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Fluoride inhibition of glucose-6-P formation in Streptococcus salivarius: Relation to glycogen synthesis and degradation
ARCHIVES
OF
BIOCHEMISTRY
Fluoride
AND
inhibition
salivarios:
Relation
JOSEPH A. KANAPKA, Department
MIOPHYSICS
144, 596402
of Glucose&P to Glycogen RAMJI
(1971)
Formation Synthesis
1,. KHANDELWAL,
in Streptococcus and
AND
IAN R. HAMILTON
of Oral Biology (Biochemistry), Faculty of Dentistry, Manitoba, Winnipeg, Canada Received
December
17, 1970; accepted
March
Degradation
University
of
1, 1971
The concentrations of glucose-6-P and ATP in intact cells of the oral microbe, Streptococcus saliva&s, were analyzed during anaerobic glucose metabolism in the presence and absence of fluoride. Addition of 2.4 mM NaF to cells actively degrading glucose resulted in an immediate decrease in the cellular glucose-6-P and ATP content concomitant with the complete inhibition of glucose uptake and glycogen synthesis. A noticeable decrease in cellular glucose-6-P concentration was produced in cells metabolizing glucose by concentrations of NaF as low as 0.06 mM, regardless of whether the inhibitor was added before or after the substrate. After the initial decline in the glucose-6-P content, the concentration of this intermediate frequently increased, resulting from the degradation of endogenous glycogen. ATP, on the other hand, remained low throughout the experimental period. Experiments with crude enzyme preparations of the organism demonstrated that the glycogen synthetic enzymes, phosphoglucomutase (EC 2.7.5. l), ADPG pyrophosphorylase, and ADPG glucan transferase (ADP-glucose:glycogen glucosyl transferase), as well as phosphorylase (EC 2.4.1 .l) in the degradative pathway, were fluoride-insensitive at NaF concentrations inhibit,ing in viva synthesis. The inability of 2.4 mu NaF to inhibit glucose-6-P formation from glycogen in &JO in the absence of exogenous glucose confirms that phosphoglucomutase and phosphorylase in this organism are not inhibited by fluoride. The results strongly suggest that fluoride interacts, in some manner, with the sugar transport system in the organism as phosphorylation by hexokinase (EC 2.7.1.1) was not fluoride-sensitive. The “apparent” inhibition of glycogen synthesis is discussed in relation to the availability of ATP and glucose-6-P.
Anaerobic glucose metabolism by resting cells of the oral microbe, Xt~eptococcus saliva&s, results in the formation of lactic acid and large quantities of intracellular polyglucose glycogen (1). Fluoride has been shown to inhibit exogenous glucose metabolism, intracellular glycogen synthesis, and the degradat,ion of this polymer in the absence of an external carbon source (2, 3). Of these three parameters, glycogen synthesis was by far the most sensitive, being completely inhibited by low levels of fluoride under condit,ions of appreciable glucose degradation. On t,he other hand, the degradation of this endogenous energy source was inhibited to a much lesser degree t,han was exogenous glucose metabolism. From these studies it, was concluded that since the 596
exogenous and endogenous pathways had widely differing fluoride sensitivities, and yet shared a common glycolytic pathway from glucose-6-P to lactate, the prime site of fluoride action in intact cells of S. salivarius was at some undetermined point in the glycolytic scheme prior to glucose-6-P formation. A similar conclusion was reached in studies with S. mitis (4) and mixed oral flora (5). Evidence will be presented in this paper supporting the above supposition by demonstrating that low concentrations of fluoride had a pronounced and rapid inhibitory effect on the synthesis of glucose-6-P in whole cells of S. salivarius metabolizing exogenous glucose. Further results with crude enzyme preparations will demonstrate that the
FLUORIDE
INHIBITION
enzymes in the glycogen synthetic pathway are insensitive to fluoride at inhibitor concentrations above those required to completely inhibit ifz vivo synthesis. MATERIALS
AND
METHODS
The procedures for the growt,h, harvesting, and preparation of washed cells of Streptococcus saliv&us have been described previously (1). Cells were grown in a medium containing 170trypton‘e, 0.5% yeast extract, 0.3yo KsHP04, and 0.1% glucose, and were always harvested in the exponential phase of growth. Cell-free extracts were prepared from washed cells by sonication in a Branson sonifier (Heat Systems Ultrasonics, Plainview, New York) for 3 min at 0”. The supernatant fluid obtained after centrifugation at 30,000 g was dialyzed against 100 vol of Tris-HCl butler (50 mM, pH 7.5) for 4 hr. This dialyzed extract was used for the assay of all enzymes tested in this study. In vivo ezperintenls. The direct effect of fluoride on glycolysis in intact cells of S. salivariz~s was determined by measuring the intracellular concentration of glucose-6-P and ATP before and after the addition of NaF during glucose degradation. Washed rest,ing cells were incubated at 10 mg dry wt/ml (approximately 1.25 X lOlo cells/ml) in pot.assium phosphate buffer (0.05 M, pH 7.2) at 37” in an atmosphere of Ns + 5% Cot. After equilibration for 10 min, a zero-time sample was removed and glucose, to an approximate final concentration of 1.5 pmoles/mg cells, was added to the remaining cells to start the reaction. With the exception of one experiment, where the cells were incubated with various NaF concentrations during the equilibratjion period, the cells were exposed to fluoride after the onset of glucose metabolism. III these lat,ter cases, a portion of the incubated cells was rapidly removed at a predetermined point during metabolism and added to NaF under the same condit.ions in a separate reaction flask. Samples (0.5-1.0 ml) were removed periodically in both the control and fluoride-containing flasks and added to an equal volume of 1 N perchloric acid to stop the reaction and to extract the cells. Extraction proceeded for 30 min at room temperature, followed by neutralization of the samples to pH 7.G with KOH-triethanolamine buffer (6). After rapid freezing and thawing, cell debris and precipitated KC104 were removed by centrifugation at 3000 g at 4”. The supernatant fractions were stored frozen until analyzed, with the assays for glucose-6-P and ATP always completed within 48 hr after sampling. When glycogen synthesis was measured, glucose-U-l% was employed as the substrate and additional samples (0.2 ml) were removed and added to 1.0 ml of 50yo ethanol in 0.1 x HCI to stop the reaction.
OF GLUCOSE-6-P
,597
Metabolic analyses. The concentration of glucose-6-P and ATP extracted from the cells was assayed by t’he direct enzymatic-fluoromet,ric methods outlined by Maitra and Estabrook (7). Glucose-6-P was determined by its conversion to Gphosphogluconate with NADP+ and commercial glucose-6-P dehydrogenase. The same system was used to assay ATP by the addition of hexokinase and glucose. The quantity of these metabolites in the samples was determined by the addition of an internal standard of glucose-G-P which had been standardized both gravimetrically and spectrophotometrically. Fluorescence of the rcsultant NADPH was measured in a Farrand Ratio Fluorometer equipped with a modified Heath Model EU-BOB Servo Recorder (Farrand Optical Co., Mt. Vernon, New York). A mercury vapor lamp H85 A3/UV served as the radiation source, and the light for excitation was isolated with a Farrand interference filter with peak transmission at 342 rng, while the fluorescent light was isolated with a 435.rnp interference filter. Extracellular glucose was determined by the glucose oxidase method (8). Glycogen synthesis was determined by the incorporation of glucose-U14C into intact cells as described previously (I). Enzyme assays. Phosphoglucomutase activity in cell-free extracts was determined in a coupled enzyme assay by the conversion of glucose-l-P to glucose-6-P in the presence of catalytic amounts of glucose-1,6-diphosphate. Glucose-6-P was further converted to 6-phosphogluconic acid with commercial glucose-6-phosphate dehydrogenase and NADP. The formation of the resulting NADPH was measured spectrophotometrically at 340 rns. The reaction mixture consisted of 10 mM Tris-HCl (pH 7.5), 0.5 mM MgCls, 0.5 rnM glucose-l-P, 4 PM glucose-1,6-diphosphate, 0.2 mg NADP+, 20 pg commercial glucose-6-phosphate dehydrogenase, and 5.3 rg extract protein in a total volume of 1.0 ml. ADPG pyrophosphorylase act,ivity in the extract was determined by the incorporation of glucose-U-14C from glucose-‘%-l-P (1 X lo6 dpm/ pmole) into ADP-glucose (9). The reaction mixture consisted of 50 mM Tris-HCl (pH 7.8)) 0.5 mM glucose-14C-l-P, 1 mM ATP, 10 mM MgC12, 0.9 rg inorganic pyrophosphatase, and 250 pg extract protein in a total volume of 0.2 ml. ADPG glucan transferase was determined by the incorporation of glucose-UJ4C from ADP-glucose1% (0.2 X IO6 dpm/pmole) into glycogen (10). The reaction mixture contained 50 mM Tris-HCl (pH 7.8), 0.5 mM ADP-glucosel%!, 25 mM KCl, 10 mM mercaptoethanol, 0.5 mg glycogen, 0.2 mg bovine serum albumin, and 25 rg extract protein in a total volume of 0.2 ml. Phosphorylase activity was determined by the
598
KANAPKA,
KHANDELWAL,
AND
HAMILTON
incorporat,ion of glucose-U-r4C from glucoseJ4C1-P into glycogen in the absence of ATP (11). The reaction mixture contained 100 mM fi-glycerophosphate buffer (pH 6.8), 10 mM glucose-W-l-P, 0.5 mg glycogen, and 89 pg extract protein in a total volume of 0.1 ml. Materials. All enzymes, co-factors, and glycolytic intermediates used in these experiments were purchased from Boehringer Mannheim Corp. (New York). All other chemicals were of reagent grade. RESULTS Efect oj jhoride on in vivo glucose mefabolism. As shown previously (1,2), the addition of exogenous glucose to the nonproliferating intact celts of S. salivarius resulted in the immediate uptake of this substrate with concomitant glycogen synthesis. Confirmation of these results is shown in Fig. 1 along with the cellular concentrations of glucose6-P and ATP. Upon glucose addition, the intracellular content of glucose-6-P and of ATP rose rapidly from low endogenous levels to higher levels characteristic of exogenous glucose metabolism. At the point of glucose 8exhaustion (3.6 min), the intracellular glycogen content was maximum, and the glucose-6-P concentration began to decline dramatically to a low level characteristic of endogenous metabolism. The ATP content 0, at this point, however, continued at a 0 2 relatively constant level probably because 4 6 MINUTES the cells had started to degrade the newly formed cellular polysaccharide for energy. FIG. 1. The effect of 2.4 mM NaF on exogenous The addition of 2.4 mM NaF to the cells glucose uptake, glycogen synthesis and degradacontent of glucose-6-P 1.5 min after the addition of the glucose tion, and the intracellular and ATP, during anaerobic glucose metabolism profoundly altered this metabolic pattern. by washed cells of 8. salivarius. Control cells Exogenous glucose uptake from the medium (0); fluoride-treated cells (0). was immediately stopped, as was glycogen synthesis, confirming our earlier results glucose-6-P in the fluoride-treated cells was (2, 3). Furthermore, the addition of fluoride also resulted in a rapid decrease in the cellu- not associated with an increase in the cellular concentration of ATP. The continued low lar concentrations of ATP and glucose-6-P, concomitant with the effects on glucose synthesis of both ATP and glucose-6-P in the presence of fluoride during the experiuptake and glycogen synthesis. Although glucose-6-P initially dropped to 15% of the mental period has been shown elsewhere (21) to be the result of the degradation of control value 15 set after the fluoride addition, the concentration increased to 50% of small quantities of cellular glycogen. The fluoride effect on the cellular levels of this value before the end of the 6-min exATP and glucose-6-P was a sustained one perimental period. ATP, on the other hand, since prolonged incubation of the cells (up dropped to 20 % of the control value initially and remained at about this level in the to 40 min) demonstrated that as long as remaining samples. Thus, the increase in fluoride was present in the medium the
FLUORIDE
INHIBITION
cellular content of both ATP and glucose-6-P remained at greatly reduced levels. Unlike the ATP concentration which remained low throughout the experimental period, the cellular content of glucose-6-P in these cases often rose to levels slightly above the steadystate concentration maintained in the control cells in the absence of exogenous glucose. In the control cells, on the other hand, the glucose-6-P concentration dropped immediately upon depletion of the exogenous glucose, while the ATE’ concentration declined very gradually, requiring almost 35 min to reach the ATP level observed in the fluoride-treated cells. This indicates that the endogenous degradation of intracellular glycogen in this organism (1) can maintain the ATP content of the cells at a relatively high level for a considerable amount of time after the depletion of the exogenous carbon source. E$ect of fluoride comentration on intracellular glucose-6-P concentration. Previous studies have demonstrated that 0.96 ml% NaF completely inhibited the metabolism of exogenous glucose by S. salivarius (2,3). Glycogen synthesis, on the other hand, was completely inhibited by fluoride at half of this concentration, while a level as low as 0.06 rnwl produced a significant inhibitory effect. Therefore, an experiment was designed to investigate the effect of low fluoride levels 011 the intracellular concentration of glucose-6-P, the first apparent intracellular precursor in glycogen synthesis. Furthermore, in order to test for the so-called substrate-protective effect (12)) fluoride was added to the cells before and after the substrate. When the cells were preincubated at 37” with NaF (O-l.2 mlr) followed by the addition of 15 mzr glucose, a progressive decrease in the maximum intracellular glucose-6-P concentration was observed (Table I). As the NaF concentration increased to 1.2 m&f, the maximum glucose-6-P level decreased from 7.1 to 1.0 nmoles/mg dry wt cells. A more dramatic effect was observed under the same conditions when these NaF concentrations were added after the onset of glucose metabolism. In this experiment, the inhibitor was added 30 set after the addition of the glucose and again resulted in an im-
599
OF GLUCOSE-BP TABLE
I
EFFBXT OF PRBINCUB.ITION WITH NaF ON THE CELLULAR CONCENTRATION OF GILMAXIMUM cosn-6-P IN WASHED CELLS OF S. saliaarius DURING:SIYBSEQUENT GLUCOSE METABOLISM N\'aF mn 0
0.06 0.12 0.25 0.60 1.20 B nmoles/mg
Glucose-6-Pa
Percent of control
7.1 4.3 3.1 2.4 1.6 1.0
100 61 44 34 23 14
dry weight
OY 0
of cells.
1.0
2.0
MINUTES FIG. 2. The effect of varying concentrations of NaF on glucose-6-P levels in cells of S. salivariusmetabolizing glucose. NaF was added at 30 set to give the following final concentrations (mM): 0 (0); 0.06 (0); 0.12 (A); 0.25 (A); 0.60 (a);
1.2 (ml. mediate, rapid decrease in the intracellular glucose-6-P concentration (Fig. 2). KaF, as low as 0.06 mill, produced an initial slight drop in the glucose-6-P content followed by a rapid recovery to levels similar to those in the control cells. However, at higher NaF concentrations (0.12-1.2 mM) a much more pronounced initial drop in glucose-6-P levels was observed without a subsequent increase during the 2-min experimental period. As this figure indicates, the decrease in the
600
KANAPKA,
KHANDELWAL,
AND
TABLE INFLUENCE
OF
NaF
0
1.2
2.4
4.8
1474 147 876 106” 235d
159
159 90 107 363
171 147
147 147
glucose-6-P formed/mg prot,ein/min. ADP-glucose formed/mg protein/min. glycogen formed/mg protein/min. glucose-l-P incorporated into glycogen/mg
cellular glucose-6-P concentration was very rapid, occurring within 15 set of the fluoride addition. This represented the minimum time necessary for accurate sampling in the system employed. Fluoride e$ect on the enzymes involved in glycogen metabolism. Two possibilities exist to explain the in vivo inhibition of glycogen synthesis in S. saliva&us by fluoride: (a) fluoride interacts directly with one or more of the enzymes involved in the synthetic process, or (b) that insufficient glucose-6-P is available for synthesis. Phosphoglucomutase, in the glycogen synthetic pathway, generally has been cited as the point of fluoride inhibition in microbes (13, 14), while in. vitro fluoride inhibition of this enzyme from rabbit muscle (15) and higher plants (16) has been observed. To test for the first possibility, dialyzed crude extract preparations of S. salivarzus were prepared, and the activities of phosphoglucomutase, ADPG pyrophosphorylase, and ADPG glucan transferase in the synthetic pathway, as well as phosphorylase in the degradative pathway, were assayed in the presence and absence of fluoride. The results (Table II) indicate that none of these enzymes was sensitive to fluoride &a z&o, at concentrations equal to, or higher those
which
OF GLYCOGEN
?;aF (IIIM)
Phosphoglucomutase Preincubation No preincubation ADPG Pyrophosphorylase ADPG Glucan transferase Phosphorylase
than,
II
ON THE ENZYMES INVOLVED IN THE SYNTHESIS AND DEGRBD.ITION IN CRUDE EXTRACTS OF 8. sakivarius
Enzyme
a nmoles b pmoles c nmoles d pmoles
HAMILTON
completely
inhibited
in vivo glycogen synthesis. Phosphoglucomutase was resistant to NaF up to 9.6 m&f regardless of whether the enzyme was preincubated with fluoride, or whether the
7.2
9.6
-
171 147
-
97
85
-
111
108
-
-
407
-
450
-
protein/min.
inhibitor was added after the reaction had started. ADPG pyrophosphorylase and ADPG glucan transferase were not significantly affected by concentrations to 4.8 mm NaF. Phosphorylase activity, on the other hand, was actually stimulated by concentrations of NaF up to 7.2 rnb1. In vivo e$ect of Jluoride on glycogen degradation. Since small intracellular glucose-l-P concentrations exist in cells of S. salivarius during exogenous glucose metabolism, it was difficult to measure accurately the amount of this compound in the presence of the normally high levels of glucose-6-P. Therefore, evidence that phosphoglucomutase was fluoride-insensitive in the direction of glycogen synthesis was difficult to confirm in vivo in the presence of exogenous glucose. However, the effect of fluoride on this enzyme and on phosphorylase could be measured accurately by monitoring the cellular content of glucose-6-P during glycogen degradation in the absence of external glucose. This was tested in an experiment where whole cells were incubated with glucose to permit the synthesis of intracellular glycogen. Approximately 1 min after the exogenous glucose had been depleted, 2.4 mM NaF was added to a portion of the cells. The glucose-6-P level in the control cells rose upon the addition of the substrate immediately in the characteristic manner and dropped again once the exogenous substrate was exhausted (Fig. 3). Under these conditions of glycogen degradation, glucose-
FLUORIDE
IXHIBITION
Glucose
MINUTES FIG. 3. The effect of 2.4 rnM NaF on glucose-6-P levels in cells of S. salioarius-metabolizing glycogen. Control cells (0) ; fluoride-treated cells (0).
6-P formation was initially stimulated by the addition of the fluoride, resulting in concentrations higher than those in the controls but which then decreased to the control level by the end of the experimental period. As inhibition of phosphoglucomutase and/or glycogen phosphorylase would have resulted in a decrease in the glucose-6-P concentration, it is clear that these enzymes are not fluoride-sensitive under in viva conditions thereby confirming the in vitro enzyme assays. DISCUSSION
Fluoride effect on glucose-6-P formation. The fluoride inhibition of glucose-6-P formation in whole cells of S. salivarius confirms the indirect evidence obtained in previous studies which indicated that the site of inhibition was at some point in glycolysis prior t’o glucose-B-P formation. Although this fluoride-sensitive component is unknown, possible sites of inhibition would include the glucose phosphorylating enzyme, hexokinase, the reactions involved in the glucose transport process, or associated processes, such as bhe availability of ATP for phosphorylat’ion.
OF GLUCOSE-O-I’
GO1
In order to delineate the site of fluoride action in 8. salivarius, hexokinase activity in crude extracts was assayed by the formation of glucose-6-P from glucose and ,4Tl’ in the presence and absence of fluoride. As no loss of activity \vas observed at Sal? concentrations as high as 24 mnr, one must conclude that hexokinase is not the fluoridesensitive component. The role of ATP is more difficult to assess. Although the cellular ATP content decreases rapidly upon fluoride addition (Fig. l), it was not possible from these results to determine whether the decrease was the cause or the effect of the decrease in the cellular glucose-&P concentration. One might assume that ATI’ would be required for the phosphorylation of glucose, either within the cell by the hexokinase reaction, or at the membrane during transport (17, IS). However, the fact that glucose transport in many bacterial species is mediated by the phosphoenolpyruvate-phosphotransferase (I’EPPT) system (see review, 19) indicates that ATl’ may not be involved in active glucose transport. Indeed, the fact that sugars are concentrated in membrane vesicles as their phosphorylated derivatives by this transferase system (LO), raises the question as to the physiological role of hexokinase in these cells. Data presented elsewhere (21) have demonstrated the existence of the PEP-PT system in 8. saliva&s as well :\s the in viva inhibition of enolase by fluoride. These results have lead to the conclusion that the fluoride inhibition of glucose6-P formation is caused, at least in part, by the decreased synthesis of the P-enolpyruvate required for glucose transport. Fluoride inhibition of glycogen synthesis. Regardless of the actual site of fluoride action which leads to the observed decline in the cellular glucose-&P content, the consequences of this decrease will be considerable. Obviously, the flux of carbon through the glycolytic pathway will be severely curtailed, making less glucose-6-P available for synthesis and catabolism. The rapid decline of both the cellular ATP concentration and glycogen synthesis attest to this fact. The finding that the glycogen-synthesizing enzymes are not fluoride-sensitive, however,
602
KANAPKA,
KHANDELWAL,
raises the question as to how glycogen formation can be prevented by fluoride at concentrations permitting appreciable exogenous glucose uptake (2, 3). Obviously, an inadequate supply of either glucose-6-P and/or ATP would limit glycogen synthesis. Under conditions of fluoride inhibition, where entry of glucose into the cell is restricted, one would expect that glucose-6-P would be required preferentially for energy production. Supporting this suggestion are the theoretical considerations proposed by Atkinson (22) who has suggested that one mode of control of biosynthetic reactions is through the “energy charge” of the adenylate system, defined as [ATP + 1/2ADP]/[AMP + ADP + ATP] (23). A high-energy charge in a cell will allow biosynthetic reactions to proceed, while low-energy charge would inhibit such reactions. Thus, bacterial glycogen will not be synthesized when the demand for ATP utilization is equivalent to ATP production (24). In this condition, the intracellular steady-state concentration of ATP, and hence the energy charge, would be low. Therefore, the ATP required as a substrate for the ADP-glucose pyrophosphorylase reaction in the glycogen synthetic pathway, as well as the required glucose-l-P (from glucose-6-P), would not be available. Furthermore, when the cellular ATP concentration is low, the concentration of the total of ADP, AMP, and Pi must necessarily be high. These substances are generally feedback inhibitors of bacterial ADP-glucose pyrophosphorylase (25), although enzymes from different species are inhibited by different members, or combinations of members, of this group. Thus, even though some glucose6-P may accumulate in NaF-treated cells of S. saliva&s, the low concentration of ATP existing in the cell under these conditions would make it unavailable for glycogen synthesis. ACKNOWLEDGMENT The research reported here was supported by a grant from the Medical Research Council of Canada (MA-3546).
AND
HAMILTON REFERENCES
1. HAMILTON, I. R., Can. J. Microbial. 14, 65 (1968). 2. HAMILTON, I. R., Can. J. Microbial. 16, 1013 (1969). 3. HAMILTON, I. R., Can. J. Microbial. 16, 1021 (1969). R. C., 4. WEISS, S., KING, W. J., KESTENBAUM, AND DONOHUE, J. J., Ann. N. Y. Acad. Sci. 131, 839 (1965). H. J., AND KLEINBERG, I., Arch. 5. SANDHAM, Oral Biol. 14, 619 (1969). R. W., AND MAITRA, P. K., Anal. 6. ESTABROOK, Biochem. 3, 369 (1962). 7. MAITRA, P. K., AND ESTBBROOK, R. W., Anal. Biochem. 7, 472 (1964). G. R., AND GETCHELL, C., Clin. 8. KINGSLEY, Chem. 6, 466 (1960). 9. SHEN, L., AND PREISS, J., Biochem. Biophys. Res Commun. 17, 424 (1964). 10. GRE~;NBERG, E., .XND PREISS, J., J. Biol. Chem. 240, 2341 (1965). 11. CHEN, G. S., AND SEGEL, I. H., Arch. Biochem. Biophys. 127, 164 (1968). J., GURNEY, R., AND SPERBER, 12. RUNNSTROM, E., Enzymologia 10, 1 (1941). W. J., .%ND CHUNG, C. W., Ann. 13. NICKERSON, J. Botany 39, 669 (1952). 14. CHUNG, C. W., AND NICKERSON, W. J., J. Biol Chem. 208, 395 (1954). 15. NAJJAR,~. A., J. Biol. Chem. 176,281 (1948). 16. YANQ, S. F., AND MILLER, G. W., Biochem. J. 88, 509 (1963). 17. SCARBOROUGH, G. A., RCMLEY, M. K., AND KENNEDY, E. P., Proc. Nat. Acad. Sci. U. S. A. 80, 951 (1968). 18. WEST, I. C., Fed. Eur. Biol. Sot. Lett. 4, 69 (1969). 19. KAB.~CK, H. R., in “Annual Review of Biochemistry” (E. E. Snell, ed.), Vol. 39, p. 561. Annual Reviews, Inc., Palo Alto, California (1970). 20. K?LB.~cK, H. R., J. Biol. Chem. 243,371l (1968). 21. KANAPKA, J. A., AND HAMILTON, I. R., to be published in Arch. Biochem. Biophys. 22. ATKINSON, D. E., Biochemistry 7,403O (1968). 23. ATKINSON, D. E., AND WALTON, G. M., J. Biol. Chem. 242, 3239 (1967). 24. EIDELS, L., EDELMANN, P. L., AND PREISS, J., Arch. Biochem. Biophys. 140, 60 (1970). 25. PREISS, J., in “Current Topics of Cellular Regulation” (B. L. Horecker and E. R. Stadtman, eds.), Vol. 1, p. 125. Academic Press, New York (1969).
OF
BIOCHEMISTRY
Fluoride
AND
inhibition
salivarios:
Relation
JOSEPH A. KANAPKA, Department
MIOPHYSICS
144, 596402
of Glucose&P to Glycogen RAMJI
(1971)
Formation Synthesis
1,. KHANDELWAL,
in Streptococcus and
AND
IAN R. HAMILTON
of Oral Biology (Biochemistry), Faculty of Dentistry, Manitoba, Winnipeg, Canada Received
December
17, 1970; accepted
March
Degradation
University
of
1, 1971
The concentrations of glucose-6-P and ATP in intact cells of the oral microbe, Streptococcus saliva&s, were analyzed during anaerobic glucose metabolism in the presence and absence of fluoride. Addition of 2.4 mM NaF to cells actively degrading glucose resulted in an immediate decrease in the cellular glucose-6-P and ATP content concomitant with the complete inhibition of glucose uptake and glycogen synthesis. A noticeable decrease in cellular glucose-6-P concentration was produced in cells metabolizing glucose by concentrations of NaF as low as 0.06 mM, regardless of whether the inhibitor was added before or after the substrate. After the initial decline in the glucose-6-P content, the concentration of this intermediate frequently increased, resulting from the degradation of endogenous glycogen. ATP, on the other hand, remained low throughout the experimental period. Experiments with crude enzyme preparations of the organism demonstrated that the glycogen synthetic enzymes, phosphoglucomutase (EC 2.7.5. l), ADPG pyrophosphorylase, and ADPG glucan transferase (ADP-glucose:glycogen glucosyl transferase), as well as phosphorylase (EC 2.4.1 .l) in the degradative pathway, were fluoride-insensitive at NaF concentrations inhibit,ing in viva synthesis. The inability of 2.4 mu NaF to inhibit glucose-6-P formation from glycogen in &JO in the absence of exogenous glucose confirms that phosphoglucomutase and phosphorylase in this organism are not inhibited by fluoride. The results strongly suggest that fluoride interacts, in some manner, with the sugar transport system in the organism as phosphorylation by hexokinase (EC 2.7.1.1) was not fluoride-sensitive. The “apparent” inhibition of glycogen synthesis is discussed in relation to the availability of ATP and glucose-6-P.
Anaerobic glucose metabolism by resting cells of the oral microbe, Xt~eptococcus saliva&s, results in the formation of lactic acid and large quantities of intracellular polyglucose glycogen (1). Fluoride has been shown to inhibit exogenous glucose metabolism, intracellular glycogen synthesis, and the degradat,ion of this polymer in the absence of an external carbon source (2, 3). Of these three parameters, glycogen synthesis was by far the most sensitive, being completely inhibited by low levels of fluoride under condit,ions of appreciable glucose degradation. On t,he other hand, the degradation of this endogenous energy source was inhibited to a much lesser degree t,han was exogenous glucose metabolism. From these studies it, was concluded that since the 596
exogenous and endogenous pathways had widely differing fluoride sensitivities, and yet shared a common glycolytic pathway from glucose-6-P to lactate, the prime site of fluoride action in intact cells of S. salivarius was at some undetermined point in the glycolytic scheme prior to glucose-6-P formation. A similar conclusion was reached in studies with S. mitis (4) and mixed oral flora (5). Evidence will be presented in this paper supporting the above supposition by demonstrating that low concentrations of fluoride had a pronounced and rapid inhibitory effect on the synthesis of glucose-6-P in whole cells of S. salivarius metabolizing exogenous glucose. Further results with crude enzyme preparations will demonstrate that the
FLUORIDE
INHIBITION
enzymes in the glycogen synthetic pathway are insensitive to fluoride at inhibitor concentrations above those required to completely inhibit ifz vivo synthesis. MATERIALS
AND
METHODS
The procedures for the growt,h, harvesting, and preparation of washed cells of Streptococcus saliv&us have been described previously (1). Cells were grown in a medium containing 170trypton‘e, 0.5% yeast extract, 0.3yo KsHP04, and 0.1% glucose, and were always harvested in the exponential phase of growth. Cell-free extracts were prepared from washed cells by sonication in a Branson sonifier (Heat Systems Ultrasonics, Plainview, New York) for 3 min at 0”. The supernatant fluid obtained after centrifugation at 30,000 g was dialyzed against 100 vol of Tris-HCl butler (50 mM, pH 7.5) for 4 hr. This dialyzed extract was used for the assay of all enzymes tested in this study. In vivo ezperintenls. The direct effect of fluoride on glycolysis in intact cells of S. salivariz~s was determined by measuring the intracellular concentration of glucose-6-P and ATP before and after the addition of NaF during glucose degradation. Washed rest,ing cells were incubated at 10 mg dry wt/ml (approximately 1.25 X lOlo cells/ml) in pot.assium phosphate buffer (0.05 M, pH 7.2) at 37” in an atmosphere of Ns + 5% Cot. After equilibration for 10 min, a zero-time sample was removed and glucose, to an approximate final concentration of 1.5 pmoles/mg cells, was added to the remaining cells to start the reaction. With the exception of one experiment, where the cells were incubated with various NaF concentrations during the equilibratjion period, the cells were exposed to fluoride after the onset of glucose metabolism. III these lat,ter cases, a portion of the incubated cells was rapidly removed at a predetermined point during metabolism and added to NaF under the same condit.ions in a separate reaction flask. Samples (0.5-1.0 ml) were removed periodically in both the control and fluoride-containing flasks and added to an equal volume of 1 N perchloric acid to stop the reaction and to extract the cells. Extraction proceeded for 30 min at room temperature, followed by neutralization of the samples to pH 7.G with KOH-triethanolamine buffer (6). After rapid freezing and thawing, cell debris and precipitated KC104 were removed by centrifugation at 3000 g at 4”. The supernatant fractions were stored frozen until analyzed, with the assays for glucose-6-P and ATP always completed within 48 hr after sampling. When glycogen synthesis was measured, glucose-U-l% was employed as the substrate and additional samples (0.2 ml) were removed and added to 1.0 ml of 50yo ethanol in 0.1 x HCI to stop the reaction.
OF GLUCOSE-6-P
,597
Metabolic analyses. The concentration of glucose-6-P and ATP extracted from the cells was assayed by t’he direct enzymatic-fluoromet,ric methods outlined by Maitra and Estabrook (7). Glucose-6-P was determined by its conversion to Gphosphogluconate with NADP+ and commercial glucose-6-P dehydrogenase. The same system was used to assay ATP by the addition of hexokinase and glucose. The quantity of these metabolites in the samples was determined by the addition of an internal standard of glucose-G-P which had been standardized both gravimetrically and spectrophotometrically. Fluorescence of the rcsultant NADPH was measured in a Farrand Ratio Fluorometer equipped with a modified Heath Model EU-BOB Servo Recorder (Farrand Optical Co., Mt. Vernon, New York). A mercury vapor lamp H85 A3/UV served as the radiation source, and the light for excitation was isolated with a Farrand interference filter with peak transmission at 342 rng, while the fluorescent light was isolated with a 435.rnp interference filter. Extracellular glucose was determined by the glucose oxidase method (8). Glycogen synthesis was determined by the incorporation of glucose-U14C into intact cells as described previously (I). Enzyme assays. Phosphoglucomutase activity in cell-free extracts was determined in a coupled enzyme assay by the conversion of glucose-l-P to glucose-6-P in the presence of catalytic amounts of glucose-1,6-diphosphate. Glucose-6-P was further converted to 6-phosphogluconic acid with commercial glucose-6-phosphate dehydrogenase and NADP. The formation of the resulting NADPH was measured spectrophotometrically at 340 rns. The reaction mixture consisted of 10 mM Tris-HCl (pH 7.5), 0.5 mM MgCls, 0.5 rnM glucose-l-P, 4 PM glucose-1,6-diphosphate, 0.2 mg NADP+, 20 pg commercial glucose-6-phosphate dehydrogenase, and 5.3 rg extract protein in a total volume of 1.0 ml. ADPG pyrophosphorylase act,ivity in the extract was determined by the incorporation of glucose-U-14C from glucose-‘%-l-P (1 X lo6 dpm/ pmole) into ADP-glucose (9). The reaction mixture consisted of 50 mM Tris-HCl (pH 7.8)) 0.5 mM glucose-14C-l-P, 1 mM ATP, 10 mM MgC12, 0.9 rg inorganic pyrophosphatase, and 250 pg extract protein in a total volume of 0.2 ml. ADPG glucan transferase was determined by the incorporation of glucose-UJ4C from ADP-glucose1% (0.2 X IO6 dpm/pmole) into glycogen (10). The reaction mixture contained 50 mM Tris-HCl (pH 7.8), 0.5 mM ADP-glucosel%!, 25 mM KCl, 10 mM mercaptoethanol, 0.5 mg glycogen, 0.2 mg bovine serum albumin, and 25 rg extract protein in a total volume of 0.2 ml. Phosphorylase activity was determined by the
598
KANAPKA,
KHANDELWAL,
AND
HAMILTON
incorporat,ion of glucose-U-r4C from glucoseJ4C1-P into glycogen in the absence of ATP (11). The reaction mixture contained 100 mM fi-glycerophosphate buffer (pH 6.8), 10 mM glucose-W-l-P, 0.5 mg glycogen, and 89 pg extract protein in a total volume of 0.1 ml. Materials. All enzymes, co-factors, and glycolytic intermediates used in these experiments were purchased from Boehringer Mannheim Corp. (New York). All other chemicals were of reagent grade. RESULTS Efect oj jhoride on in vivo glucose mefabolism. As shown previously (1,2), the addition of exogenous glucose to the nonproliferating intact celts of S. salivarius resulted in the immediate uptake of this substrate with concomitant glycogen synthesis. Confirmation of these results is shown in Fig. 1 along with the cellular concentrations of glucose6-P and ATP. Upon glucose addition, the intracellular content of glucose-6-P and of ATP rose rapidly from low endogenous levels to higher levels characteristic of exogenous glucose metabolism. At the point of glucose 8exhaustion (3.6 min), the intracellular glycogen content was maximum, and the glucose-6-P concentration began to decline dramatically to a low level characteristic of endogenous metabolism. The ATP content 0, at this point, however, continued at a 0 2 relatively constant level probably because 4 6 MINUTES the cells had started to degrade the newly formed cellular polysaccharide for energy. FIG. 1. The effect of 2.4 mM NaF on exogenous The addition of 2.4 mM NaF to the cells glucose uptake, glycogen synthesis and degradacontent of glucose-6-P 1.5 min after the addition of the glucose tion, and the intracellular and ATP, during anaerobic glucose metabolism profoundly altered this metabolic pattern. by washed cells of 8. salivarius. Control cells Exogenous glucose uptake from the medium (0); fluoride-treated cells (0). was immediately stopped, as was glycogen synthesis, confirming our earlier results glucose-6-P in the fluoride-treated cells was (2, 3). Furthermore, the addition of fluoride also resulted in a rapid decrease in the cellu- not associated with an increase in the cellular concentration of ATP. The continued low lar concentrations of ATP and glucose-6-P, concomitant with the effects on glucose synthesis of both ATP and glucose-6-P in the presence of fluoride during the experiuptake and glycogen synthesis. Although glucose-6-P initially dropped to 15% of the mental period has been shown elsewhere (21) to be the result of the degradation of control value 15 set after the fluoride addition, the concentration increased to 50% of small quantities of cellular glycogen. The fluoride effect on the cellular levels of this value before the end of the 6-min exATP and glucose-6-P was a sustained one perimental period. ATP, on the other hand, since prolonged incubation of the cells (up dropped to 20 % of the control value initially and remained at about this level in the to 40 min) demonstrated that as long as remaining samples. Thus, the increase in fluoride was present in the medium the
FLUORIDE
INHIBITION
cellular content of both ATP and glucose-6-P remained at greatly reduced levels. Unlike the ATP concentration which remained low throughout the experimental period, the cellular content of glucose-6-P in these cases often rose to levels slightly above the steadystate concentration maintained in the control cells in the absence of exogenous glucose. In the control cells, on the other hand, the glucose-6-P concentration dropped immediately upon depletion of the exogenous glucose, while the ATE’ concentration declined very gradually, requiring almost 35 min to reach the ATP level observed in the fluoride-treated cells. This indicates that the endogenous degradation of intracellular glycogen in this organism (1) can maintain the ATP content of the cells at a relatively high level for a considerable amount of time after the depletion of the exogenous carbon source. E$ect of fluoride comentration on intracellular glucose-6-P concentration. Previous studies have demonstrated that 0.96 ml% NaF completely inhibited the metabolism of exogenous glucose by S. salivarius (2,3). Glycogen synthesis, on the other hand, was completely inhibited by fluoride at half of this concentration, while a level as low as 0.06 rnwl produced a significant inhibitory effect. Therefore, an experiment was designed to investigate the effect of low fluoride levels 011 the intracellular concentration of glucose-6-P, the first apparent intracellular precursor in glycogen synthesis. Furthermore, in order to test for the so-called substrate-protective effect (12)) fluoride was added to the cells before and after the substrate. When the cells were preincubated at 37” with NaF (O-l.2 mlr) followed by the addition of 15 mzr glucose, a progressive decrease in the maximum intracellular glucose-6-P concentration was observed (Table I). As the NaF concentration increased to 1.2 m&f, the maximum glucose-6-P level decreased from 7.1 to 1.0 nmoles/mg dry wt cells. A more dramatic effect was observed under the same conditions when these NaF concentrations were added after the onset of glucose metabolism. In this experiment, the inhibitor was added 30 set after the addition of the glucose and again resulted in an im-
599
OF GLUCOSE-BP TABLE
I
EFFBXT OF PRBINCUB.ITION WITH NaF ON THE CELLULAR CONCENTRATION OF GILMAXIMUM cosn-6-P IN WASHED CELLS OF S. saliaarius DURING:SIYBSEQUENT GLUCOSE METABOLISM N\'aF mn 0
0.06 0.12 0.25 0.60 1.20 B nmoles/mg
Glucose-6-Pa
Percent of control
7.1 4.3 3.1 2.4 1.6 1.0
100 61 44 34 23 14
dry weight
OY 0
of cells.
1.0
2.0
MINUTES FIG. 2. The effect of varying concentrations of NaF on glucose-6-P levels in cells of S. salivariusmetabolizing glucose. NaF was added at 30 set to give the following final concentrations (mM): 0 (0); 0.06 (0); 0.12 (A); 0.25 (A); 0.60 (a);
1.2 (ml. mediate, rapid decrease in the intracellular glucose-6-P concentration (Fig. 2). KaF, as low as 0.06 mill, produced an initial slight drop in the glucose-6-P content followed by a rapid recovery to levels similar to those in the control cells. However, at higher NaF concentrations (0.12-1.2 mM) a much more pronounced initial drop in glucose-6-P levels was observed without a subsequent increase during the 2-min experimental period. As this figure indicates, the decrease in the
600
KANAPKA,
KHANDELWAL,
AND
TABLE INFLUENCE
OF
NaF
0
1.2
2.4
4.8
1474 147 876 106” 235d
159
159 90 107 363
171 147
147 147
glucose-6-P formed/mg prot,ein/min. ADP-glucose formed/mg protein/min. glycogen formed/mg protein/min. glucose-l-P incorporated into glycogen/mg
cellular glucose-6-P concentration was very rapid, occurring within 15 set of the fluoride addition. This represented the minimum time necessary for accurate sampling in the system employed. Fluoride e$ect on the enzymes involved in glycogen metabolism. Two possibilities exist to explain the in vivo inhibition of glycogen synthesis in S. saliva&us by fluoride: (a) fluoride interacts directly with one or more of the enzymes involved in the synthetic process, or (b) that insufficient glucose-6-P is available for synthesis. Phosphoglucomutase, in the glycogen synthetic pathway, generally has been cited as the point of fluoride inhibition in microbes (13, 14), while in. vitro fluoride inhibition of this enzyme from rabbit muscle (15) and higher plants (16) has been observed. To test for the first possibility, dialyzed crude extract preparations of S. salivarzus were prepared, and the activities of phosphoglucomutase, ADPG pyrophosphorylase, and ADPG glucan transferase in the synthetic pathway, as well as phosphorylase in the degradative pathway, were assayed in the presence and absence of fluoride. The results (Table II) indicate that none of these enzymes was sensitive to fluoride &a z&o, at concentrations equal to, or higher those
which
OF GLYCOGEN
?;aF (IIIM)
Phosphoglucomutase Preincubation No preincubation ADPG Pyrophosphorylase ADPG Glucan transferase Phosphorylase
than,
II
ON THE ENZYMES INVOLVED IN THE SYNTHESIS AND DEGRBD.ITION IN CRUDE EXTRACTS OF 8. sakivarius
Enzyme
a nmoles b pmoles c nmoles d pmoles
HAMILTON
completely
inhibited
in vivo glycogen synthesis. Phosphoglucomutase was resistant to NaF up to 9.6 m&f regardless of whether the enzyme was preincubated with fluoride, or whether the
7.2
9.6
-
171 147
-
97
85
-
111
108
-
-
407
-
450
-
protein/min.
inhibitor was added after the reaction had started. ADPG pyrophosphorylase and ADPG glucan transferase were not significantly affected by concentrations to 4.8 mm NaF. Phosphorylase activity, on the other hand, was actually stimulated by concentrations of NaF up to 7.2 rnb1. In vivo e$ect of Jluoride on glycogen degradation. Since small intracellular glucose-l-P concentrations exist in cells of S. salivarius during exogenous glucose metabolism, it was difficult to measure accurately the amount of this compound in the presence of the normally high levels of glucose-6-P. Therefore, evidence that phosphoglucomutase was fluoride-insensitive in the direction of glycogen synthesis was difficult to confirm in vivo in the presence of exogenous glucose. However, the effect of fluoride on this enzyme and on phosphorylase could be measured accurately by monitoring the cellular content of glucose-6-P during glycogen degradation in the absence of external glucose. This was tested in an experiment where whole cells were incubated with glucose to permit the synthesis of intracellular glycogen. Approximately 1 min after the exogenous glucose had been depleted, 2.4 mM NaF was added to a portion of the cells. The glucose-6-P level in the control cells rose upon the addition of the substrate immediately in the characteristic manner and dropped again once the exogenous substrate was exhausted (Fig. 3). Under these conditions of glycogen degradation, glucose-
FLUORIDE
IXHIBITION
Glucose
MINUTES FIG. 3. The effect of 2.4 rnM NaF on glucose-6-P levels in cells of S. salioarius-metabolizing glycogen. Control cells (0) ; fluoride-treated cells (0).
6-P formation was initially stimulated by the addition of the fluoride, resulting in concentrations higher than those in the controls but which then decreased to the control level by the end of the experimental period. As inhibition of phosphoglucomutase and/or glycogen phosphorylase would have resulted in a decrease in the glucose-6-P concentration, it is clear that these enzymes are not fluoride-sensitive under in viva conditions thereby confirming the in vitro enzyme assays. DISCUSSION
Fluoride effect on glucose-6-P formation. The fluoride inhibition of glucose-6-P formation in whole cells of S. salivarius confirms the indirect evidence obtained in previous studies which indicated that the site of inhibition was at some point in glycolysis prior t’o glucose-B-P formation. Although this fluoride-sensitive component is unknown, possible sites of inhibition would include the glucose phosphorylating enzyme, hexokinase, the reactions involved in the glucose transport process, or associated processes, such as bhe availability of ATP for phosphorylat’ion.
OF GLUCOSE-O-I’
GO1
In order to delineate the site of fluoride action in 8. salivarius, hexokinase activity in crude extracts was assayed by the formation of glucose-6-P from glucose and ,4Tl’ in the presence and absence of fluoride. As no loss of activity \vas observed at Sal? concentrations as high as 24 mnr, one must conclude that hexokinase is not the fluoridesensitive component. The role of ATP is more difficult to assess. Although the cellular ATP content decreases rapidly upon fluoride addition (Fig. l), it was not possible from these results to determine whether the decrease was the cause or the effect of the decrease in the cellular glucose-&P concentration. One might assume that ATI’ would be required for the phosphorylation of glucose, either within the cell by the hexokinase reaction, or at the membrane during transport (17, IS). However, the fact that glucose transport in many bacterial species is mediated by the phosphoenolpyruvate-phosphotransferase (I’EPPT) system (see review, 19) indicates that ATl’ may not be involved in active glucose transport. Indeed, the fact that sugars are concentrated in membrane vesicles as their phosphorylated derivatives by this transferase system (LO), raises the question as to the physiological role of hexokinase in these cells. Data presented elsewhere (21) have demonstrated the existence of the PEP-PT system in 8. saliva&s as well :\s the in viva inhibition of enolase by fluoride. These results have lead to the conclusion that the fluoride inhibition of glucose6-P formation is caused, at least in part, by the decreased synthesis of the P-enolpyruvate required for glucose transport. Fluoride inhibition of glycogen synthesis. Regardless of the actual site of fluoride action which leads to the observed decline in the cellular glucose-&P content, the consequences of this decrease will be considerable. Obviously, the flux of carbon through the glycolytic pathway will be severely curtailed, making less glucose-6-P available for synthesis and catabolism. The rapid decline of both the cellular ATP concentration and glycogen synthesis attest to this fact. The finding that the glycogen-synthesizing enzymes are not fluoride-sensitive, however,
602
KANAPKA,
KHANDELWAL,
raises the question as to how glycogen formation can be prevented by fluoride at concentrations permitting appreciable exogenous glucose uptake (2, 3). Obviously, an inadequate supply of either glucose-6-P and/or ATP would limit glycogen synthesis. Under conditions of fluoride inhibition, where entry of glucose into the cell is restricted, one would expect that glucose-6-P would be required preferentially for energy production. Supporting this suggestion are the theoretical considerations proposed by Atkinson (22) who has suggested that one mode of control of biosynthetic reactions is through the “energy charge” of the adenylate system, defined as [ATP + 1/2ADP]/[AMP + ADP + ATP] (23). A high-energy charge in a cell will allow biosynthetic reactions to proceed, while low-energy charge would inhibit such reactions. Thus, bacterial glycogen will not be synthesized when the demand for ATP utilization is equivalent to ATP production (24). In this condition, the intracellular steady-state concentration of ATP, and hence the energy charge, would be low. Therefore, the ATP required as a substrate for the ADP-glucose pyrophosphorylase reaction in the glycogen synthetic pathway, as well as the required glucose-l-P (from glucose-6-P), would not be available. Furthermore, when the cellular ATP concentration is low, the concentration of the total of ADP, AMP, and Pi must necessarily be high. These substances are generally feedback inhibitors of bacterial ADP-glucose pyrophosphorylase (25), although enzymes from different species are inhibited by different members, or combinations of members, of this group. Thus, even though some glucose6-P may accumulate in NaF-treated cells of S. saliva&s, the low concentration of ATP existing in the cell under these conditions would make it unavailable for glycogen synthesis. ACKNOWLEDGMENT The research reported here was supported by a grant from the Medical Research Council of Canada (MA-3546).
AND
HAMILTON REFERENCES
1. HAMILTON, I. R., Can. J. Microbial. 14, 65 (1968). 2. HAMILTON, I. R., Can. J. Microbial. 16, 1013 (1969). 3. HAMILTON, I. R., Can. J. Microbial. 16, 1021 (1969). R. C., 4. WEISS, S., KING, W. J., KESTENBAUM, AND DONOHUE, J. J., Ann. N. Y. Acad. Sci. 131, 839 (1965). H. J., AND KLEINBERG, I., Arch. 5. SANDHAM, Oral Biol. 14, 619 (1969). R. W., AND MAITRA, P. K., Anal. 6. ESTABROOK, Biochem. 3, 369 (1962). 7. MAITRA, P. K., AND ESTBBROOK, R. W., Anal. Biochem. 7, 472 (1964). G. R., AND GETCHELL, C., Clin. 8. KINGSLEY, Chem. 6, 466 (1960). 9. SHEN, L., AND PREISS, J., Biochem. Biophys. Res Commun. 17, 424 (1964). 10. GRE~;NBERG, E., .XND PREISS, J., J. Biol. Chem. 240, 2341 (1965). 11. CHEN, G. S., AND SEGEL, I. H., Arch. Biochem. Biophys. 127, 164 (1968). J., GURNEY, R., AND SPERBER, 12. RUNNSTROM, E., Enzymologia 10, 1 (1941). W. J., .%ND CHUNG, C. W., Ann. 13. NICKERSON, J. Botany 39, 669 (1952). 14. CHUNG, C. W., AND NICKERSON, W. J., J. Biol Chem. 208, 395 (1954). 15. NAJJAR,~. A., J. Biol. Chem. 176,281 (1948). 16. YANQ, S. F., AND MILLER, G. W., Biochem. J. 88, 509 (1963). 17. SCARBOROUGH, G. A., RCMLEY, M. K., AND KENNEDY, E. P., Proc. Nat. Acad. Sci. U. S. A. 80, 951 (1968). 18. WEST, I. C., Fed. Eur. Biol. Sot. Lett. 4, 69 (1969). 19. KAB.~CK, H. R., in “Annual Review of Biochemistry” (E. E. Snell, ed.), Vol. 39, p. 561. Annual Reviews, Inc., Palo Alto, California (1970). 20. K?LB.~cK, H. R., J. Biol. Chem. 243,371l (1968). 21. KANAPKA, J. A., AND HAMILTON, I. R., to be published in Arch. Biochem. Biophys. 22. ATKINSON, D. E., Biochemistry 7,403O (1968). 23. ATKINSON, D. E., AND WALTON, G. M., J. Biol. Chem. 242, 3239 (1967). 24. EIDELS, L., EDELMANN, P. L., AND PREISS, J., Arch. Biochem. Biophys. 140, 60 (1970). 25. PREISS, J., in “Current Topics of Cellular Regulation” (B. L. Horecker and E. R. Stadtman, eds.), Vol. 1, p. 125. Academic Press, New York (1969).