activity is significant the levels of mevalonolactone recovered in the assay might be higher than in the absence of such activity. Consequently kinase activity will appear to be lower than the true value. Conditions should be sought to minimize this activity (duration of incubation, protein concentration, etc.). Under the MVA kinase assay conditions, we have already demonstrated that prenols arising from hy-
MEVALONATE
KINASE
135
ASSAY
drolysis of the corresponding pyrophosphates do not accumulate in the diethyl ether extract. Consequently, for phosphatase to interfere with the assay, only the hydrolysis of MVAP back to MVA need be considered. In a separate study we measured the specific activity of phosphomevalonate kinase in the pea seed extract according to the method of Tchen (11). An excess of MVAP was then added to samples of this extract, to test for phosphatase interference over different periods of time. The protein concentration used was the same as that for the MVA kinase assay. Paper chromatographic analysis of the incubation products in n-propanol/ ammonia/water (60 : 20: 20) revealed that no MVA accumulated during an incubation time three times the duration of the routine MVA kinase assay. First-order binding of MVA to protein would be indistinguishable from mevalonate kinase activity. The association of MVA with protein may take place during the enzymic incubation period and/or during the isolation process following termination of the assay. In either case an anomalously high MVA kinase activity would be measured. Binding under the conditions of isolation could conceivably arise by acid-catalyzed esterification of mevalonic acid to a number of residues on protein (ser, thr, asp, glu, etc.). That this does not occur is evidenced by complete recovery of mevalonolactone at zero time incubation (Fig. 2). The absence of any significant binding during incubation was shown by incubating a typical enzyme preparation for approximately 10 min with a known amount of MVA, and determining the proportion of W recoverable by dialysis. According to the lactone assay, 10 min is sufficient time to convert 92% of the R-MVA into products (Fig. 1). Even under very mild conditi,ons of termination with 0.1 M EDTA and subsequent dialysis, essentially all of the W was recovered in the dialyzate (Table 1). From these results, it appears that binding of MVA to protein does not interfere with the kinase assay. In the present study, stereospecificity for the substrate was observed, Influence Initia,l MVA-2-W X 10e6 dpm 0.706
of Protein
TABLE 1 Binding on Recovery Nondialyzable Wb X 10e6 dpm 0.016
of Radioactivity” y0 dialyzed 1% 98
a Refers to dialysable 1°C from 10 min incubation in presence of MVA-2-W. b Label remaining in dialysis bag after 18 hr dialysis against several changes of water with intermittent changes against saturated NaCl solution. The incubated sample was terminated with 3.0 ml 0.1 M EDTA instead of KOH solution.
136
GREEN AND BAISTED
although the actual enantiomer metabolized was not determined. Metabolism of MVA ceased when 50% of the racemic substrate initially added had been consumed (Fig. 1j . A number of authors have isolated mevalonate kinase from various plant and animal sources and have shown it to be stereospecific for the phosphorylation of racemic MVA (2, 10, 22). The absolute stereochemistry of the utilizable enantiomer has been established as n-mevalonate (23). Accepting the available substrate to be one-half the total added to the enzyme preparation, changes in the concentration of the utilizable entaniomer during the period of the assay can be calculated. The first-order equation used for the initially racemic substrate is:2 log, 0.5&/(/l,
- 0.54
= kt
Figure 2 shows the linear relationship between reaction time and a logarithmic function of substrate disappearance relative to initial substrate, and Figure 3, the linear relationship between the first-order rate constants and enzyme concentration.
TIME
(min)
FIG. 2. First-order kinetic assay of MVA kinsse. Aliquots of the enzyme preparation were incubated as described in Figure 1 and results plotted in terms of the fist-order kinetic equation for racemic substrate.2 SUMMARY
A suitable assay has been developed for accurate measurements of mevalonate kinase in a crude enzyme preparation. The assay is particu* log. 0.5&/(At - 0.5Ao)= log. &a*, o,, = total available substrate at t = 0, at = available substrate remaining at so that the typical ratio of initial to
ObAo/(At -00.5Ao).
where A0= total racemic substrate at t = 0, At = total substrate remaining at time t, and time t; then ao = 0.5A0and at = At -0.5A. final substrate
concentration,
so/at, becomes
MEVALONATE
2 2
0.16-
is z
0.12-
F
0.08 -
2
0.04-
KINASE
ASSAY
137
O%i?G-Yr PROTEIN
(mg/ml)
FIG. 3. Demonstration of linear response of assay to changes in enzyme concentration. Aliquots of enzyme solution were diluted with phosphate buffer to 3.0 ml to give the range of protein concentrations shown. Each assay was conducted with 159 mpmoles MVAS-“C for 2.0 min. Corresponding first-order rate constants were calculated and plotted against enzyme concentration.
larly useful where comparative, tissue-specific, or developmental studies are to be made on mevalonate kinase. We have found this method of assay of mevalonate kinase to be particularly suitable for our own studies of kinase activity in crude homogenates of germinated pea seeds. In the present study, the specific activity of the kinase was calculated to be 0.4 mpmole R-MVA min-l mg-I. This represents a lower limit for the activity due to the first-order conditions used for the assay. As a general assay for the kinase, the procedure seems promising in terms of accuracy, simplicity, and labor. ACKNOWLEDGMENTS This work was supported by a Research Grant AM 09265 from the National Institutes of Health and was conducted during the tenure of a Research Career Development Award held by D.J.B. One of us (T.R.G.) is supported by an NDEA fellowship. This work forms part of the thesis requirement for the Ph.D. at Oregon State University. REFERENCES 19, 168, 201 (1965).
1. CLAYTON, R. B., Quart. Rev. 2. TCHEN, T. T., J. Biol. Chem. 233, 1100 (1958). 3. BLOCH, K., CHAYKIN, S., PHILIPS, A. H., AND DE WAARD, A., J. Biol. Chem. 234, 2595 (1959). 4. LYNEN, F., AGRANOFF, B. W., EQCERER, H., HENNING, U., AND MOSLEIN, E. M., Angew. Chem. 71, 657 (1959). 5. AGRANOFF, B., EGGQIEB, H., HENNING, U., AND LYNEN, F., J. Biol. Chem. 235, 326 (1960).
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6. KIRSCHNEB, K., PhD. thesis, Ludwig-Maximiliano-UniversitLt, Munich, Germany, 1961. 7. RICHARDS, J. H., AND HENDRICKSON, J. W., in “The Biosynthesis of Steroids, Terpenes and Acetogenins.” Benjamin, New York, 1964. S. LEVY, H. R., AND POPJAK, G., B&hem. J. 75,417 (1960). 9. MARKLEY, K., AND SMALLMAN, E., Biochim. Biophys. Acta 47, 327 (1961). 10. LOOMIS, W. D., AND BATTAILE, J., Biochim. Biophys. Acta 67, 54 (1963). 11. TCHEN, T. T., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. V, p. 489. Academic Press, New York, 1962. 12. DEWAARD, A., AND POPJAK, G., Biochem. J. 73,410 (1959). 13. POPJAK, G., in “Methods of Enzymology” (R. B. Clayton, ed.), Vol. 15, p. 393. Academic Press, New York, 1969. 14. PORTER, J. W., AND GUCHHAIT, R. B., Anal. Biochem. 15, 509 (1966). 15. SHAH, D. H., CLELAND, W. W., AND PORTER, J. W., J. Bid. Chem. 240, 1946 (1965). 16. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem. 177, 751 ( 1949). 17. BRAY, G. A., Anal. Biochem. 1,279 (1960). 18. HORNING, E. C., VANDENHEUVEL, W. J. A., AND CREECH, B. G., in “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. 11, p. 69. Interscience, New York, 1963. 19. DUGAN, R. E., RASSON, E., AND PORTER, J. W., Anal. Biochem. 22, 249 (1968). 20. WITTING, L. A., AND PORTER, J. W., .I. BioZ. Chem. 234, 2841 (1959). 21. HURLBERT, R. B., SCIIMITZ, H., BRUM, A. F., AND POTTER, V. R., J. BioZ. Chem. 209, 23 (1954). 22. FOLKERS, K., AND WAGNER, A. F., in “Advances in Enzymology” (F. F. Nord, ed.), Vol. 23, p. 471. Interscience, New York, 1961. 23. CORNFORTH, R. H., FLETCHER, K., HELLIG, H., AND POPJAK, G., Nature 185, 923 (1960).
BIOCHEMISTRY
38, 130-138
(1970)
A Mevalonate
Kinase
T. R. GREEN Department
Received
D. J. BAISTED
AND
of Biochemistry and Cowallis,
Assay
Biophysics, Oregon Oregon 97331
February
State
University,
26, 1970
Mevalonic acid (MVA) is the precursor of numerous terpenoids in animals, plants, and microorganisms (1). Implicated in the biosynthesis of these compounds are several phosphorylated intermediates arising from MVA: 5-phosphomevalonate (MVAP) (2, 3), 5pyrophosphomevalonate (MVAPP) (3)) A3-isopentenyl pyrophosphate (IPP) (3)) y,ydimethylallyl pyrophosphate (DMAPP) (4, 5), geranyl pyrophosphate (GPP) (4), farnesyl pyrophosphate (FPP) (4), and geranylgeranyl pyrophosphate (GGPP) (6)) in the order that they are formed from MVA. Further isopentenylation can lead to higher acyclic terpenoids while further condensations and cyclizations of these intermediates can lead to a great variety of terpenoids and .also sterols (7). The initial phosphorylation of mevalonic acid is catalyzed by an enzyme, mevalonate kinase (EC 2.7.1.36), which has been isolated from yeast (2), liver (8,9) and several higher plants (10). The kinase has been assayed in three ways: (1) radiochromatographically using Dowex-formate columns (2) for the measurement of the rate of disappearance of mevalonic .acid-2-W; (2) by paper chromatography (11, 12) and thin-layer chromatography (13) for the measurement of the appearance of phosphorylated products; (3) spectrophotometrically (11) as a coupled reaction with pyruvate kinase and lactate dehydrogenase to measure NADH consumption. This consumption ultimately depends upon the formation of ADP during the reaction. There are certain disadvantages with all of the assay procedures. The spectrophotometric assay may only be used with relatively pure preparations of the kinase as it is ultimately dependent upon the measurement of ADP formed during conversion of MVA to MVAP by ATP. However, reactions such as the further conversion of MVAP to MVAPP or of MVAPP to IPP utilize ATP and also produce ADP in the process. Furthermore, preparations of mevalonic kinase are frequently contaminated by enzymes catalyzing these two reactions and as a consequence MVA 130
MEVALONATE
KINASE
ASSAY
131
kinase activities of such preparations measured by this procedure must be high. Other difficulties encountered using this assay for crude preparations have also been discussed (13). The assay involving paper and thin-layer chromatography of the extract for quantitation of the products of MVA metabolism must be carried out on small volumes to avoid overloading of the paper or plate. Although they have the advantage of requiring very small amounts of substrate, the paper chromatographic assay, at least, suffers from a relatively large inaccuracy (11). The Dowex-formate column chromatography of the assay mixture is the most rigorous method for the measurement of disappearance of substrate but it is also the most laborious. In this paper we describe a procedure for the assay of MVA kinase in a 40,OOOg supernatant from homogenates of germinated pea seeds. The assay is based upon measurement of the rate of disappearance of MVA2-14C under conditions of first-order kinetics with respect to the substrate. The residual MVA in the extracts is removed by a modification of a procedure of Porter and Guchhait for the isolation of MVA lactone (14). That the residual radioactivity is associated only with MVA lactone is confirmed by paper and gas chromatography. In addition, all radioactive metabolites of MVA in the extract were identified by ion-exchange and paper chromatography and confirmed the fact that disappearance of MVA must have occurred only through the kinase. Phosphatase activity in the extract was established as having no influence on the assay. MATERIALS
RS-MVA-~-I~C was obtained from New England Nuclear Corporation as the N,N’-dibenzylethylenediamine salt1 The salt was converted to the sodium salt in aqueous solution and diluted to appropriate activity with water. Solution activities normally ranged between 0.707 and 2.095 X lo6 dpm/lO ,ul (specific activity 5.9 pCi/pmole). Adenosine 5’triphosphate was obtained from P-L Biochemicals, Inc. Chromosorb W (60-80 mesh) and butanediol succinate polyester were both purchased from Perco Supplies. Pea seeds (Pisum sativum) were the Blue Bantam strain of the W. Atlee Burpee Seed Company. An Fii ammonium sulfate fraction of pork liver prepared according to the method of Popjak (13) was used for the synthesis of R-MVAP-~-~C from RS-MVA-~-~~C. The product was isolated and identified by elution from a Dowex-formate ionexchange resin (3). The identity of the product and its purity were confirmed by its paper chromatographic behavior in an n-propanol/ammonia/ ‘Symbols
R
and s according to Cahn-Ingold-Prelog nomenclature.
132
GREEN
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BASTED
water (60: 20: 20) system (15). Radiochromatographic scanning of the paper indicated better than 99% of the radioactivity applied to the paper was associated with a peak, the Rf of which coincided with that of MVAP. METHODS
Protein Determinations. All protein measurements were made by the biuret method at 540 nm according to the method of Gornall et al. (16). Enzyme Preparation. Batches of 100 seeds were germinated in the dark for 9 hr with distilled water in glass Petri dishes. After washing them thoroughly, they were transferred to a Waring blender and mixed with 75 ml ice-cold 0.1 M phosphate buffer, pH 6.8, which was 0.01 M in MgS04, 0.01 M in glutathione, and 0.45 M in sucrose. After 30 set homogenization, the homogenate was strained through four layers of cheese cloth, and then centrifuged at 40,OOOg for 10 min to give a clear cell-free supernatant. This supernatant was used as the source of mevalonate kinase. Protein concentrations normally ranged between 40 and 45 mg/ml. Where necessary, the protein concentrations were decreased by dilution of the supernatant with phosphate buffer. Enzyme preparations were carried out at cold room temperatures. Mevalonate Kinase Assay. A series of tubes, each containing 3.0 ml of the crude 40,OOOg supernatant containing 0.18 pmole ATP, were thermally equilibrated in a water bath to 24” and 10 ~1 MVA-2-14C solution was added to each tube. After incubating the tubes for various intervals of time, 2.0 ml hot 20% KOH was added to each tube to terminate the reaction and then 2 mg cold MVA lactone was added to each sample as carrier. Each ,sample was transferred quantitatively to a continuous ether extractor; the solutions were acidified to pH 1 with 12 N H,SO, and allowed to stand at 37” for 15-30 min to lactonize nonmetabolized MVA. The incubations were terminated with 20% KOH rather than acid because it was found that protein precipitated in the presence of acid and made the quantitative transfer of the solution to the extractor difficult. The alkali treatment maintained a clear solution. Following lactonization, the samples were continuously extracted with diethyl ether for 12-14 hr under reflux. The ether extracts were concentrated to a small volume on a steam bath under a hood, transferred to graduated test tubes in tetrahydrofuran (5-10 ml), and aliquots (l/100) removed for scintillation counting on a Packard Tri-Carb scintillation counter. Bray scintillation fluor (17) was used and each sample aliquot counted and internally spiked with a toluene-14C standard in order to detect quenching. The decrease in radioactivity of the ether extract with time of reaction provided the basis of the mevalonate kinase assay. For routine measurements of kinase activity in pea seed homogenates an incubation time of 2.0 min was used.
MEVALONATE
KINASE
ASSAY
133
RESULTS AND DISCUSSION For the assay to be valid it was necessary to establish the following: (1) that under the conditions of the assay the 14C content of the ether extract was truly mevalonolactone, (2) that the enzyme assayed was mevalonate kinase, (3) that the assay was not influenced by phosphatase, and (4) that the recovery of mevalonolactone was not impaired by binding to protein. To substantiate that the 14C content of the ether extract represented MVA-2-l% lactone, aliquots from incubations allowed to proceed for different lengths of time were subjected to gas and paper chromatography. Gas chromatography was conducted on a 6’ X 1/4#’ column of Chromosorb W coated with 20% butanediol succinate polyester according to the procedure of VandenHeuvel (18). The column was run at 195” and a helium flow rate of 40-50 ml/min in a Beckman GC 4 instrument equipped with a flame ionization detector. The effluent was split 10: 1 in favor of passage through maNuclear-Chicago gas ionization chamber, so that carrier MVA lactone and radioactivity were measured simultaneously on a two-channel recorder equipped with Disc integrators for peak area measurement. (If no gas ionization chamber is available, the lactone can be trapped and counted in a scintillation vial as described by Porter and Guchhait.) In our experiments no labeled compounds other than mevalonolactone were detected in the diethyl ether extract until more than 90% of the R-MVA had been metabolized. From Figure 1 it can be seen that this represents an incubation time greater than 6 min. Where other labeled compounds are found in the ether extract, internal normalization of the radiochromatographic tracings with respect to mevalonolactone can be used to determine the proportion of 14C associated with mevalonolactone. Sensitivity of our counting equipment permitted the detection of less than 2% of the emerging radioactivity in a single peak. In cases in which internal normalization cannot be used due to selective adsorption of labeled compounds to the column, the acidified samples can be extracted with petroleum ether prior to diethyl ether extraction (13, 14). Ascending paper chromatography of the extract in a n-propanol/ammonia/water (60:20: 20) system followed by radiochromatographic scanning on a Packard model 7201 scanner also demonstrated no other contaminating component to be present until 90% of the R-MVA had been metabolized. The measure of the 14C content of the diethyl ether extract under the conditions of the assay was then concluded to represent the nonmetabolized MVA. It was necessary to correlate the disappearance of MVA with the appearance of products resulting from metabolism through the kinase. An incubate in which 32% of the RS-MVA had metabolized was subjected to the following procedures:
134
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TIME (min) FIG. 1. Stereospecificity of MVA kinase for one enantiomer of RS-MVA-2-%. 3.0 ml aliquots of the 40,OOOg supernatant were incubated with 159 mamoles of the racemic substrate as described in the text. Disappearance was followed versus incubation period.
(a) DEAE-cellulose chromatography under conditions that separated MVA from the other radioactive components (19). These metabolites were subjected to paper chromatography in three systems: n-propanol/ ammonia/water (60: 20: 20) (15) ; tert-butanol/formic acid/water (40 : 10: 16) (3)) and n-butanol/formic acid/water (77: 10: 13) (2,20). (5) Dowex-formate chromatography of the acid-stable intermediates (3, 21). (c) Acid hydrolysis and evaporation of volatile compounds for determination of ally1 derivatives (11). By these methods the distribution of radioactivity in the incubate was found to be: MVA (68%), MVAP (6-7%), MVAPP (9-10%/o), IPP (4-5%), DMAPP (11%) and IP (~1%). No higher sally1 phosphates or pyrophosphates beyond dimethylallyl pyrophosphate were observed. By these procedures we were able to account for all of the radioactivity in the incubate as products resulting from metabolism of the MVA through MVA kinase. If phosphatase activity is significant the levels of mevalonolactone recovered in the assay might be higher than in the absence of such activity. Consequently kinase activity will appear to be lower than the true value. Conditions should be sought to minimize this activity (duration of incubation, protein concentration, etc.). Under the MVA kinase assay conditions, we have already demonstrated that prenols arising from hy-
MEVALONATE
KINASE
135
ASSAY
drolysis of the corresponding pyrophosphates do not accumulate in the diethyl ether extract. Consequently, for phosphatase to interfere with the assay, only the hydrolysis of MVAP back to MVA need be considered. In a separate study we measured the specific activity of phosphomevalonate kinase in the pea seed extract according to the method of Tchen (11). An excess of MVAP was then added to samples of this extract, to test for phosphatase interference over different periods of time. The protein concentration used was the same as that for the MVA kinase assay. Paper chromatographic analysis of the incubation products in n-propanol/ ammonia/water (60 : 20: 20) revealed that no MVA accumulated during an incubation time three times the duration of the routine MVA kinase assay. First-order binding of MVA to protein would be indistinguishable from mevalonate kinase activity. The association of MVA with protein may take place during the enzymic incubation period and/or during the isolation process following termination of the assay. In either case an anomalously high MVA kinase activity would be measured. Binding under the conditions of isolation could conceivably arise by acid-catalyzed esterification of mevalonic acid to a number of residues on protein (ser, thr, asp, glu, etc.). That this does not occur is evidenced by complete recovery of mevalonolactone at zero time incubation (Fig. 2). The absence of any significant binding during incubation was shown by incubating a typical enzyme preparation for approximately 10 min with a known amount of MVA, and determining the proportion of W recoverable by dialysis. According to the lactone assay, 10 min is sufficient time to convert 92% of the R-MVA into products (Fig. 1). Even under very mild conditi,ons of termination with 0.1 M EDTA and subsequent dialysis, essentially all of the W was recovered in the dialyzate (Table 1). From these results, it appears that binding of MVA to protein does not interfere with the kinase assay. In the present study, stereospecificity for the substrate was observed, Influence Initia,l MVA-2-W X 10e6 dpm 0.706
of Protein
TABLE 1 Binding on Recovery Nondialyzable Wb X 10e6 dpm 0.016
of Radioactivity” y0 dialyzed 1% 98
a Refers to dialysable 1°C from 10 min incubation in presence of MVA-2-W. b Label remaining in dialysis bag after 18 hr dialysis against several changes of water with intermittent changes against saturated NaCl solution. The incubated sample was terminated with 3.0 ml 0.1 M EDTA instead of KOH solution.
136
GREEN AND BAISTED
although the actual enantiomer metabolized was not determined. Metabolism of MVA ceased when 50% of the racemic substrate initially added had been consumed (Fig. 1j . A number of authors have isolated mevalonate kinase from various plant and animal sources and have shown it to be stereospecific for the phosphorylation of racemic MVA (2, 10, 22). The absolute stereochemistry of the utilizable enantiomer has been established as n-mevalonate (23). Accepting the available substrate to be one-half the total added to the enzyme preparation, changes in the concentration of the utilizable entaniomer during the period of the assay can be calculated. The first-order equation used for the initially racemic substrate is:2 log, 0.5&/(/l,
- 0.54
= kt
Figure 2 shows the linear relationship between reaction time and a logarithmic function of substrate disappearance relative to initial substrate, and Figure 3, the linear relationship between the first-order rate constants and enzyme concentration.
TIME
(min)
FIG. 2. First-order kinetic assay of MVA kinsse. Aliquots of the enzyme preparation were incubated as described in Figure 1 and results plotted in terms of the fist-order kinetic equation for racemic substrate.2 SUMMARY
A suitable assay has been developed for accurate measurements of mevalonate kinase in a crude enzyme preparation. The assay is particu* log. 0.5&/(At - 0.5Ao)= log. &a*, o,, = total available substrate at t = 0, at = available substrate remaining at so that the typical ratio of initial to
ObAo/(At -00.5Ao).
where A0= total racemic substrate at t = 0, At = total substrate remaining at time t, and time t; then ao = 0.5A0and at = At -0.5A. final substrate
concentration,
so/at, becomes
MEVALONATE
2 2
0.16-
is z
0.12-
F
0.08 -
2
0.04-
KINASE
ASSAY
137
O%i?G-Yr PROTEIN
(mg/ml)
FIG. 3. Demonstration of linear response of assay to changes in enzyme concentration. Aliquots of enzyme solution were diluted with phosphate buffer to 3.0 ml to give the range of protein concentrations shown. Each assay was conducted with 159 mpmoles MVAS-“C for 2.0 min. Corresponding first-order rate constants were calculated and plotted against enzyme concentration.
larly useful where comparative, tissue-specific, or developmental studies are to be made on mevalonate kinase. We have found this method of assay of mevalonate kinase to be particularly suitable for our own studies of kinase activity in crude homogenates of germinated pea seeds. In the present study, the specific activity of the kinase was calculated to be 0.4 mpmole R-MVA min-l mg-I. This represents a lower limit for the activity due to the first-order conditions used for the assay. As a general assay for the kinase, the procedure seems promising in terms of accuracy, simplicity, and labor. ACKNOWLEDGMENTS This work was supported by a Research Grant AM 09265 from the National Institutes of Health and was conducted during the tenure of a Research Career Development Award held by D.J.B. One of us (T.R.G.) is supported by an NDEA fellowship. This work forms part of the thesis requirement for the Ph.D. at Oregon State University. REFERENCES 19, 168, 201 (1965).
1. CLAYTON, R. B., Quart. Rev. 2. TCHEN, T. T., J. Biol. Chem. 233, 1100 (1958). 3. BLOCH, K., CHAYKIN, S., PHILIPS, A. H., AND DE WAARD, A., J. Biol. Chem. 234, 2595 (1959). 4. LYNEN, F., AGRANOFF, B. W., EQCERER, H., HENNING, U., AND MOSLEIN, E. M., Angew. Chem. 71, 657 (1959). 5. AGRANOFF, B., EGGQIEB, H., HENNING, U., AND LYNEN, F., J. Biol. Chem. 235, 326 (1960).
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6. KIRSCHNEB, K., PhD. thesis, Ludwig-Maximiliano-UniversitLt, Munich, Germany, 1961. 7. RICHARDS, J. H., AND HENDRICKSON, J. W., in “The Biosynthesis of Steroids, Terpenes and Acetogenins.” Benjamin, New York, 1964. S. LEVY, H. R., AND POPJAK, G., B&hem. J. 75,417 (1960). 9. MARKLEY, K., AND SMALLMAN, E., Biochim. Biophys. Acta 47, 327 (1961). 10. LOOMIS, W. D., AND BATTAILE, J., Biochim. Biophys. Acta 67, 54 (1963). 11. TCHEN, T. T., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. V, p. 489. Academic Press, New York, 1962. 12. DEWAARD, A., AND POPJAK, G., Biochem. J. 73,410 (1959). 13. POPJAK, G., in “Methods of Enzymology” (R. B. Clayton, ed.), Vol. 15, p. 393. Academic Press, New York, 1969. 14. PORTER, J. W., AND GUCHHAIT, R. B., Anal. Biochem. 15, 509 (1966). 15. SHAH, D. H., CLELAND, W. W., AND PORTER, J. W., J. Bid. Chem. 240, 1946 (1965). 16. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem. 177, 751 ( 1949). 17. BRAY, G. A., Anal. Biochem. 1,279 (1960). 18. HORNING, E. C., VANDENHEUVEL, W. J. A., AND CREECH, B. G., in “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. 11, p. 69. Interscience, New York, 1963. 19. DUGAN, R. E., RASSON, E., AND PORTER, J. W., Anal. Biochem. 22, 249 (1968). 20. WITTING, L. A., AND PORTER, J. W., .I. BioZ. Chem. 234, 2841 (1959). 21. HURLBERT, R. B., SCIIMITZ, H., BRUM, A. F., AND POTTER, V. R., J. BioZ. Chem. 209, 23 (1954). 22. FOLKERS, K., AND WAGNER, A. F., in “Advances in Enzymology” (F. F. Nord, ed.), Vol. 23, p. 471. Interscience, New York, 1961. 23. CORNFORTH, R. H., FLETCHER, K., HELLIG, H., AND POPJAK, G., Nature 185, 923 (1960).