Vol. 128, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Nov. 1976, p. 673-676 Copyright X) 1976 American Society for Microbiology
Ribonucleotide Reductase Activity in Ether-Treated Cells of Agmenellum quadruplicatum FLORENCE K. GLEASON* AND JOHN M. WOOD Freshwater Biological Institute, University of Minnesota, Navarre, Minnesota 55392
Received for publication 12 May 1976
Ribonucleotide (cytidine 5'-diphosphate) reductase activity can be detected in situ in cells of the blue-green alga Agmenellum quadruplicatum, strain PR-6, after the cells are made permeable by treatment with ether. The Agmenellum reductase resembles the enzyme from Escherichia coli.
Ribonucleotide reductase catalyzes the reduction of the four ribonucleotides to the corresponding deoxyribonucleotides. In most organisms, this reaction is the chief source of deoxyribonucleotides. Thus, the enzyme plays a key role in deoxyribonucleic acid (DNA) synthesis and subsequent cell division. There are two known types of ribonucleotide reductase which differ principally in their cofactor requirements. In Escherichia coli the ribonucleotide reductase is a nonheme iron-containing protein which requires adenosine 5'-triphosphate (ATP) and Mg2+ as cofactors. It reduces ribonucleoside diphosphates (13). Similar enzymes have been described in yeast (14) and mammalian tissues (8, 11). The enzyme isolated from Lactobacillus leichmannii uses ribonucleoside triphosphates as substrates and adenosylcobalamin as cofactor (2). Published reports indicate that the cobalamin-dependent reductase is probably more common among bacteria (5). In this paper we describe some of the properties of the ribonucleotide reductase in the blue-green alga (bacterium) Agmenellum quadruplicatum. Enzyme activity was assayed in situ in ethertreated cells and resembles the reductase from E. coli. A. quadruplicatum strain PR-6 was obtained in axenic culture from C. Van Baalen (The University of Texas, Marine Science Institute, Port Aransas, Tex.). The organism was maintained at room temperature in liquid medium, ASP-2 (12). Cells used for enzyme assays were grown in the same medium supplemented with 1 g of Na2CO3 per liter. A gas mixture consisting of 5% Co2 in N2 was bubbled through the medium. Cells were grown in continuous light from Sylvania cool white lamps at an intensity of 670 /iEinsteins/m2 per s. Culture growth was monitored by direct counting in a Petroff-Hauser counting chamber. All of the following operations were performed at 4°C. Cells were harvested in the late
logarithmic phase by centrifugation for 10 min at 7,000 x g in a Sorvall RC-5 refrigerated centrifuge. Cells were resuspended in the following medium: 0.04 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.4, 0.08 M KCl, 0.007 M magnesium acetate, 0.002 M ethyleneglycol-bis-(,3-aminoethyl ether)N,N'tetraacetic acid, 0.5 M sucrose. The cells were washed once by recentrifuging and resuspended in a small volume of the above medium (1/100 to 1/200 of the original culture volume). The procedure for ether treatment was modified from that used for E. coli as described by Vosberg and Hoffman-Berling (15). An equal volume of ether was added to resuspended cells, and the mixture was rocked gently for 1 min. The cells were then layered onto a sample of the above medium that contained an additional 0.1 g of sucrose per ml. A 4-ml layer was used for each 1 ml of ether-treated cells. The tubes were centrifuged for 10 min at 10,000 x g. The upper layers were removed, and the pelleted cells were resuspended in the same amount of medium as above. Samples were frozen for future assay. Ribonucleotide reductase activity was stable for 2 weeks in frozen, ether-treated cells. Ribonucleotide diphosphate reductase (EC 1.17.4.1) was assayed by a procedure modified from that of Fuchs and Warner (4). The reaction mixture contained 80 mM HEPES (N-2-
hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, pH 8.2, 2.8 mM ATP, 12 mM MgCl2, 2 mM ethylenediaminetetraacetate, 32 mM dithiothreitol, 2.3 mM nicotinamide adenine dinucleotide phosphate (reduced), 6 ,umol of [3H]cytidine 5'-diphosphate (CDP) (specific activity, 0.4 ,Ci/,umol), and cells in a final volume of 0.050 ml. The reaction was initiated by addition of cells to the reaction mixture, and the tubes were incubated at 37°C. The reaction was terminated by boiling the tubes for 2 min. The following additions were then made: 0.002 ml of deoxycytidine 5'-monophosphate (0.1 M), 673
674
NOTES
J. BACTERIOL.
0.002 ml of cytidine 5'-monophosphate (0.025 M), and 0.005 ml of potato apyrase (10 mg/ml). The reaction tubes were incubated at 37°C for an additional 30 min. The apyrase reaction was stopped by boiling the vessels for 2 min. The tubes were then centrifuged at 1,000 x g for 5 min to sediment the cells. A 0.015-ml amount of the supernatant was spotted on a polyethyleneimine thin-layer chromatography plate (Brinkmann Instruments Inc., Westbury, N.Y.). Plates were developed in the following solvent: 6 g of Na2B4O7, 3 g of H3BO3, 30 ml of ethylene glycol, 70 ml of distilled water. Spots of deoxycytidine 5'-monophosphate were cut out of the plate, and the nucleotide was eluted by soaking for 30 min in 1 ml of 0.7 M MgCl2 in 0.02 M
tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.4. Scintillation fluid, 10 ml of PCS solubilizer (Amersham/Searle Corp., Arlington Heights, Ill.), was added, and the samples were counted in a Searle Mark II scintillation spectrometer. Protein was determined on sonicated cell samples (15 min with a Heat Systems sonifier) by the biuret method (6), using bovine serum albumin as a standard. All nucleotides and dithiothreitol were purchased from P. L. Biochemicals, Milwaukee, 0 30 20 10 Wis. [3H]CDP (23 Ci/mmol) was a product of MINUTES Amersham/Searle, Arlington Heights, Ill. FIG. 1. Ribonucleotide reductase reaction in the HEPES and potato apyrase were from the presence of ether-treated cells. Assay conditions are Sigma Chemical Co., St. Louis, Mo. described in the text. Previous results had shown that unlike the majority of blue-green algae, extracts of A. TABLE 1. Requirements for CDP reductase activity quadruplicatum had no ribonucleotide reducReduction tase activity in the exchange reaction with 3HReaction system (nmol of dCDP/mg labeled adenosylcobalamin (5a). Attempts to of cell protein) demonstrate reduction in crude extracts using 81.1 system ........ ..... 3H-labeled CDP were hampered by inadequate Complete 13.8 No incubation ................ separation of substrate and product. Much Boiled cells .................. 9.9 more consistent results were obtained using Omissions. ether-treated cells. After the reaction, the cells ATP ....................... 22.8 30.0 Mg2 ...................... and most of their high-molecular-weight com9.7 ATP, Mg2 . ................. ponents are easily removed from the assay mix5.7 Dithiothreitol ......... ..... ture by centrifugation. The nucleotides in the 41.8 NADPHa ................ . supernatant can then be separated by thin11.0 cells ....................... layer chromatography. Like typical bacterial cells (16), the cyanophyte cells remain intact a NADPH, Nicotinamide adenine dinucleotide after ether treatment but are rendered permea- phosphate, reduced form. ble to nucleotides and other components of the In addition, reaction in ether-treated cells rereductase reaction. Figure 1 shows that ribonucleotide reduction quires a reducing agent, dithiothreitol, and is in the presence of cells is proportional to time; stimulated by nicotinamide adenine dinucleothe reaction is linear up to 30 min at 37°C. tide phosphate, reduced form. The latter is Requirements for CDP reductase activity are probably used to reduce thioredoxin, a small shown in Table 1. Like the E. coli enzyme, sulfhydryl-containing protein which is the ribonucleotide reductase in A. quadruplicatum physiological reducing agent in ribonucleotide requires ATP and Mg2+ for maximal activity. reduction (9).
NOTES
VOL. 128, 1976
675
Table 2 describes the effects of iron-chelating agents on reductase activity. Hydroxyurea and z 80 hydroxylamine are known to inhibit ribonucleotide reduction in E. coli by apparently pre- 0 venting free radical formation at the iron-con- X- 60 -J taining active site of the enzyme (1). These w agents are also effective inhibitors of the reaction inA. quadruplicatum. Thus, the Agmenel"'40 * lum reductase probably contains iron at the E active site. Presumably, DNA synthesis in this organism could also be inhibited by addition of X 20 hydroxyurea. Although it has been reported v) that hydroxyurea is not effective in this capac- 00 ity in Anacystis nidulans (10), recent results in E our laboratory show that this organism con- C n 80 60 40 20 0 tains the cobalamin-dependent reductase dATP (PM) (5a). Thus, hydroxyurea would not be expected to be an effective inhibitor in A. nidulans but FIG. 2. Effect of dATP on ribonucleotide reducshould be in A. quadruplicatum, assuming it is tion. The assay mixture contained all the components incorporated into the cell. listed in the text. Varying amounts of dATP were The ribonucleotide reductase from E. coli is added, and the reaction tubes were incubated at 37°C known to be subject to allosteric regulation by for 30 min. deoxynucleotides, principally dATP (3). dATP has a dual role, being stimulatory at concentrations below 10 ,tM and inhibitory at higher walls, like those of many bacteria, can be made concentrations. The reductase activity in A. permeable by treatment with ether. This techquadruplicatum can also be altered by addition nique can be quite useful for the study of in situ of dATP. The results in Fig. 2 show that unlike enzymatic reactions in these organiisms. the E. coli enzyme, the reaction in the cyanoWe wish to thank H. R. Warner for suggesting the ether phyte is inhibited by all concentrations of dATP treatment and assisting with the assays. This research was supported by Public Health Service tested. The effect of dATP on reductase activity grant 7R01-AM 18101-01 from the National Institute of Arcould explain some of the results obtained by thritis, Metabolism, and Digestive Diseases. Ingram and Fisher (7). These workers described the inhibition of DNA synthesis in A. LITERATURE CITED quadruplicatum by addition of deoxyadenosine 1. Atkin, C. L., L. Thelander, P. Reichard, and G. Lang. to their cultures. We suggest that when it is 1973. Iron and free radical in ribonucleotide reductase. Exchange of iron and Mossbauer spectroscopy of incorporated into the cell, the nucleotide is the protein B2 subunit of the E. coli enzyme. J. Biol. probably readily phosphorylated to the triphosChem. 248:7464-7472. riphate and becomes an effective inhibitor of 2. Blakley, R. L. 1965. Cobamides and ribonucleotide rebonucleotide reduction. According to the data duction. I. Cobamide stimulation of ribonucleotide reduction in extracts of Lactobacillus leichmannii. J. in Fig. 2, at low concentrations the deoxynuBiol. Chem. 240:2173-2180. cleotide would inhibit only slightly and lead to 3. Brown, N. C., Z. N. Canellakis, B. Lundin, P. Reisome slowing of the growth rate. At higher chard, and L. Thelander. 1969. Ribonucleoside diconcentrations, above 50 ,AM, enzyme activity phosphate reductase. Purification of the two subunits, proteins Bi and B2. Eur. J. Biochem. 9:561is less than half-maximal, thus providing little 573. substrate for DNA synthesis and leading to the 4. Fuchs, J. A., and H. R. Warner. 1975. Isolation of an DNA degradation noted by Ingram and Fisher. Escherichia coli mutant deficient in glutathione synThese results show that blue-green algal cell thesis. J. Bacteriol. 124:140-148. -
a-
Hogenkamp. 1972. 5'Deoxyadenosylcobalamin-dependent ribonucleotide
5. Gleason, F. K., and H. P. C.
TABLE 2. Effect of iron-chelating agents on CDP reductase activity Reduction (nmol of dCDP/mg of cell protein) 29.5 Complete system ............. Reaction system
Additions 9 mM hydroxyurea 9 mM hydroxylamine
......... .......
8.2 4.6
reductase: a survey of its distribution. Biochim. Biophys. Acta 277:466-470. 5a.Gleason, F. K., and J. M. Wood. 1976. Ribonucleotide reductase in blue-green algae: dependence on adenosylcobalamin. Science 192:1343-1344. 6. Gornall, A. G., C. J. Bardawill, and M. M. David. 1949. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177:751-766. 7. Ingram, L. O., and W. D. Fisher. 1972. Selective inhibition of deoxyribonucleic acid synthesis by 2'-deoxyadenosine in the blue-green bacterium Agmenellum
676
NOTES
quadruplicatum. J. Bacteriol. 112:170-175. 8. Larsson, A. 1969. Ribonucleotide reductase from regenerating rat liver. Eur. J. Biochem. 11:113-121. 9. Laurent, T. C., E. C. Moore, and P. Reichard. 1964. Enzymatic synthesis of deoxyribonucleotides. IV. Isolation and characterization of thioredoxin, the hydrogen donor from Escherichia coli B. J. Biol. Chem. 239:3436-3444. 10. Madan, V., and H. D. Kumar. 1971. Action of nalidixic acid and hydroxyurea on two blue-green algae. Z. Allg. Mikrobiol. 11:495-499. 11. Moore, E. C., and P. Reichard. 1964. Enzymatic synthesis of deoxyribonucleotides. VI. The cytidine diphosphate reductase system from Novikoffhepatoma. J. Biol. Chem. 239:3453-3456.
J. BACTERIOL.
Provasoli, L., J. J. A. McLaughlin, and M. R. Droop. 1957. The development of artificial media for marine algae. Arch. Mikrobiol. 25:392-428. 13. Reichard, P. 1968. The biosynthesis of deoxyribonucleotides. Eur. J. Biochem. 3:259-266. 14. Vitols, E., V. A. Bauer, and E. C. Stanbrough. 1970. Ribonucleotide reductase from Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 41:71-77. 15. Vosberg, H., and H. Hoffman-Berling. 1971. DNA synthesis in nucleotide-permeable Escherichia coli cells. I. Preparation and properties of ether-treated cells. J. 12.
Mol. Biol. 58:739-753. 16. Warner, H. R. 1973. Properties of ribonucleoside diphosphate reductase in nucleotide-permeable cells. J. Bacteriol. 115:18-22.
JOURNAL OF BACTERIOLOGY, Nov. 1976, p. 673-676 Copyright X) 1976 American Society for Microbiology
Ribonucleotide Reductase Activity in Ether-Treated Cells of Agmenellum quadruplicatum FLORENCE K. GLEASON* AND JOHN M. WOOD Freshwater Biological Institute, University of Minnesota, Navarre, Minnesota 55392
Received for publication 12 May 1976
Ribonucleotide (cytidine 5'-diphosphate) reductase activity can be detected in situ in cells of the blue-green alga Agmenellum quadruplicatum, strain PR-6, after the cells are made permeable by treatment with ether. The Agmenellum reductase resembles the enzyme from Escherichia coli.
Ribonucleotide reductase catalyzes the reduction of the four ribonucleotides to the corresponding deoxyribonucleotides. In most organisms, this reaction is the chief source of deoxyribonucleotides. Thus, the enzyme plays a key role in deoxyribonucleic acid (DNA) synthesis and subsequent cell division. There are two known types of ribonucleotide reductase which differ principally in their cofactor requirements. In Escherichia coli the ribonucleotide reductase is a nonheme iron-containing protein which requires adenosine 5'-triphosphate (ATP) and Mg2+ as cofactors. It reduces ribonucleoside diphosphates (13). Similar enzymes have been described in yeast (14) and mammalian tissues (8, 11). The enzyme isolated from Lactobacillus leichmannii uses ribonucleoside triphosphates as substrates and adenosylcobalamin as cofactor (2). Published reports indicate that the cobalamin-dependent reductase is probably more common among bacteria (5). In this paper we describe some of the properties of the ribonucleotide reductase in the blue-green alga (bacterium) Agmenellum quadruplicatum. Enzyme activity was assayed in situ in ethertreated cells and resembles the reductase from E. coli. A. quadruplicatum strain PR-6 was obtained in axenic culture from C. Van Baalen (The University of Texas, Marine Science Institute, Port Aransas, Tex.). The organism was maintained at room temperature in liquid medium, ASP-2 (12). Cells used for enzyme assays were grown in the same medium supplemented with 1 g of Na2CO3 per liter. A gas mixture consisting of 5% Co2 in N2 was bubbled through the medium. Cells were grown in continuous light from Sylvania cool white lamps at an intensity of 670 /iEinsteins/m2 per s. Culture growth was monitored by direct counting in a Petroff-Hauser counting chamber. All of the following operations were performed at 4°C. Cells were harvested in the late
logarithmic phase by centrifugation for 10 min at 7,000 x g in a Sorvall RC-5 refrigerated centrifuge. Cells were resuspended in the following medium: 0.04 M tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.4, 0.08 M KCl, 0.007 M magnesium acetate, 0.002 M ethyleneglycol-bis-(,3-aminoethyl ether)N,N'tetraacetic acid, 0.5 M sucrose. The cells were washed once by recentrifuging and resuspended in a small volume of the above medium (1/100 to 1/200 of the original culture volume). The procedure for ether treatment was modified from that used for E. coli as described by Vosberg and Hoffman-Berling (15). An equal volume of ether was added to resuspended cells, and the mixture was rocked gently for 1 min. The cells were then layered onto a sample of the above medium that contained an additional 0.1 g of sucrose per ml. A 4-ml layer was used for each 1 ml of ether-treated cells. The tubes were centrifuged for 10 min at 10,000 x g. The upper layers were removed, and the pelleted cells were resuspended in the same amount of medium as above. Samples were frozen for future assay. Ribonucleotide reductase activity was stable for 2 weeks in frozen, ether-treated cells. Ribonucleotide diphosphate reductase (EC 1.17.4.1) was assayed by a procedure modified from that of Fuchs and Warner (4). The reaction mixture contained 80 mM HEPES (N-2-
hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, pH 8.2, 2.8 mM ATP, 12 mM MgCl2, 2 mM ethylenediaminetetraacetate, 32 mM dithiothreitol, 2.3 mM nicotinamide adenine dinucleotide phosphate (reduced), 6 ,umol of [3H]cytidine 5'-diphosphate (CDP) (specific activity, 0.4 ,Ci/,umol), and cells in a final volume of 0.050 ml. The reaction was initiated by addition of cells to the reaction mixture, and the tubes were incubated at 37°C. The reaction was terminated by boiling the tubes for 2 min. The following additions were then made: 0.002 ml of deoxycytidine 5'-monophosphate (0.1 M), 673
674
NOTES
J. BACTERIOL.
0.002 ml of cytidine 5'-monophosphate (0.025 M), and 0.005 ml of potato apyrase (10 mg/ml). The reaction tubes were incubated at 37°C for an additional 30 min. The apyrase reaction was stopped by boiling the vessels for 2 min. The tubes were then centrifuged at 1,000 x g for 5 min to sediment the cells. A 0.015-ml amount of the supernatant was spotted on a polyethyleneimine thin-layer chromatography plate (Brinkmann Instruments Inc., Westbury, N.Y.). Plates were developed in the following solvent: 6 g of Na2B4O7, 3 g of H3BO3, 30 ml of ethylene glycol, 70 ml of distilled water. Spots of deoxycytidine 5'-monophosphate were cut out of the plate, and the nucleotide was eluted by soaking for 30 min in 1 ml of 0.7 M MgCl2 in 0.02 M
tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 7.4. Scintillation fluid, 10 ml of PCS solubilizer (Amersham/Searle Corp., Arlington Heights, Ill.), was added, and the samples were counted in a Searle Mark II scintillation spectrometer. Protein was determined on sonicated cell samples (15 min with a Heat Systems sonifier) by the biuret method (6), using bovine serum albumin as a standard. All nucleotides and dithiothreitol were purchased from P. L. Biochemicals, Milwaukee, 0 30 20 10 Wis. [3H]CDP (23 Ci/mmol) was a product of MINUTES Amersham/Searle, Arlington Heights, Ill. FIG. 1. Ribonucleotide reductase reaction in the HEPES and potato apyrase were from the presence of ether-treated cells. Assay conditions are Sigma Chemical Co., St. Louis, Mo. described in the text. Previous results had shown that unlike the majority of blue-green algae, extracts of A. TABLE 1. Requirements for CDP reductase activity quadruplicatum had no ribonucleotide reducReduction tase activity in the exchange reaction with 3HReaction system (nmol of dCDP/mg labeled adenosylcobalamin (5a). Attempts to of cell protein) demonstrate reduction in crude extracts using 81.1 system ........ ..... 3H-labeled CDP were hampered by inadequate Complete 13.8 No incubation ................ separation of substrate and product. Much Boiled cells .................. 9.9 more consistent results were obtained using Omissions. ether-treated cells. After the reaction, the cells ATP ....................... 22.8 30.0 Mg2 ...................... and most of their high-molecular-weight com9.7 ATP, Mg2 . ................. ponents are easily removed from the assay mix5.7 Dithiothreitol ......... ..... ture by centrifugation. The nucleotides in the 41.8 NADPHa ................ . supernatant can then be separated by thin11.0 cells ....................... layer chromatography. Like typical bacterial cells (16), the cyanophyte cells remain intact a NADPH, Nicotinamide adenine dinucleotide after ether treatment but are rendered permea- phosphate, reduced form. ble to nucleotides and other components of the In addition, reaction in ether-treated cells rereductase reaction. Figure 1 shows that ribonucleotide reduction quires a reducing agent, dithiothreitol, and is in the presence of cells is proportional to time; stimulated by nicotinamide adenine dinucleothe reaction is linear up to 30 min at 37°C. tide phosphate, reduced form. The latter is Requirements for CDP reductase activity are probably used to reduce thioredoxin, a small shown in Table 1. Like the E. coli enzyme, sulfhydryl-containing protein which is the ribonucleotide reductase in A. quadruplicatum physiological reducing agent in ribonucleotide requires ATP and Mg2+ for maximal activity. reduction (9).
NOTES
VOL. 128, 1976
675
Table 2 describes the effects of iron-chelating agents on reductase activity. Hydroxyurea and z 80 hydroxylamine are known to inhibit ribonucleotide reduction in E. coli by apparently pre- 0 venting free radical formation at the iron-con- X- 60 -J taining active site of the enzyme (1). These w agents are also effective inhibitors of the reaction inA. quadruplicatum. Thus, the Agmenel"'40 * lum reductase probably contains iron at the E active site. Presumably, DNA synthesis in this organism could also be inhibited by addition of X 20 hydroxyurea. Although it has been reported v) that hydroxyurea is not effective in this capac- 00 ity in Anacystis nidulans (10), recent results in E our laboratory show that this organism con- C n 80 60 40 20 0 tains the cobalamin-dependent reductase dATP (PM) (5a). Thus, hydroxyurea would not be expected to be an effective inhibitor in A. nidulans but FIG. 2. Effect of dATP on ribonucleotide reducshould be in A. quadruplicatum, assuming it is tion. The assay mixture contained all the components incorporated into the cell. listed in the text. Varying amounts of dATP were The ribonucleotide reductase from E. coli is added, and the reaction tubes were incubated at 37°C known to be subject to allosteric regulation by for 30 min. deoxynucleotides, principally dATP (3). dATP has a dual role, being stimulatory at concentrations below 10 ,tM and inhibitory at higher walls, like those of many bacteria, can be made concentrations. The reductase activity in A. permeable by treatment with ether. This techquadruplicatum can also be altered by addition nique can be quite useful for the study of in situ of dATP. The results in Fig. 2 show that unlike enzymatic reactions in these organiisms. the E. coli enzyme, the reaction in the cyanoWe wish to thank H. R. Warner for suggesting the ether phyte is inhibited by all concentrations of dATP treatment and assisting with the assays. This research was supported by Public Health Service tested. The effect of dATP on reductase activity grant 7R01-AM 18101-01 from the National Institute of Arcould explain some of the results obtained by thritis, Metabolism, and Digestive Diseases. Ingram and Fisher (7). These workers described the inhibition of DNA synthesis in A. LITERATURE CITED quadruplicatum by addition of deoxyadenosine 1. Atkin, C. L., L. Thelander, P. Reichard, and G. Lang. to their cultures. We suggest that when it is 1973. Iron and free radical in ribonucleotide reductase. Exchange of iron and Mossbauer spectroscopy of incorporated into the cell, the nucleotide is the protein B2 subunit of the E. coli enzyme. J. Biol. probably readily phosphorylated to the triphosChem. 248:7464-7472. riphate and becomes an effective inhibitor of 2. Blakley, R. L. 1965. Cobamides and ribonucleotide rebonucleotide reduction. According to the data duction. I. Cobamide stimulation of ribonucleotide reduction in extracts of Lactobacillus leichmannii. J. in Fig. 2, at low concentrations the deoxynuBiol. Chem. 240:2173-2180. cleotide would inhibit only slightly and lead to 3. Brown, N. C., Z. N. Canellakis, B. Lundin, P. Reisome slowing of the growth rate. At higher chard, and L. Thelander. 1969. Ribonucleoside diconcentrations, above 50 ,AM, enzyme activity phosphate reductase. Purification of the two subunits, proteins Bi and B2. Eur. J. Biochem. 9:561is less than half-maximal, thus providing little 573. substrate for DNA synthesis and leading to the 4. Fuchs, J. A., and H. R. Warner. 1975. Isolation of an DNA degradation noted by Ingram and Fisher. Escherichia coli mutant deficient in glutathione synThese results show that blue-green algal cell thesis. J. Bacteriol. 124:140-148. -
a-
Hogenkamp. 1972. 5'Deoxyadenosylcobalamin-dependent ribonucleotide
5. Gleason, F. K., and H. P. C.
TABLE 2. Effect of iron-chelating agents on CDP reductase activity Reduction (nmol of dCDP/mg of cell protein) 29.5 Complete system ............. Reaction system
Additions 9 mM hydroxyurea 9 mM hydroxylamine
......... .......
8.2 4.6
reductase: a survey of its distribution. Biochim. Biophys. Acta 277:466-470. 5a.Gleason, F. K., and J. M. Wood. 1976. Ribonucleotide reductase in blue-green algae: dependence on adenosylcobalamin. Science 192:1343-1344. 6. Gornall, A. G., C. J. Bardawill, and M. M. David. 1949. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177:751-766. 7. Ingram, L. O., and W. D. Fisher. 1972. Selective inhibition of deoxyribonucleic acid synthesis by 2'-deoxyadenosine in the blue-green bacterium Agmenellum
676
NOTES
quadruplicatum. J. Bacteriol. 112:170-175. 8. Larsson, A. 1969. Ribonucleotide reductase from regenerating rat liver. Eur. J. Biochem. 11:113-121. 9. Laurent, T. C., E. C. Moore, and P. Reichard. 1964. Enzymatic synthesis of deoxyribonucleotides. IV. Isolation and characterization of thioredoxin, the hydrogen donor from Escherichia coli B. J. Biol. Chem. 239:3436-3444. 10. Madan, V., and H. D. Kumar. 1971. Action of nalidixic acid and hydroxyurea on two blue-green algae. Z. Allg. Mikrobiol. 11:495-499. 11. Moore, E. C., and P. Reichard. 1964. Enzymatic synthesis of deoxyribonucleotides. VI. The cytidine diphosphate reductase system from Novikoffhepatoma. J. Biol. Chem. 239:3453-3456.
J. BACTERIOL.
Provasoli, L., J. J. A. McLaughlin, and M. R. Droop. 1957. The development of artificial media for marine algae. Arch. Mikrobiol. 25:392-428. 13. Reichard, P. 1968. The biosynthesis of deoxyribonucleotides. Eur. J. Biochem. 3:259-266. 14. Vitols, E., V. A. Bauer, and E. C. Stanbrough. 1970. Ribonucleotide reductase from Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 41:71-77. 15. Vosberg, H., and H. Hoffman-Berling. 1971. DNA synthesis in nucleotide-permeable Escherichia coli cells. I. Preparation and properties of ether-treated cells. J. 12.
Mol. Biol. 58:739-753. 16. Warner, H. R. 1973. Properties of ribonucleoside diphosphate reductase in nucleotide-permeable cells. J. Bacteriol. 115:18-22.
