Archives Internationales de Physiologie, de Biochimie et cle Biophysique, 1992, 100, 203-205
203
ReCu le 15 juin 1991.
In vitro effects of divalent metal ions and metabolic modulators on purified glutamine synthetase from brain of teleostean fish. BY
R. A. SINGH
(I)
and S. N. SINGH (')
[('I Department of Zoology, S. G. R. Post Graduate College, Dobhi, Jaunpur, Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
(1. P.; (') Biochemistry & Molecular Biology Laboratory, Centre of Advanced Study in Zoology, Banaras Hindu University, Varanasi, India]
(1 figure)
The purified brain glutamine synthetase of a teleostean fish, Clarias batrachus has been examined under the influence of divalent metal ions activation and in vitro studies using different amino acids and metabolic modulators. All observations of the enzyme activity are based on transferase reaction at optimum temperature and pH. The enzyme exhibits an absolute requirement for Mn2+(most potent) Mg2+ and Co2+.The activity is markedly inhibited by leucine, asparagine, isoleucine, carbamyl phosphate, uridine monophosphate and glutamate and activated by a-ketoglutarate. The significance of these results has been discussed in brief with emphasis on its involvement in teleostean physiology and metabolism.
Introduction Ammonia detoxification and glutamate recycling in neural tissue is mediated primarily by the enzyme glutamine synthetase (GS; EC 6.3.1.2) (SHANK& A~RISON, 1981). GS requires a divalent metal ion for activity (WEDLERet al., 1982; DENMAN& WEDLER, 1986) and its regulation 1984; MAURIZI& GINSBURG, is brought about by a variety of control mechanisms & GINSBURG, 1974). The im(MISTER,1974; STADMAN portant one is the feedback regulation of the enzyme by several metabolites. However, such changes on regulatory mechanism have not been carried out on GS of freshwater teleostean fishes which are primarily ammonotelic and may be subjected to a rather severe ammonia load. Recently we have reported the GS association with ammonia detoxication and nitrogen metabolism in brain tissue (SWGH& SINGH,1989). In the present attempt, the purified enzyme preparation is subjected to observe the effects of some divalent metal ions and metabolic modulators with a view to see the alterations (if any) in the enzyme activity. The results obtained from the present investigation are discussed with particular reference to their possible physiological impacts.
Materials and Methods All the chemicals (analytical grade) and biochemicals used were purchased from Sigma Chemicals Co. USA. Adult Clarias batrachus weighing 80-85 g, their acclimation to the laboratory conditions, brain tissues removal and enzyme extraction were performed (SINGH& SINGH,1990). The purified enzyme
samples were obtained after isolation of GS from brain tissue extract (SINGH& SINGH,1989). The procedures involved acid precipitation at pH 4.3 followed by chromatography on hydroxylapatite and DEAEcellulose. Trace amounts of contaminants were eliminated by rechromatography on DEAE-cellulose. Spectrophotometric assay procedures for determination of GS activity by transferase reaction was the same (ROWEet al., 1970) with minor modifications. The modified standard reaction mixture contained imidazole (20 mM; pH 7.2), MnCl, (3 mM), sodium arsenate (20 mM), ADP (0.4mM), NH,OH (60 m; pH 7) and L-glutamine (50 mM). The reaction was initiated by adding the appropriately diluted enzyme and after 15 min of incubation it was stopped with a ferric et al., 1962). The activity chloride reagent (PAMILJANS of GS was determined by measuring the amount of yglutamyl hydroxamate formed per min per mg protein after measuring the protein content (LOWRYet al., 1951). Calibration curve was determined by the use of y-glutamyl hydroxamate standard. Zero-time assay mixture was used as reaction blanks. All the enzyme assays were carried out in duplicate at optimum pH (7.2) and temperature (35°C). Effects of different divalent metal ions, viz. Mn2+, Mg2+and Coz+on Gs activity were observed by adding them to the suitably diluted purified enzyme, not previously treated with the metal ions. Activities of the enzyme were determined by transferase reaction at different concentrations (0-40 mM) of these ions. The appropriate GS assays were also made with the metabolites (20 mM), viz., leucine, asparagine, isoleucine, carbamyl phosphate, uridine monophosphate, glutamate and a-ketoglutarate.
204
R . A . SINGH AND S. N. SMGH
TABLEI. In vitro effect of divalent cations on the activity of glutamine synthetase purified from brain of Clarias batrachus. Enzyme activity
Concentration
I
Per cent activity*
Units/mg protein Mn2+
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
1
2 3 5 10 20 40
*
0.638 f 0.033 0.825 f 0.053 1.000 f 0.035 0.988 f 0.061 0.963 f 0.056 0.938 & 0.033 0.875 t 0.036
0.255 0.338 0.475 0.550 0.563 0.525 0.338
f 0.019 f 0.037 f 0.045
0.067 rt 0.040 +_ 0.029 f 0.027 ?
0.063 0.088 0.124 0.213 0.375 0.563 0.500
t 0.016 k
t t f f f
I
CO’+
Mg’+
0.089 0.015 0.037 0.050 0.028 0.028
63.8 f 2.708 82.5 f 5.261 98.8 f 6.086 96.3 5.453 93.8 f 3.259 87.5 3.571
* *
22.5 33.8 47.5 55.0 56.3 52.5 33.8
f
1.930
f 3.663 f 4.515
f 6.671 f 4.008 t 2.872 & 2.716
Mgz’ 6.3 8.8 13.5 21.3 37.5 56.3 50.0
k
1.640
f 8.874 f 1.500 f 3.678 5.000 f 2.773 f 2.817
*
Percentage of enzyme activities are calculated as against the highest activity obtained with 3 mM Mn’’.
“t
Results Table I shows in vitro effects of divalent cations (Mn2+,Mgz+and Co2+)known to function as cofactors for the GS activity. The enzyme exhibits an absolute requirement for these ions. However, MnZ+is found to be highly required and its 3 m~ concentration causes 100% enzyme activity and higher concentrations are rather inhibitory. Mg2+and Coz+activate the enzyme up to 56% only at 20 m~ and 10 m~ respectively but further increase in concentrations inhibits the enzyme activity which is more pronounced in the case of Co2+ at its 40 mM concentration. Figure 1 shows the effects of different metabolites (20 mM) on transferase reaction of Mn2+(3 mM) activated GS activity. The enzyme activity is significantly inhibited in presence of all the metabolites used except that of a-ketoglutarate which significantly activates (1 16%)the enzyme as against the control value. The inhibition by each metabolite shows some variations and a maximum (50%) is observed in the presence of glutamate. The per cent inhibition values are represented in the order of glutamate > uridine monophosphate > carbamyl phosphate > isoleucine > asparagine > and leucine. Discussion
The aim of the present investigation is to study the effects of divalent metal ion ligands on GS activity, its in vitro regulation and involvement in teleostean physiology and metabolism at molecular level, with emphasis on brain cells. GS requires a divalent metal ion for activity and the studies (WEDLERet al., 1982; DENMAN & WEDLER,1984) with the enzyme from different sources indicate that it has been satisfied mainly by Mn2+,Mg2+and Coz+.In the present study (Table I) it appears that MnZ+plays a major role to fulfil such an ionic requirement in the regulation of GS. However, the enzyme is also activated by Mg2+and Co2+.This is of interest because there are instances (ELLIOTT, 1951; LEPO et al., 1982; DOWTON & KENNEDY, 1985) illustrating that Mg” most potentially activates GS while Mn2+and Co” strongly inhibit the Mg2+ activated
i
I 20 > N Z UJ
0
A
B
C
D
E
F
G
H
FIG.1. Effects of various metabolites (20 mM) on purified glutamine synthetase activity from brain of Clarias batrachus. A - control, B - leucine, C - asparagine, D - isoleucine, E - carbamyl phosphate, F - uridine monophosphate, G - glutamate and H - aketoglutarate.
enzyme. Recently (WEDLERet al., 1982) it has been reported that both Mn2+and Mg” bound to the enzyme to have different binding sites but Mn2+may be the true divalent metal ion required for the enzyme activity. Thus, the present findings reveal that like glutamide synthetases from different sources, teleostean brain GS requires certain divalent metal ions (Mn2+most potent) that play a crucial role in regulation of enzyme activity that may regulate the enzyme in vivo. Observations comprising activation of GS by aketoglutarate and inhibition in presence of various metabolites, viz. , leucine, asparagine, isoleucine,
205
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
GLUTAMINE SYNTHETASE FROM BRAIN IN TELEOSTEAN FISH
carbamyl phosphate, uridine monophosphate and glutamate (Fig. 1) support the reported findings in avian (SATOH& ~ ~ A T S U N1983) O , and mammalian (TATE& MEISTER,1971; PAHUJA & REID, 1985) tissues. It appears, therefore, the teleostean GS has most of the properties similar to those reported for avain and mammalian glutamine synthetases. This may reveal that the gene, responsible for synthesis of the enzyme apparently does not undergo any structural change during the course of vertebrate evolution. However, the in vitro regulation by various effectors used may regulate the enzyme activity in teleostean brain in vivo which may be helpful for the physiological and metabolic view point. Acknowledgement. - Authors thank Prof. M.S. KANNUNGO for helpful suggestions, RAS is grateful to Dr. B. P. SINGH,Principal, S. G. R. Post-Graduate College, Dobhi, Jaunpur, U. P. for encouragement and UGC, New Delhi, India for financial assistance.
References DENMAN,R. B. & WEDLER,F. C. (1984) Arch. Biochem. Biophys. 232, 427-440. DOWTON,M. & KENNEDY,I. (1985) Insect Biochem. 15, 763-770. ELLIOTT,W. H. (1951) Biochem. J. 49, 106-112. LEPO,J. E., WYSS,0. & TABITA,F. R. (1982) Biochim. Biophys. Acta 704, 414-421.
LOWRY,0. H., KOSEBROUGH, N. J., FARR,A. L. & RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. MAURIZI,M. R. & GINSBURG, A. (1986) Biochemistry 25, 131-140. MEISTER,A. (1974) The Enzyme (BOYER,P. D. ed.) Vol. 10, pp. 699-754. Academic Press, New York. PAHUJA,S. L. & REID, T. W. (1985) Exp. Eye. Res. 40, 75-83. PAMILJANS, V., KRISHNASWAMY, R., DUMVILLE, G . & MEISTER,A. (1962) Biochemistry 1, 153-158. ROWE,W. B., RONZIO,R. P., WELNER,V. P. & MEISTER, A . (1970) Methods in Enzymology (TABOR,H. &TABOR,C. W. eds.) Vol. 17, pp. 900-910, Academic Press, New York. SATOH,T . & MATSUNO,T. (1983) Comp. Biochem. Physiol. 7 5 8 , 655-658. SHANK,R. P. & APRISON, M. H . (1981) Life Sci. 28, 837-842. SINGH,R. A. & STNGH,S. N. (1989) Arch. Int. Physiol. Biochim. 97, 145-152. SINGH,R. A. & SINGH,S. N. (1990) Arch. Int. Physiol. Biochim. 98, 95-101. STADMAN,E. R. & GINSBURG, A. (1974) T h e E n z y m e ( B ~ Y EP. ~ , D. ed.) Vol. 10, pp. 755-807, Academic Press, New York. TATE,S. S. & MEISTER,A. (1971) Proc. Natl. Acad. Sci., U.S.A. 68, 781-785. WEDLER,F. C., DENMAN,R. B. & ROBY, W. G. (1982) Biochemistry 21, 6389-6396.
S. N. SINGH Biochemistry and Molecular Biology, Department of Zoology, Banaras Hindu University, Varanasi, 221005 India.
203
ReCu le 15 juin 1991.
In vitro effects of divalent metal ions and metabolic modulators on purified glutamine synthetase from brain of teleostean fish. BY
R. A. SINGH
(I)
and S. N. SINGH (')
[('I Department of Zoology, S. G. R. Post Graduate College, Dobhi, Jaunpur, Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
(1. P.; (') Biochemistry & Molecular Biology Laboratory, Centre of Advanced Study in Zoology, Banaras Hindu University, Varanasi, India]
(1 figure)
The purified brain glutamine synthetase of a teleostean fish, Clarias batrachus has been examined under the influence of divalent metal ions activation and in vitro studies using different amino acids and metabolic modulators. All observations of the enzyme activity are based on transferase reaction at optimum temperature and pH. The enzyme exhibits an absolute requirement for Mn2+(most potent) Mg2+ and Co2+.The activity is markedly inhibited by leucine, asparagine, isoleucine, carbamyl phosphate, uridine monophosphate and glutamate and activated by a-ketoglutarate. The significance of these results has been discussed in brief with emphasis on its involvement in teleostean physiology and metabolism.
Introduction Ammonia detoxification and glutamate recycling in neural tissue is mediated primarily by the enzyme glutamine synthetase (GS; EC 6.3.1.2) (SHANK& A~RISON, 1981). GS requires a divalent metal ion for activity (WEDLERet al., 1982; DENMAN& WEDLER, 1986) and its regulation 1984; MAURIZI& GINSBURG, is brought about by a variety of control mechanisms & GINSBURG, 1974). The im(MISTER,1974; STADMAN portant one is the feedback regulation of the enzyme by several metabolites. However, such changes on regulatory mechanism have not been carried out on GS of freshwater teleostean fishes which are primarily ammonotelic and may be subjected to a rather severe ammonia load. Recently we have reported the GS association with ammonia detoxication and nitrogen metabolism in brain tissue (SWGH& SINGH,1989). In the present attempt, the purified enzyme preparation is subjected to observe the effects of some divalent metal ions and metabolic modulators with a view to see the alterations (if any) in the enzyme activity. The results obtained from the present investigation are discussed with particular reference to their possible physiological impacts.
Materials and Methods All the chemicals (analytical grade) and biochemicals used were purchased from Sigma Chemicals Co. USA. Adult Clarias batrachus weighing 80-85 g, their acclimation to the laboratory conditions, brain tissues removal and enzyme extraction were performed (SINGH& SINGH,1990). The purified enzyme
samples were obtained after isolation of GS from brain tissue extract (SINGH& SINGH,1989). The procedures involved acid precipitation at pH 4.3 followed by chromatography on hydroxylapatite and DEAEcellulose. Trace amounts of contaminants were eliminated by rechromatography on DEAE-cellulose. Spectrophotometric assay procedures for determination of GS activity by transferase reaction was the same (ROWEet al., 1970) with minor modifications. The modified standard reaction mixture contained imidazole (20 mM; pH 7.2), MnCl, (3 mM), sodium arsenate (20 mM), ADP (0.4mM), NH,OH (60 m; pH 7) and L-glutamine (50 mM). The reaction was initiated by adding the appropriately diluted enzyme and after 15 min of incubation it was stopped with a ferric et al., 1962). The activity chloride reagent (PAMILJANS of GS was determined by measuring the amount of yglutamyl hydroxamate formed per min per mg protein after measuring the protein content (LOWRYet al., 1951). Calibration curve was determined by the use of y-glutamyl hydroxamate standard. Zero-time assay mixture was used as reaction blanks. All the enzyme assays were carried out in duplicate at optimum pH (7.2) and temperature (35°C). Effects of different divalent metal ions, viz. Mn2+, Mg2+and Coz+on Gs activity were observed by adding them to the suitably diluted purified enzyme, not previously treated with the metal ions. Activities of the enzyme were determined by transferase reaction at different concentrations (0-40 mM) of these ions. The appropriate GS assays were also made with the metabolites (20 mM), viz., leucine, asparagine, isoleucine, carbamyl phosphate, uridine monophosphate, glutamate and a-ketoglutarate.
204
R . A . SINGH AND S. N. SMGH
TABLEI. In vitro effect of divalent cations on the activity of glutamine synthetase purified from brain of Clarias batrachus. Enzyme activity
Concentration
I
Per cent activity*
Units/mg protein Mn2+
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
1
2 3 5 10 20 40
*
0.638 f 0.033 0.825 f 0.053 1.000 f 0.035 0.988 f 0.061 0.963 f 0.056 0.938 & 0.033 0.875 t 0.036
0.255 0.338 0.475 0.550 0.563 0.525 0.338
f 0.019 f 0.037 f 0.045
0.067 rt 0.040 +_ 0.029 f 0.027 ?
0.063 0.088 0.124 0.213 0.375 0.563 0.500
t 0.016 k
t t f f f
I
CO’+
Mg’+
0.089 0.015 0.037 0.050 0.028 0.028
63.8 f 2.708 82.5 f 5.261 98.8 f 6.086 96.3 5.453 93.8 f 3.259 87.5 3.571
* *
22.5 33.8 47.5 55.0 56.3 52.5 33.8
f
1.930
f 3.663 f 4.515
f 6.671 f 4.008 t 2.872 & 2.716
Mgz’ 6.3 8.8 13.5 21.3 37.5 56.3 50.0
k
1.640
f 8.874 f 1.500 f 3.678 5.000 f 2.773 f 2.817
*
Percentage of enzyme activities are calculated as against the highest activity obtained with 3 mM Mn’’.
“t
Results Table I shows in vitro effects of divalent cations (Mn2+,Mgz+and Co2+)known to function as cofactors for the GS activity. The enzyme exhibits an absolute requirement for these ions. However, MnZ+is found to be highly required and its 3 m~ concentration causes 100% enzyme activity and higher concentrations are rather inhibitory. Mg2+and Coz+activate the enzyme up to 56% only at 20 m~ and 10 m~ respectively but further increase in concentrations inhibits the enzyme activity which is more pronounced in the case of Co2+ at its 40 mM concentration. Figure 1 shows the effects of different metabolites (20 mM) on transferase reaction of Mn2+(3 mM) activated GS activity. The enzyme activity is significantly inhibited in presence of all the metabolites used except that of a-ketoglutarate which significantly activates (1 16%)the enzyme as against the control value. The inhibition by each metabolite shows some variations and a maximum (50%) is observed in the presence of glutamate. The per cent inhibition values are represented in the order of glutamate > uridine monophosphate > carbamyl phosphate > isoleucine > asparagine > and leucine. Discussion
The aim of the present investigation is to study the effects of divalent metal ion ligands on GS activity, its in vitro regulation and involvement in teleostean physiology and metabolism at molecular level, with emphasis on brain cells. GS requires a divalent metal ion for activity and the studies (WEDLERet al., 1982; DENMAN & WEDLER,1984) with the enzyme from different sources indicate that it has been satisfied mainly by Mn2+,Mg2+and Coz+.In the present study (Table I) it appears that MnZ+plays a major role to fulfil such an ionic requirement in the regulation of GS. However, the enzyme is also activated by Mg2+and Co2+.This is of interest because there are instances (ELLIOTT, 1951; LEPO et al., 1982; DOWTON & KENNEDY, 1985) illustrating that Mg” most potentially activates GS while Mn2+and Co” strongly inhibit the Mg2+ activated
i
I 20 > N Z UJ
0
A
B
C
D
E
F
G
H
FIG.1. Effects of various metabolites (20 mM) on purified glutamine synthetase activity from brain of Clarias batrachus. A - control, B - leucine, C - asparagine, D - isoleucine, E - carbamyl phosphate, F - uridine monophosphate, G - glutamate and H - aketoglutarate.
enzyme. Recently (WEDLERet al., 1982) it has been reported that both Mn2+and Mg” bound to the enzyme to have different binding sites but Mn2+may be the true divalent metal ion required for the enzyme activity. Thus, the present findings reveal that like glutamide synthetases from different sources, teleostean brain GS requires certain divalent metal ions (Mn2+most potent) that play a crucial role in regulation of enzyme activity that may regulate the enzyme in vivo. Observations comprising activation of GS by aketoglutarate and inhibition in presence of various metabolites, viz. , leucine, asparagine, isoleucine,
205
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by University of Sydney on 01/02/15 For personal use only.
GLUTAMINE SYNTHETASE FROM BRAIN IN TELEOSTEAN FISH
carbamyl phosphate, uridine monophosphate and glutamate (Fig. 1) support the reported findings in avian (SATOH& ~ ~ A T S U N1983) O , and mammalian (TATE& MEISTER,1971; PAHUJA & REID, 1985) tissues. It appears, therefore, the teleostean GS has most of the properties similar to those reported for avain and mammalian glutamine synthetases. This may reveal that the gene, responsible for synthesis of the enzyme apparently does not undergo any structural change during the course of vertebrate evolution. However, the in vitro regulation by various effectors used may regulate the enzyme activity in teleostean brain in vivo which may be helpful for the physiological and metabolic view point. Acknowledgement. - Authors thank Prof. M.S. KANNUNGO for helpful suggestions, RAS is grateful to Dr. B. P. SINGH,Principal, S. G. R. Post-Graduate College, Dobhi, Jaunpur, U. P. for encouragement and UGC, New Delhi, India for financial assistance.
References DENMAN,R. B. & WEDLER,F. C. (1984) Arch. Biochem. Biophys. 232, 427-440. DOWTON,M. & KENNEDY,I. (1985) Insect Biochem. 15, 763-770. ELLIOTT,W. H. (1951) Biochem. J. 49, 106-112. LEPO,J. E., WYSS,0. & TABITA,F. R. (1982) Biochim. Biophys. Acta 704, 414-421.
LOWRY,0. H., KOSEBROUGH, N. J., FARR,A. L. & RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. MAURIZI,M. R. & GINSBURG, A. (1986) Biochemistry 25, 131-140. MEISTER,A. (1974) The Enzyme (BOYER,P. D. ed.) Vol. 10, pp. 699-754. Academic Press, New York. PAHUJA,S. L. & REID, T. W. (1985) Exp. Eye. Res. 40, 75-83. PAMILJANS, V., KRISHNASWAMY, R., DUMVILLE, G . & MEISTER,A. (1962) Biochemistry 1, 153-158. ROWE,W. B., RONZIO,R. P., WELNER,V. P. & MEISTER, A . (1970) Methods in Enzymology (TABOR,H. &TABOR,C. W. eds.) Vol. 17, pp. 900-910, Academic Press, New York. SATOH,T . & MATSUNO,T. (1983) Comp. Biochem. Physiol. 7 5 8 , 655-658. SHANK,R. P. & APRISON, M. H . (1981) Life Sci. 28, 837-842. SINGH,R. A. & STNGH,S. N. (1989) Arch. Int. Physiol. Biochim. 97, 145-152. SINGH,R. A. & SINGH,S. N. (1990) Arch. Int. Physiol. Biochim. 98, 95-101. STADMAN,E. R. & GINSBURG, A. (1974) T h e E n z y m e ( B ~ Y EP. ~ , D. ed.) Vol. 10, pp. 755-807, Academic Press, New York. TATE,S. S. & MEISTER,A. (1971) Proc. Natl. Acad. Sci., U.S.A. 68, 781-785. WEDLER,F. C., DENMAN,R. B. & ROBY, W. G. (1982) Biochemistry 21, 6389-6396.
S. N. SINGH Biochemistry and Molecular Biology, Department of Zoology, Banaras Hindu University, Varanasi, 221005 India.
