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A simple method to purify ribonucleotide reductase
ANALYTICAL
BIOCHEMISTRY
l%i,467-470
(1983)
A Simple Method to Purify Ribonucleotide THOMASSPECTOR Wellcome
Resenrch
Laboratories,
Reductase
AND DEVRON R. AVERETT Research
Triangle
Park,
North
Carolina
27709
Received May 4, 1983 Assays of ribonucleotide reductase in extracts of Detroit 98 (human) cells were found to be complicated by the rapid depletion of the substrate (CDP) by nucleoside diphosphate kinase. Assays of either lOO,OOOgsupematants or ammonium sulfate-fractionated extracts resulted in the conversion of >90% of the substrate to CTP within 2 min. It was therefore desirable to separate nucleoside diphosphate kinase from ribonucleotide reductase. Chromatography of the fractionated extract on an ATP-agarose column resulted in the delivery of nondissociated ribonucleotide reductase in the void volume and the retention of >99.9% of the nucleoside diphosphate kinase. The kinase could be eluted by 2 mM ATP. The ribonucleotide reductase was recovered from this commercially available gel with an apparent yield of >200%. It could be accurately assayed with only minimal extraneous depletion of substrate. Furthermore, it was stable to storage at -80°C. Tris-HCl was found to inhibit the enzyme. When HEPES (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid)-Na buffer was used in place of Tris-HCl, the rate of CDP reduction was increased by 2.5-fold. Since the above procedure selectively removes nucleoside diphosphate kinase from crude preparations of ribonucleotide reductase, it should have general applicability for purifying ribonucleotide reductase from other sources.
The close association between nucleoside diphosphate (NDP)’ kinase (EC 2.7.4.6) and ribonucleotide reductase (EC 1.17.4.1) ( l-4) has been a problem to investigators attempting to study the latter enzyme. In the presence of Mg+‘, NDP kinase catalyzes the transfer of phosphate from a nucleoside triphosphate donor to a nucleoside diphosphate acceptor. The problem arises because ribonucleotide reductase catalyzes the convemion of ribonucleoside diphosphates to 2’deoxyribonucleoside diphosphates and usually requires the presence of Mg+’ and a nucleoside triphosphate activator. Thus, the donor, the acceptor, and the Mgf2 requirements of NDP kinase are all satisfied by the components of the ribonucleotide reductase assay mixture. Furthermore, the apparent levels of NDP kinase (5-7) in mammalian cell extracts are 103- to 106-fold higher
’ Abbreviations used: NDP, nucleoside diphosphate; DTT, dithiothreitol; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
than those of ribonucleotide reductase (8- 14). The net result is the very rapid conversion of the radiolabeled substrate of ribonucleotide reductase to a nonsubstrate nucleoside triphosphate and the concomitant degradation of the nucleoside triphosphate activator to form an unlabeled nucleoside diphosphate that may compete for reduction. Therefore, if meaningful assays of ribonucleotide reductase are to be obtained, it is imperative that the NDP kinase be removed from the preparation. Previously, this purification has required extensive procedures that significantly decrease the yields and often dissociate the reductase into subunits ( 11,13,15. 16). We recently described a single-step affinity-chromatographic procedure that selectively adsorbed NDP kinase and CDP phosphatase from crude preparations of the ribonucleotide reductase that is induced by herpes simplex virus (17). Presently, the same procedure was used to purify eucaryotic ribonudeotide reductase. It resulted in very high recoveries of nondissociated reductase which is purified to 467
0003-2697183 $3.00 Copyright (c‘) 1981 by Academic Prcsr. Inc. All nghts of reproductmn ,n an\ form rewrved
468
SPECTOR
AND
a state that allows accurate assaysof nucleotide reduction. This method therefore promises to have general applicability. MATERIALS
AND
METHODS
Materials. [14C]CDP (450 Ci/mol) was purchased from New England Nuclear. It was further purified as described elsewhere (17). ATP-agarose Type 3 (C-8 linked) athnity resin was purchased from P-L Biochemicals. Ribonucleotide reductase assays.The standard 0.05-ml reaction mixture for ribonucleotide reductase contained 0.1 mrvi [ 14C]CDP (0.09 &i), 4.5 mM ATP, 5.5 mM MgClz, 5 mM DTT, 100 mM Tris-HCl, pH 7.5, and enzyme. The reaction mixtures were incubated at 37°C for 20 min and the reactions were terminated by the addition of 10 ~1 of 100 mM hydroxyurea and 60 mM EDTA ( 17), followed by incubating at 100°C for 4 min. Product formation was determined by the modified Dowex- 1-borate method previously described (17). One unit of enzyme catalyzes the reduction of 1 nmol GDP/h. NDP kinase assays. The fractions containing ribonucleotide reductase were assayed for NDP kinase radiochemically. The reaction mixture described for the [14C]CDP reductase assay was used. The product, [14C]CTP, was separated from the substrate by the thin-layer chromatographic system previously described (17). NDP kinase assays of all other fractions were assayed spectrophotometrically according to the coupled-enzyme method of Cheng et al. (18). The blank rate obtained with NDP kinase omitted and dTDP present was subtracted from the rates of the complete reaction. The blank rates obtained with NDP kinase present and dTDP omitted were negligible. Distribution ofthe radioactivity derivedfrom
[ 14C]CDP. Prior to the snake venom-digestion step, samples of the ribonucleotide reductase reaction mixtures were analyzed for the extraneous diversion of CDP as previously described ( 17). Cell culture and extract preparation. Detroit 98 cells (human sternal marrow) were grown
AVERETT
in monolayers in Eagle’s minimum essential medium with Earle’s salts (Gibco) which was supplemented with 10% heat-inactivated horse serum and 0.29 mg glutamine/ml. The exponentially growing cells were washed with phosphate-buffered saline, scraped from the flasks, and collected by centrifugation at 8OOg. The cells were stored at -80°C until use. Six milliliters of packed cells were thawed at 4°C by the addition of 3 ml of 100 mM Hepes-Na buffer, pH 7.6, containing 2 mM DTT and 2 mM MgCl*, and then lysed with a Dounce homogenizer. The lysates were centrifuged at 100,OOOg for 90 min. Ribonucleotide reductase was precipitated from the supematant by the addition of 0.29 g ammonium sulfate/ml of solution (50% saturation). The precipitate was collected by centrifugation, dissolved in a minimal volume of 20 mM Tris-HCl buffer, pH 7.6, containing 2 mM DTT and 2 mM MgCl* (Buffer A), and then dialyzed for 2 h against two l-liter changes of the same buffer. The retentate was clarified by centrifugation and chromatographed on the ATP-agarose affinity column as described in the text. RESULTS
AND
DISCUSSION
Eucaryotic ribonucleotide reductase. The ammonium sulfate-fractionated extract (see Materials and Methods) of the Detroit 98 cells was chromatographed on the ATP-agarose affinity column. The ribonucleotide reductase was eluted as a symmetrical peak in the void volume and was not detected in the other fractions (Fig. 1). Approximately 10 units (17 mg protein) of reductase were applied to the column and 22 units were recovered. Although this apparent recovery of 220% was much higher than yields obtained by other methods (11,13,15,16), it was not an accurate estimation due to the probable underestimation of the ribonucleotide reductase (1) in the sample applied to the column. The ribonucleotide reductase in the peak fractions was purified 3.4-fold by this chromatography step and a total of 9. l-fold overall. The enzyme was
PURIFICATION
OF RIBONUCLEOTIDE
REDUCTASE
469
I\ i
8.0
FRACTION
FIG. 1. The ammonium sulfate-fractionated eucaryotic ribonucleotide reductase (0.6 ml) was applied to the 0.7 X 14-cm ATP affinity column (equilibrated in Buffer A) at a rate of 3 ml/h. The column was then washed with the same buffer. The fraction size was 1.0 ml. At fraction 15, the rate was increased to 14 ml/h and the fraction size to 2.4 ml. A buffer of 20 mM Tris-HCl, pH 7.5, containing the reagents indicated at the arrows was used to elute fractions 15-30.
stored at -80°C and retained >95% of its activity for at least 1 month. It catalyzed the reduction of CDP at a rate that was constant for at least 20 min when assayed at a concentration of 5.6 units/ml. HPLC analysis (17) of the dephosphorylated reaction product confirmed that 2’-deoxycytidine and not cytosine was actually being measured. It became apparent that the 100 mM TrisHCl buffer used in the assay mixtures was inhibiting the enzyme. When the buffer was switched to either 50 or 150 mM Hepes-Na, the reaction rate increased by 2.5-fold. Inhibition by Tris-HCl (probably caused by the Cl- anion) was previously observed in studies of the ribonucleotide reductase which is induced by herpes simplex virus (17). It may be prudent to avoid the use of this buffer in all ribonucleotide reductase assays. The ribonucleotide reductase reaction mixtures were analyzed for possible extraneous depletion of the [‘4C]CDP substrate. Within
2 min, the CDP content of the reaction mixtures containing either the 100,OOOg supernatant or the ammonium sulfate fraction was reduced to 8% of its original concentration and remained at this level for the remaining 18 min of the reaction. The CDP was converted mainly to CTP. A trace amount of cytidine was also formed. Because the concentration of CDP was rapidly depleted and then remained at a low, but relatively constant, level, the rate of ribonucleotide reduction in these fractions appeared to be linear with respect to time. However, this rate was probably significantly lower than would be expected if the CDP had not been depleted and ADP had not been concomitantly generated ( 1). As noted above, the removal of NDP kinase resulted in an apparent 220% increase in the amount of CDP reductase. The measurement of substrate stability is thus a valuable adjunct to linearity measurements. In contrast to the results with the crude
470
SPECTOR AND AVERETT
fractions, the CDP substrate was considerably less depleted in reaction mixtures containing ribonucleotide reductase which had been chromatographed on the ATP column. After the 20-min reaction, 92-97% of the [14C]CDP was recovered intact when 10 ~1 of the ribonucleotide reductase-containing fractions were assayed. The recovery was 82-91% when 30 ~1 was assayed. The radiolabel that was diverted was distributed between CTP, CMP, and @dine. NDP kinase. NDP kinase was retained on the affinity column. It was not eluted by 0.5 M KCl, but was eluted by 2 mM ATP. Although approximately 90% of the total NDP kinase was separated from the reductase by ammonium sulfate fractionation prior to chromatography, the level of NDP kinase in the sample applied to the column was still four orders of magnitude greater than the level of reductase. It can be seen in Fig. 1 that greater than 99.9% ofthe NDP kinase recovered from the affinity column was well separated from the ribonucleotide reductase fractions. However, traces of NDP kinase did coelute with the reductase. Even though the contaminating kinase was less than 0.1% of the total, its level was of similar magnitude as the level of reductase. This undoubtedly contributed to the small depletion of CDP in the reductase assay mixtures. In conclusion, this simple chromatographic procedure removes the vast majority of NDP kinase, an enzyme that would significantly deplete the concentration of CDP. It therefore provides preparations of ribonucleotide reductase which can be accurately assayed.
ACKNOWLEDGMENT The authors gratefully appreciate N. K. Cohn for providing the Detroit 98 cells and T. E. Jones for performing some of the assaysof this study.
REFERENCES 1. Ikenaka, K., Fukushima, M., Shirasaka, T., and Fujii, S. (1981) Gann 72, 8-18. 2. von Diibeln, U. ( 1976) Biochem. Eiophys. Rex Commun. 72, 1160-I 168. 3. Reddy, G. P. V., and Pardee, A. B. ( 1980) Proc. Nufl. Acad. Sci. USA 77, 3312-3316. 4. Holmgren, A. (198 1) Curr. Top. Cell Regal. 19,4776. 5. Parks, R. E., Jr., and Agarwal, R. P. (1973) in The Enzymes (Bayer, P. D., ed.), Vol. 8, pp. 307-333, Academic Press, New York. 6. Colomb, M. G., Cheruy, A., and Vignais, P. V. (1972) Biochemistry 11, 3370-3378. 7. Lascu, I., Due, M., and Cristea, A. (198 1) Anal. Biochem. 113, 207-2 11. 8. Moore, E. C. (1977) Advan. Enz. Reg. 15, 101-114. 9. Larsson, A. (1969) Eur. J. Biochem. 11, 113-121. 10. Hopper, S. ( 1978) in Methods in Enzymology (Ho&e., P. A., and Jones, M. E., eds.), Vol. 51, pp. 237246, Academic Press, New York. 11. Engstriim, Y., Eriksson, S., Thelander, L., and Akerman, M. (1979) Biochemistry 18, 2941-2948. 12. Cory, J. G., and Mansell, M. M. (1975) Cancer Rex 35,2327-233 1. 13. Chang, C.-H., and Cheng, Y.-C. (1979) Cancer Res. 39,436-442. 14. Takeda, E., and Weber, G. (198 1) Life Sci. 28, 10071014. 15. Thelander, L., Eriksson, S., and Akerman, M. (1980) J. Biol. Chem. 255, 7426-7432. 16. Thelander, L., and Reichard, P. (1979) Annu. Rev. Biochem. 48, 133-158. 17. Averett, D. R., Lubbers, C. A., Elion, G. B., and Spector, T. (1983) J. Biol. Chem. 258,983 l-9838. 18. Cheng, Y.-C., Agarwal, R. P., and Parks, R. E., Jr. (1971) Biochemistry 10, 2139-2143.
BIOCHEMISTRY
l%i,467-470
(1983)
A Simple Method to Purify Ribonucleotide THOMASSPECTOR Wellcome
Resenrch
Laboratories,
Reductase
AND DEVRON R. AVERETT Research
Triangle
Park,
North
Carolina
27709
Received May 4, 1983 Assays of ribonucleotide reductase in extracts of Detroit 98 (human) cells were found to be complicated by the rapid depletion of the substrate (CDP) by nucleoside diphosphate kinase. Assays of either lOO,OOOgsupematants or ammonium sulfate-fractionated extracts resulted in the conversion of >90% of the substrate to CTP within 2 min. It was therefore desirable to separate nucleoside diphosphate kinase from ribonucleotide reductase. Chromatography of the fractionated extract on an ATP-agarose column resulted in the delivery of nondissociated ribonucleotide reductase in the void volume and the retention of >99.9% of the nucleoside diphosphate kinase. The kinase could be eluted by 2 mM ATP. The ribonucleotide reductase was recovered from this commercially available gel with an apparent yield of >200%. It could be accurately assayed with only minimal extraneous depletion of substrate. Furthermore, it was stable to storage at -80°C. Tris-HCl was found to inhibit the enzyme. When HEPES (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid)-Na buffer was used in place of Tris-HCl, the rate of CDP reduction was increased by 2.5-fold. Since the above procedure selectively removes nucleoside diphosphate kinase from crude preparations of ribonucleotide reductase, it should have general applicability for purifying ribonucleotide reductase from other sources.
The close association between nucleoside diphosphate (NDP)’ kinase (EC 2.7.4.6) and ribonucleotide reductase (EC 1.17.4.1) ( l-4) has been a problem to investigators attempting to study the latter enzyme. In the presence of Mg+‘, NDP kinase catalyzes the transfer of phosphate from a nucleoside triphosphate donor to a nucleoside diphosphate acceptor. The problem arises because ribonucleotide reductase catalyzes the convemion of ribonucleoside diphosphates to 2’deoxyribonucleoside diphosphates and usually requires the presence of Mg+’ and a nucleoside triphosphate activator. Thus, the donor, the acceptor, and the Mgf2 requirements of NDP kinase are all satisfied by the components of the ribonucleotide reductase assay mixture. Furthermore, the apparent levels of NDP kinase (5-7) in mammalian cell extracts are 103- to 106-fold higher
’ Abbreviations used: NDP, nucleoside diphosphate; DTT, dithiothreitol; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
than those of ribonucleotide reductase (8- 14). The net result is the very rapid conversion of the radiolabeled substrate of ribonucleotide reductase to a nonsubstrate nucleoside triphosphate and the concomitant degradation of the nucleoside triphosphate activator to form an unlabeled nucleoside diphosphate that may compete for reduction. Therefore, if meaningful assays of ribonucleotide reductase are to be obtained, it is imperative that the NDP kinase be removed from the preparation. Previously, this purification has required extensive procedures that significantly decrease the yields and often dissociate the reductase into subunits ( 11,13,15. 16). We recently described a single-step affinity-chromatographic procedure that selectively adsorbed NDP kinase and CDP phosphatase from crude preparations of the ribonucleotide reductase that is induced by herpes simplex virus (17). Presently, the same procedure was used to purify eucaryotic ribonudeotide reductase. It resulted in very high recoveries of nondissociated reductase which is purified to 467
0003-2697183 $3.00 Copyright (c‘) 1981 by Academic Prcsr. Inc. All nghts of reproductmn ,n an\ form rewrved
468
SPECTOR
AND
a state that allows accurate assaysof nucleotide reduction. This method therefore promises to have general applicability. MATERIALS
AND
METHODS
Materials. [14C]CDP (450 Ci/mol) was purchased from New England Nuclear. It was further purified as described elsewhere (17). ATP-agarose Type 3 (C-8 linked) athnity resin was purchased from P-L Biochemicals. Ribonucleotide reductase assays.The standard 0.05-ml reaction mixture for ribonucleotide reductase contained 0.1 mrvi [ 14C]CDP (0.09 &i), 4.5 mM ATP, 5.5 mM MgClz, 5 mM DTT, 100 mM Tris-HCl, pH 7.5, and enzyme. The reaction mixtures were incubated at 37°C for 20 min and the reactions were terminated by the addition of 10 ~1 of 100 mM hydroxyurea and 60 mM EDTA ( 17), followed by incubating at 100°C for 4 min. Product formation was determined by the modified Dowex- 1-borate method previously described (17). One unit of enzyme catalyzes the reduction of 1 nmol GDP/h. NDP kinase assays. The fractions containing ribonucleotide reductase were assayed for NDP kinase radiochemically. The reaction mixture described for the [14C]CDP reductase assay was used. The product, [14C]CTP, was separated from the substrate by the thin-layer chromatographic system previously described (17). NDP kinase assays of all other fractions were assayed spectrophotometrically according to the coupled-enzyme method of Cheng et al. (18). The blank rate obtained with NDP kinase omitted and dTDP present was subtracted from the rates of the complete reaction. The blank rates obtained with NDP kinase present and dTDP omitted were negligible. Distribution ofthe radioactivity derivedfrom
[ 14C]CDP. Prior to the snake venom-digestion step, samples of the ribonucleotide reductase reaction mixtures were analyzed for the extraneous diversion of CDP as previously described ( 17). Cell culture and extract preparation. Detroit 98 cells (human sternal marrow) were grown
AVERETT
in monolayers in Eagle’s minimum essential medium with Earle’s salts (Gibco) which was supplemented with 10% heat-inactivated horse serum and 0.29 mg glutamine/ml. The exponentially growing cells were washed with phosphate-buffered saline, scraped from the flasks, and collected by centrifugation at 8OOg. The cells were stored at -80°C until use. Six milliliters of packed cells were thawed at 4°C by the addition of 3 ml of 100 mM Hepes-Na buffer, pH 7.6, containing 2 mM DTT and 2 mM MgCl*, and then lysed with a Dounce homogenizer. The lysates were centrifuged at 100,OOOg for 90 min. Ribonucleotide reductase was precipitated from the supematant by the addition of 0.29 g ammonium sulfate/ml of solution (50% saturation). The precipitate was collected by centrifugation, dissolved in a minimal volume of 20 mM Tris-HCl buffer, pH 7.6, containing 2 mM DTT and 2 mM MgCl* (Buffer A), and then dialyzed for 2 h against two l-liter changes of the same buffer. The retentate was clarified by centrifugation and chromatographed on the ATP-agarose affinity column as described in the text. RESULTS
AND
DISCUSSION
Eucaryotic ribonucleotide reductase. The ammonium sulfate-fractionated extract (see Materials and Methods) of the Detroit 98 cells was chromatographed on the ATP-agarose affinity column. The ribonucleotide reductase was eluted as a symmetrical peak in the void volume and was not detected in the other fractions (Fig. 1). Approximately 10 units (17 mg protein) of reductase were applied to the column and 22 units were recovered. Although this apparent recovery of 220% was much higher than yields obtained by other methods (11,13,15,16), it was not an accurate estimation due to the probable underestimation of the ribonucleotide reductase (1) in the sample applied to the column. The ribonucleotide reductase in the peak fractions was purified 3.4-fold by this chromatography step and a total of 9. l-fold overall. The enzyme was
PURIFICATION
OF RIBONUCLEOTIDE
REDUCTASE
469
I\ i
8.0
FRACTION
FIG. 1. The ammonium sulfate-fractionated eucaryotic ribonucleotide reductase (0.6 ml) was applied to the 0.7 X 14-cm ATP affinity column (equilibrated in Buffer A) at a rate of 3 ml/h. The column was then washed with the same buffer. The fraction size was 1.0 ml. At fraction 15, the rate was increased to 14 ml/h and the fraction size to 2.4 ml. A buffer of 20 mM Tris-HCl, pH 7.5, containing the reagents indicated at the arrows was used to elute fractions 15-30.
stored at -80°C and retained >95% of its activity for at least 1 month. It catalyzed the reduction of CDP at a rate that was constant for at least 20 min when assayed at a concentration of 5.6 units/ml. HPLC analysis (17) of the dephosphorylated reaction product confirmed that 2’-deoxycytidine and not cytosine was actually being measured. It became apparent that the 100 mM TrisHCl buffer used in the assay mixtures was inhibiting the enzyme. When the buffer was switched to either 50 or 150 mM Hepes-Na, the reaction rate increased by 2.5-fold. Inhibition by Tris-HCl (probably caused by the Cl- anion) was previously observed in studies of the ribonucleotide reductase which is induced by herpes simplex virus (17). It may be prudent to avoid the use of this buffer in all ribonucleotide reductase assays. The ribonucleotide reductase reaction mixtures were analyzed for possible extraneous depletion of the [‘4C]CDP substrate. Within
2 min, the CDP content of the reaction mixtures containing either the 100,OOOg supernatant or the ammonium sulfate fraction was reduced to 8% of its original concentration and remained at this level for the remaining 18 min of the reaction. The CDP was converted mainly to CTP. A trace amount of cytidine was also formed. Because the concentration of CDP was rapidly depleted and then remained at a low, but relatively constant, level, the rate of ribonucleotide reduction in these fractions appeared to be linear with respect to time. However, this rate was probably significantly lower than would be expected if the CDP had not been depleted and ADP had not been concomitantly generated ( 1). As noted above, the removal of NDP kinase resulted in an apparent 220% increase in the amount of CDP reductase. The measurement of substrate stability is thus a valuable adjunct to linearity measurements. In contrast to the results with the crude
470
SPECTOR AND AVERETT
fractions, the CDP substrate was considerably less depleted in reaction mixtures containing ribonucleotide reductase which had been chromatographed on the ATP column. After the 20-min reaction, 92-97% of the [14C]CDP was recovered intact when 10 ~1 of the ribonucleotide reductase-containing fractions were assayed. The recovery was 82-91% when 30 ~1 was assayed. The radiolabel that was diverted was distributed between CTP, CMP, and @dine. NDP kinase. NDP kinase was retained on the affinity column. It was not eluted by 0.5 M KCl, but was eluted by 2 mM ATP. Although approximately 90% of the total NDP kinase was separated from the reductase by ammonium sulfate fractionation prior to chromatography, the level of NDP kinase in the sample applied to the column was still four orders of magnitude greater than the level of reductase. It can be seen in Fig. 1 that greater than 99.9% ofthe NDP kinase recovered from the affinity column was well separated from the ribonucleotide reductase fractions. However, traces of NDP kinase did coelute with the reductase. Even though the contaminating kinase was less than 0.1% of the total, its level was of similar magnitude as the level of reductase. This undoubtedly contributed to the small depletion of CDP in the reductase assay mixtures. In conclusion, this simple chromatographic procedure removes the vast majority of NDP kinase, an enzyme that would significantly deplete the concentration of CDP. It therefore provides preparations of ribonucleotide reductase which can be accurately assayed.
ACKNOWLEDGMENT The authors gratefully appreciate N. K. Cohn for providing the Detroit 98 cells and T. E. Jones for performing some of the assaysof this study.
REFERENCES 1. Ikenaka, K., Fukushima, M., Shirasaka, T., and Fujii, S. (1981) Gann 72, 8-18. 2. von Diibeln, U. ( 1976) Biochem. Eiophys. Rex Commun. 72, 1160-I 168. 3. Reddy, G. P. V., and Pardee, A. B. ( 1980) Proc. Nufl. Acad. Sci. USA 77, 3312-3316. 4. Holmgren, A. (198 1) Curr. Top. Cell Regal. 19,4776. 5. Parks, R. E., Jr., and Agarwal, R. P. (1973) in The Enzymes (Bayer, P. D., ed.), Vol. 8, pp. 307-333, Academic Press, New York. 6. Colomb, M. G., Cheruy, A., and Vignais, P. V. (1972) Biochemistry 11, 3370-3378. 7. Lascu, I., Due, M., and Cristea, A. (198 1) Anal. Biochem. 113, 207-2 11. 8. Moore, E. C. (1977) Advan. Enz. Reg. 15, 101-114. 9. Larsson, A. (1969) Eur. J. Biochem. 11, 113-121. 10. Hopper, S. ( 1978) in Methods in Enzymology (Ho&e., P. A., and Jones, M. E., eds.), Vol. 51, pp. 237246, Academic Press, New York. 11. Engstriim, Y., Eriksson, S., Thelander, L., and Akerman, M. (1979) Biochemistry 18, 2941-2948. 12. Cory, J. G., and Mansell, M. M. (1975) Cancer Rex 35,2327-233 1. 13. Chang, C.-H., and Cheng, Y.-C. (1979) Cancer Res. 39,436-442. 14. Takeda, E., and Weber, G. (198 1) Life Sci. 28, 10071014. 15. Thelander, L., Eriksson, S., and Akerman, M. (1980) J. Biol. Chem. 255, 7426-7432. 16. Thelander, L., and Reichard, P. (1979) Annu. Rev. Biochem. 48, 133-158. 17. Averett, D. R., Lubbers, C. A., Elion, G. B., and Spector, T. (1983) J. Biol. Chem. 258,983 l-9838. 18. Cheng, Y.-C., Agarwal, R. P., and Parks, R. E., Jr. (1971) Biochemistry 10, 2139-2143.