Vol. 126, No. 3
JOURNAL OF BACTERIOLOGY, June 1976, p. 1344-1346 Copyright © 1976 American Society for Microbiology
Printed in USA.
Regulation of Lactate Dehydrogenase Activity in Rothia dentocariosa by Fructose 1,6-Diphosphate and Adenosine 5'-Triphosphate ROSELYN J. EISENBERG,* MARY ELCHISAK, AND JOANNE RUDD Department of Microbiology and Center for Oral Health Research, School ofDental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174 Received for publication 26 March 1976
The L-(+)-lactate dehydrogenase from Rothia dentocariosa strain 17931 is activated by fructose 1,6-diphosphate and inhibited by adenosine 5'-triphosphate. The enzyme has a molecular weight of 120,000. In these respects, it resembles the lactate dehydrogenase of Actinomyces viscosus.
Wolin (10) demonstrated that lactate dehydrogenases (LDHs) from certain streptococci have an absolute and specific requirement for fructose 1,6-diphosphate (FDP) for catalytic activity. Similar LDH activities have been found in other streptococci (3, 9), bifidobacteria (4), and Lactobacillus casei (5). An FDP-requiring LDH has recently been purified from Actinomyces viscosus (2). Hammond (6) recently showed that all strains of Rothia dentocariosa have deoxyribonucleic acid base compositions very similar to those of members of the genus Actinomyces. We shall present evidence that the LDH of R. dentocariosa is an FDP-requiring enzyme with certain characteristics in common with those reported for the LDH of A. viscosus. This finding provides further support for the idea that Rothia is closely related to members of the genus Actinomyces. Two observations led us to hypothesize that R. dentocariosa has an LDH that might behave similarly to the A. viscosus enzyme. First, all of the lactic acid formed by Rothia was found to be of the L-(+) form (R. Eisenberg and M. Elchisak, unpublished data). Second, no LDH activity could be demonstrated in crude extracts of R. dentocariosa in the absence of FDP (Table 1). Table 1 shows some characteristics of the LDH found in crude extracts of R. dentocariosa when the reaction was carried out from lactate to pyruvate. The reaction was specific for both L-(+)-lactate and nicotinamide adenine dinucleotide (NAD) and had an absolute requirement for FDP. Other hexose mono- and diphosphates did not substitute for FDP (data not shown). As in the case with other NAD-requiring dehydrogenases, N-acetyl-pyridine NAD was a better hydrogen acceptor than NAD. Ad-
dition of adenosine 5'-triphosphate (ATP) to the reaction mixture with either NAD or Nacetyl-pyridine NAD as coenzyme resulted in a marked inhibition of activity. This inhibition occurred in the presence or absence of Mg2+ ions. Table 2 shows that the reaction from pyruvate to lactate was stimulated by FDP and inhibited by ATP. Since there was some reduction of pyruvate in the absence of FDP, it is possible that the requirement for FDP is not absolute when the reaction goes in the forward direction. It could also reflect some other NAD-dependent reaction of pyruvate. The activation of the LDH of R. dentocariosa by FDP and inhibition by ATP are two properties characteristic of the enzymes isolated from A. viscosus (2). The A. viscosus enzyme was also found to be specific for >-(+)-lactate and NAD. The molecular weight of the R. dentocariosa LDH was estimated to be approximately 120,000 by using Sephadex G150 molecular exclusion chromatography (Fig. 1). Brown et al. (2) reported a molecular weight of approximately 100,000 for the LDH of A. viscosus. Thus the LDH from R. dentocariosa resembles the A. viscosus LDH with respect to both size and some chemical properties. It has been postulated that glycolysis is regulated in a number of microorganisms at the terminal step. Thus the LDH of fermentative organisms such as the streptococci and L. casei is activated by FDP. This action would be of positive value in such organisms, since their principle source of energy is glycolysis, and the principle product of glucose breakdown is lactic acid. The fact that an aerobic organism such as R. dentocariosa also contains this type of enzyme is rather intriguing from a biochemical standpoint. In this
1344
NOTES
VOL. 126, 1976
TABLE 1. Characteristics of LDH from Rothia dentocariosa strain 17931 a: reaction from lactate to pyruvate
TABLE 2. Characteristics of LDH from Rothia dentocariosa strain 17931: reaction from pyruvate to lactate Enzyme activity Units/mg % Complete reof proteina action
Enzyme activity
Reaction mixture
Units/mg of protein6
Complete' -FDP +NADPd +D-Lactic acide +ATP (1.0 ,umol) +N-acetyl-pyridine NAD +N-acetyl-pyridine NAD + ATP a Eight liters of Trypticase
0.42 0.0 0.0 0.048 0.164 1.13 0.493
pi9% oComec pletetionreac100 0 0 11 39 269 117
soy broth was inocu-
lated with R. dentocariosa strain 17931 (coccal form) and incubated at 37 C for 24 h. Aeration of the culture was accomplished by bubbling a mixture of 95% 02 and 5% CO2 into it. After 24 h of incubation at 37 C, the cells were harvested, washed three times with 0.01 M phosphate buffer (pH 7.0), and sonicated for 20 min in a Lab-Line sonifier. The material was centrifuged at 31,000 x g for 30 min, and the supernatant was dialyzed against phosphate buffer containing 10-3 M dithiothreitol. Twenty milliliters of extract containing 12.7 mg of protein per ml (method of Lowry et al., 7) was obtained. The growth medium was saved for analysis of total lactic acid (1) and L-(+)lactic acid (8). b LDH activity was assayed at 25 C by monitoring the L-lactate-dependent reduction of NAD at 340 nm. A unit of activity is defined as that amount of enzyme required to reduce 1 Amol of NAD per min. Reactions were initiated by addition of enzyme, and rates were proportional to enzyme concentration and time. I The complete reaction mixture contained the following components in a 1.0-ml volume: 25 umol of tris(hydroxymethyl)aminomethane buffer (pH 7.2), 100 ,umol of lithium-L-lactate, 5.8 ,mol of MgCl2, 5.0 ,umol of dithiothreitol, 1.0 ymol of NAD, and 1.0 ,umol of FDP. d Equimolar amount was substituted for NAD. I Equimolar amount was substituted for L-(+)lactate.
1345
Reaction mixture
Complete'
3.5 100 -FDP 0.151 4.3 +ATP (1 gmol) 0.96 27 +NADPHc 0 0 0 0 -Pyruvate a LDH activity was assayed at 25 C by monitoring the pyruvate-dependent oxidation of NADH at 340 nm. The reaction was initiated as in Table 1. b The complete reaction mixture contained the following components in a 1.0-ml volume: 100 ,umol of potassium phosphate buffer, pH 6.2; 5 umol of sodium pyruvate; 0.1 ,umol of NADH; 1.0 umol of FDP; 5 ,Mmol of MgCl2; and 5.0 ,umol of dithiothreitol. c 1 ,umol of NADPH instead of NAD.
100
Cy(ohhome C
90
Ovolbum.n
E 80
/
/ -
2 70 -j 60
Bovine Serum Albumin C teole Knose
60
o_Roth,o dentocorioso Loctote Dehydrogenose
z 50
-Co?olose
0 n- 40
w 30
20
10 2
3
4
5 6 7 8910
MOLECULAR WEIGHT (x10-4)
FIG. 1. Molecular weight of LDH from R. dentocariosa 17931. A column (1.5 by 83 cm) was packed with Sephadex G150 in 0.01 M phosphate buffer (pH 7.8) containing 0.2 M NaCl. The sample size was 2.0 ml, and the column was eluted at 25 C. The elution volumes of the protein standards were determined by monitoring the absorbance of column fractions at 280 nm in a Gilford 240 spectrophotometer. LDH activity was measured in the direction of lactate to pyruvate using the complete mixture described in Table 1. The LDH sample contained 25 mg of crude extract, and the protein standards contained 2.5 mg of each protein.
organism, lactic acid did not appear to be a major product of glucose catabolism when the organisms were grown aerobically. We found glycolysis. When oxygen levels were high, the that only 14% of the glucose initially present enzyme might be relatively inactive due to inin Trypticase soy broth could be accounted for hibition by ATP. as lactic acid. Yet even in this aerobic orIt is interesting that most of the organisms ganism, complex regulation of LDH activity that contain an FDP-requiring LDH fall into might be useful under varying growth condi- two major taxonomic groups: the Streptococcations. For example, under conditions of reduced ceae and the Actinomycetaceae. This might reoxygen concentrations, when ATP levels would flect an evolutionary link between the two be expected to fall and FDP levels would tend families. Serological comparisons of the FDPto rise, the LDH of R. dentocariosa would serve dependent LDH from a variety of species withan important role in regenerating NAD for in these two families might clarify this point.
1346
NOTES
It would also be useful to extend the biochemical study of LDH to other members of these families, particularly the Actinomycetaceae. This investigation was supported by Public Health Service grant DE-02623 from the National Institute of Dental Research. Roselyn J. Eisenberg is the recipient of Public Health Service Career Development Award DE70160 from the National Institute of Dental Research. We wish to express our appreciation to Gary H.. Cohen and Benjamin F. Hammond for help in preparation of this manuscript.
J.- BACTERIOL.
3.
4. 5.
6.
ADDENDUM IN PROOF We recently found an FDP-dependent L-(+)lactate dehydrogenase in extracts of Arachnia propionica, another member of the Actinomycetaceae. LITERATURE CITED 1. Barker, S. B., and W. H. Summerson. 1941. The colorimetric determination of lactic acid in biological material. J. Biol. Chem. 138:535-544. 2. Brown, A. T., C. P. Christian, and R. L. Eifert. 1975. Purification, characterization, and regulation of a nicotinamide adenine dinucleotide-dependent lactate
7. 8. 9.
10.
dehydrogenase from Actinomyces viscosus. J. Bacteriol. 122:1126-1135. Brown, A. T., and C. L. Wittenberger. 1972. Fructose 1,6-diphosphate-dependent lactate dehydrogenase from a cariogenic streptococcus: purification and properties. J. Bacteriol. 110:604-615. de Vries, W., and A. H. Stouthamer. 1968. Fermentation of glucose, lactose, galactose, mannitol and xylose by bifidobacteria. J. Bacteriol. 96:472-478. de Vries, W., W. M. C. Kapteijn, E. A. Van der Beck, and H. Stouthamer. 1970. Molar growth yields and fermentation balances of Lactobacillus casei L3 in batch cultures and in continuous cultures. J. Gen. Microbiol. 63:333-345. Hammond, B. F. 1970. Deoxyribonucleic acid base composition of Rothia dentocariosa as determined by thermal denaturation. J. Bacteriol. 104:1024-1026. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Vallee, B. L., and F. L. Hoch. 1955. Zinc, a component of yeast alcohol dehydrogenase. Proc. Natl. Acad. Sci. U.S.A. 41:327-333. Wittenberger, C. L., and N. Angelo. 1970. Purification and properties of a fructose 1,6-diphosphateactivated lactate dehydrogenase from Streptococcus faecalis. J. Bacteriol. 101:717-724. Wolin, M. J. 1964. Fructose-1,6 diphosphate requirement of streptococcal lactate dehydrogenases. Science 146:775-777.
JOURNAL OF BACTERIOLOGY, June 1976, p. 1344-1346 Copyright © 1976 American Society for Microbiology
Printed in USA.
Regulation of Lactate Dehydrogenase Activity in Rothia dentocariosa by Fructose 1,6-Diphosphate and Adenosine 5'-Triphosphate ROSELYN J. EISENBERG,* MARY ELCHISAK, AND JOANNE RUDD Department of Microbiology and Center for Oral Health Research, School ofDental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174 Received for publication 26 March 1976
The L-(+)-lactate dehydrogenase from Rothia dentocariosa strain 17931 is activated by fructose 1,6-diphosphate and inhibited by adenosine 5'-triphosphate. The enzyme has a molecular weight of 120,000. In these respects, it resembles the lactate dehydrogenase of Actinomyces viscosus.
Wolin (10) demonstrated that lactate dehydrogenases (LDHs) from certain streptococci have an absolute and specific requirement for fructose 1,6-diphosphate (FDP) for catalytic activity. Similar LDH activities have been found in other streptococci (3, 9), bifidobacteria (4), and Lactobacillus casei (5). An FDP-requiring LDH has recently been purified from Actinomyces viscosus (2). Hammond (6) recently showed that all strains of Rothia dentocariosa have deoxyribonucleic acid base compositions very similar to those of members of the genus Actinomyces. We shall present evidence that the LDH of R. dentocariosa is an FDP-requiring enzyme with certain characteristics in common with those reported for the LDH of A. viscosus. This finding provides further support for the idea that Rothia is closely related to members of the genus Actinomyces. Two observations led us to hypothesize that R. dentocariosa has an LDH that might behave similarly to the A. viscosus enzyme. First, all of the lactic acid formed by Rothia was found to be of the L-(+) form (R. Eisenberg and M. Elchisak, unpublished data). Second, no LDH activity could be demonstrated in crude extracts of R. dentocariosa in the absence of FDP (Table 1). Table 1 shows some characteristics of the LDH found in crude extracts of R. dentocariosa when the reaction was carried out from lactate to pyruvate. The reaction was specific for both L-(+)-lactate and nicotinamide adenine dinucleotide (NAD) and had an absolute requirement for FDP. Other hexose mono- and diphosphates did not substitute for FDP (data not shown). As in the case with other NAD-requiring dehydrogenases, N-acetyl-pyridine NAD was a better hydrogen acceptor than NAD. Ad-
dition of adenosine 5'-triphosphate (ATP) to the reaction mixture with either NAD or Nacetyl-pyridine NAD as coenzyme resulted in a marked inhibition of activity. This inhibition occurred in the presence or absence of Mg2+ ions. Table 2 shows that the reaction from pyruvate to lactate was stimulated by FDP and inhibited by ATP. Since there was some reduction of pyruvate in the absence of FDP, it is possible that the requirement for FDP is not absolute when the reaction goes in the forward direction. It could also reflect some other NAD-dependent reaction of pyruvate. The activation of the LDH of R. dentocariosa by FDP and inhibition by ATP are two properties characteristic of the enzymes isolated from A. viscosus (2). The A. viscosus enzyme was also found to be specific for >-(+)-lactate and NAD. The molecular weight of the R. dentocariosa LDH was estimated to be approximately 120,000 by using Sephadex G150 molecular exclusion chromatography (Fig. 1). Brown et al. (2) reported a molecular weight of approximately 100,000 for the LDH of A. viscosus. Thus the LDH from R. dentocariosa resembles the A. viscosus LDH with respect to both size and some chemical properties. It has been postulated that glycolysis is regulated in a number of microorganisms at the terminal step. Thus the LDH of fermentative organisms such as the streptococci and L. casei is activated by FDP. This action would be of positive value in such organisms, since their principle source of energy is glycolysis, and the principle product of glucose breakdown is lactic acid. The fact that an aerobic organism such as R. dentocariosa also contains this type of enzyme is rather intriguing from a biochemical standpoint. In this
1344
NOTES
VOL. 126, 1976
TABLE 1. Characteristics of LDH from Rothia dentocariosa strain 17931 a: reaction from lactate to pyruvate
TABLE 2. Characteristics of LDH from Rothia dentocariosa strain 17931: reaction from pyruvate to lactate Enzyme activity Units/mg % Complete reof proteina action
Enzyme activity
Reaction mixture
Units/mg of protein6
Complete' -FDP +NADPd +D-Lactic acide +ATP (1.0 ,umol) +N-acetyl-pyridine NAD +N-acetyl-pyridine NAD + ATP a Eight liters of Trypticase
0.42 0.0 0.0 0.048 0.164 1.13 0.493
pi9% oComec pletetionreac100 0 0 11 39 269 117
soy broth was inocu-
lated with R. dentocariosa strain 17931 (coccal form) and incubated at 37 C for 24 h. Aeration of the culture was accomplished by bubbling a mixture of 95% 02 and 5% CO2 into it. After 24 h of incubation at 37 C, the cells were harvested, washed three times with 0.01 M phosphate buffer (pH 7.0), and sonicated for 20 min in a Lab-Line sonifier. The material was centrifuged at 31,000 x g for 30 min, and the supernatant was dialyzed against phosphate buffer containing 10-3 M dithiothreitol. Twenty milliliters of extract containing 12.7 mg of protein per ml (method of Lowry et al., 7) was obtained. The growth medium was saved for analysis of total lactic acid (1) and L-(+)lactic acid (8). b LDH activity was assayed at 25 C by monitoring the L-lactate-dependent reduction of NAD at 340 nm. A unit of activity is defined as that amount of enzyme required to reduce 1 Amol of NAD per min. Reactions were initiated by addition of enzyme, and rates were proportional to enzyme concentration and time. I The complete reaction mixture contained the following components in a 1.0-ml volume: 25 umol of tris(hydroxymethyl)aminomethane buffer (pH 7.2), 100 ,umol of lithium-L-lactate, 5.8 ,mol of MgCl2, 5.0 ,umol of dithiothreitol, 1.0 ymol of NAD, and 1.0 ,umol of FDP. d Equimolar amount was substituted for NAD. I Equimolar amount was substituted for L-(+)lactate.
1345
Reaction mixture
Complete'
3.5 100 -FDP 0.151 4.3 +ATP (1 gmol) 0.96 27 +NADPHc 0 0 0 0 -Pyruvate a LDH activity was assayed at 25 C by monitoring the pyruvate-dependent oxidation of NADH at 340 nm. The reaction was initiated as in Table 1. b The complete reaction mixture contained the following components in a 1.0-ml volume: 100 ,umol of potassium phosphate buffer, pH 6.2; 5 umol of sodium pyruvate; 0.1 ,umol of NADH; 1.0 umol of FDP; 5 ,Mmol of MgCl2; and 5.0 ,umol of dithiothreitol. c 1 ,umol of NADPH instead of NAD.
100
Cy(ohhome C
90
Ovolbum.n
E 80
/
/ -
2 70 -j 60
Bovine Serum Albumin C teole Knose
60
o_Roth,o dentocorioso Loctote Dehydrogenose
z 50
-Co?olose
0 n- 40
w 30
20
10 2
3
4
5 6 7 8910
MOLECULAR WEIGHT (x10-4)
FIG. 1. Molecular weight of LDH from R. dentocariosa 17931. A column (1.5 by 83 cm) was packed with Sephadex G150 in 0.01 M phosphate buffer (pH 7.8) containing 0.2 M NaCl. The sample size was 2.0 ml, and the column was eluted at 25 C. The elution volumes of the protein standards were determined by monitoring the absorbance of column fractions at 280 nm in a Gilford 240 spectrophotometer. LDH activity was measured in the direction of lactate to pyruvate using the complete mixture described in Table 1. The LDH sample contained 25 mg of crude extract, and the protein standards contained 2.5 mg of each protein.
organism, lactic acid did not appear to be a major product of glucose catabolism when the organisms were grown aerobically. We found glycolysis. When oxygen levels were high, the that only 14% of the glucose initially present enzyme might be relatively inactive due to inin Trypticase soy broth could be accounted for hibition by ATP. as lactic acid. Yet even in this aerobic orIt is interesting that most of the organisms ganism, complex regulation of LDH activity that contain an FDP-requiring LDH fall into might be useful under varying growth condi- two major taxonomic groups: the Streptococcations. For example, under conditions of reduced ceae and the Actinomycetaceae. This might reoxygen concentrations, when ATP levels would flect an evolutionary link between the two be expected to fall and FDP levels would tend families. Serological comparisons of the FDPto rise, the LDH of R. dentocariosa would serve dependent LDH from a variety of species withan important role in regenerating NAD for in these two families might clarify this point.
1346
NOTES
It would also be useful to extend the biochemical study of LDH to other members of these families, particularly the Actinomycetaceae. This investigation was supported by Public Health Service grant DE-02623 from the National Institute of Dental Research. Roselyn J. Eisenberg is the recipient of Public Health Service Career Development Award DE70160 from the National Institute of Dental Research. We wish to express our appreciation to Gary H.. Cohen and Benjamin F. Hammond for help in preparation of this manuscript.
J.- BACTERIOL.
3.
4. 5.
6.
ADDENDUM IN PROOF We recently found an FDP-dependent L-(+)lactate dehydrogenase in extracts of Arachnia propionica, another member of the Actinomycetaceae. LITERATURE CITED 1. Barker, S. B., and W. H. Summerson. 1941. The colorimetric determination of lactic acid in biological material. J. Biol. Chem. 138:535-544. 2. Brown, A. T., C. P. Christian, and R. L. Eifert. 1975. Purification, characterization, and regulation of a nicotinamide adenine dinucleotide-dependent lactate
7. 8. 9.
10.
dehydrogenase from Actinomyces viscosus. J. Bacteriol. 122:1126-1135. Brown, A. T., and C. L. Wittenberger. 1972. Fructose 1,6-diphosphate-dependent lactate dehydrogenase from a cariogenic streptococcus: purification and properties. J. Bacteriol. 110:604-615. de Vries, W., and A. H. Stouthamer. 1968. Fermentation of glucose, lactose, galactose, mannitol and xylose by bifidobacteria. J. Bacteriol. 96:472-478. de Vries, W., W. M. C. Kapteijn, E. A. Van der Beck, and H. Stouthamer. 1970. Molar growth yields and fermentation balances of Lactobacillus casei L3 in batch cultures and in continuous cultures. J. Gen. Microbiol. 63:333-345. Hammond, B. F. 1970. Deoxyribonucleic acid base composition of Rothia dentocariosa as determined by thermal denaturation. J. Bacteriol. 104:1024-1026. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Vallee, B. L., and F. L. Hoch. 1955. Zinc, a component of yeast alcohol dehydrogenase. Proc. Natl. Acad. Sci. U.S.A. 41:327-333. Wittenberger, C. L., and N. Angelo. 1970. Purification and properties of a fructose 1,6-diphosphateactivated lactate dehydrogenase from Streptococcus faecalis. J. Bacteriol. 101:717-724. Wolin, M. J. 1964. Fructose-1,6 diphosphate requirement of streptococcal lactate dehydrogenases. Science 146:775-777.
