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Assay of Glutamine Synthetase Activity in Leaf Tissue of Vigna mungo
Biochem. Physiol. Pflanzen
in,
357-362 (1982)
Assay of Glutamine Synthetase Activity in Leaf Tissue of Vigna mungo M. H. SIDDIQUI, ANJALI MATHUR, D. MUKERJI and S. N. MATHURl) Plant Physiology Research Laboratory, Department of Botany, University of Gorakhpur, India Key Term Index: glutamine synthetase, enzyme activity determination in vivo and in vitro; Vigna mungo
Summary Freezing at -14°C in a deep freezer followed by thawing or toluene treatment of the leaf slices of l'igna mungo in Tris buffer allowed the measurement of glutamine synthetase (EC 6.3.1.2) without the need of cell-free extracts. Both the whole-tissue systems showed more than two-fold higher activity than was obtainable with cell-free extracts. Also, much reduced apparent Km values for glutamate and ATP were obtained with the whole tissue systems. Relatively, in terms of absolute activity and Km values, the toluene treated leaves proved superior to and more efficient than the freeze/thaw system. It is thus suggested that the use of toluene-treated leaves should provide a convenient and efficient alternative for the measurement of glutamine synthetase in plant tissues.
Introduction
Although measurement of enzyme activity in cell-free extracts continues to be by far the most common practice in biochemical studies, many workers have successfully measured activity of single enzymes within whole cells of numerous organism (see MUKERJI and MORRIS 1978). In whole cell assay accessibility of the enzyme to substrate and other ingredients of the assay is achieved by permeabilisation of the cell membrane either through the treatment of the cell with organic solvents or through their subjection to cycles of freeze/thaw. For accession into higher plant tissues, vacuum infiltration or treatment with dimethylsulfoxide used to be employed occasionally (KLEPPER et al. 1971; DELMER and MILLS 1969), until RHODES and STEWART (1974) made use of freeze/thaw technique for the assay of different plant enzymes, including glutamine synthetase, in leaves of aquatic plants, mainly Lemna minor and some others. The procedure included subjection of leaf slices to cycles of freezing (in liquid N2) and thawing. Alternatively permcabilisation of higher plants tissues through freezing in a deep-freeze (-14°C) or through treatment with organic solvents, if proved effective, should provide a convenient method for in vivo enzyme assays. Recently we have described a method for the assay of nitrogenase in detached root nodules of Vigna mungo (SRIVASTAVA et al. 1980). In this the permeability barrier of the nodule tissue (including bacteroids) was overcome by freezing them at -14°C in 1) To whom request for reprints be made.
358
1\1. H. SIDDIQUI et a1.
the presence of 0.05 ml of toluene/100 mg nodule. In the present investigation this possibility has been examined with respect to the assay of glutamine synthetase in leaves of a terrestrial crop plant, Vigna mungo.
Material and Methods Plant malerial Seeds of Black-gram (rigna II1UlIgO (L.) HEPPEH, cv. Type-9) were obtained locally, Culture of Rhizobium legulI1inosarum from Division of Microbiology, Indian Agricultural Research Institute, New Delhi and the chemicals from B.D. H., V.l'. Chest Institllte, New Delhi, and Sigma Chemical Co. St. Louis, U.R.A. Seeds were disinfected with 95 % ethanol for 10 min, washed several times with distilled water and were inoculated with Rhizobium by keeping them dipped in yeast extract mannitol broth (YEMA medium) for 12 h. The inoculated seeds were sown in pots containing add washed neutral sand and grown in natural ("onditions of light and temperature, with nutrient supplied on every fourth day. The leaves of 8 to 10 day old plants were taken for enzyme assay.
Enzyme assay Following KANAMORI and MATSUMOTO (1972), glutamine synthetase activity was measured by the biosynthetic assay based on glutamyl hydroxamate synthesis. The enzyme was assayed in cell-free extracts as well as in variously treated leaf tissues. For the former, freshly uprooted Vigna mungo plants were washed thoroughly with water followed by further washing of detached leaves in distilled water. One gram washed leaves were chilled for 1 h and then crushed in chilled mortar and pestle with cold 3 ml of 00 mM Tris buffer, pH 7.8 containing 1 mM mercaptoethanol, 2 mM EDTA and 2% polyvinylpyrrolidone. The homogenate was filtered through muslin cloth, the filter ate was made to 5 ml, and was then centrifuged at 20,000 rpm for 20 min at 4°C. The clear supernatant was used as the cell-free extracts of the enzyme. For whole tissue assay, the leaves were sliced into 1 to 3 mm segments. Two hundred milligram samples were place in 10 ml vaccine vial in 1 ml Tris buffer, pH 7.6. The leaf sliee suspensions so prepared were either frozen for 3 h at -14°C in a deep-freeze or were treated with 0.05 ml toluence for 80 min at 10°C followed by thawing in case of frozen samples, and were then used for enzyme assay. Cell-free extract assay was initiated by adding 0.5 ml of the enzyme extract to 1. 7 ml of the assay mixture, which was initially pre-incubated at 30°C for 2 min. In case of leaf tissue assay, 1.2 ml of the pre-incubated assay mixture was added to leaf slice preparations. The vials containing reaction mixture were allowed to incubate at 30°C in a constant temperature incubator. The assay mixture contained 50 mM Tris buffer, pH 7.6, 70 mM sodium glutamate, 2.5 mM ATP, 45 mM MgS0 4 , 4.5 mM cysteine, and 20 mM hydroxylamine (pH 7.0, freshly prepared) in a final volume of 2.2 ml. Appropriate blanks were run with cell-free extract and differentally conditioned leaf slices and they were incubated without substrate for desired time. At desired time, the reaction was terminated by transferring 1.5 ml samples from the reaction mixture to cold ferric chloride reagent (equal volumes of 5 % FeCl 3 in 0.1 N HCI, 12 % TCA and 25 % HCI were mixed together). Precipitated protein was removed by centrifugation and the intensity of the brown colour produced was read at 540 nm in a Carl Zeiss Spectrocolorimeter model Spekol 10. For single point measurements, 1.5 ml samples were transferred to 0.0 ml ferric chloride reagent after 20 min of incubation. For time course experiments (for which the total volume was adequately raised without altering relative concentration of the ingredients) 1.5 ml samples were transferred first immediately after initiation and then after every 0 min upto 30 min. For determination of Km, the rate was estimated for different substrate at different concentrations (starting from the lower level i.e. around the a.pparent Km values to optimum concentration) in all the three assay conditions and
359
UJ utumine Synthetase l'igna mungo
values l/S and l/V were plotted, and Km values were calculated by linear regression. The enzyme activity is expressed as the amount of glutamyl hydroxamate (GHA) formed per unit fresh weight of leaves. The amount of GHA produeed was calculated from the standard curve prepared with its authentic: sample. All experiments were done in triplieates and were repeated three times.
Results
Glutamine synthetase activity in differently conditioned leaf slices and in cell-free extracts Fig. 1 shows glutamine synthetase activity in cell-free extracts and in differently conditioned leaf slices of Vigna mungo. Pre-assay conditioning involved either freezing (for 3 h) of leaf slices (in buffer) at -14 DC in a deep-freezer followed by thawing at room temperature (25-30 DC) or their treatment with 0.05 ml of toluene in presence of 1 ml buffer for 80 min. It can be seen from Fig. 1 that the activity was linear with time in cell-free extracts as well as when measured in frozen/thawed or toluene treated leaf tissues. It was also found (not shown) that when the three assay were done without substrate (L-glutamate) formation of some GHA was observed, which became constant after 20 min. This formation of GHA without L-glutamate showed utilization of endogenous L-glutamate by the enzyme. Relatively, the highest activity was obtained with the toluene treated leaves while there was not much difference between the toluene treated and frozen/thawed leaves, both the whole tissue systems showed more than two times as high activity as was obtained with the cell-free extracts. J1 moles OHA . g-l fr. wI.
81
--,------------,
""
5
r----------+------------+-------A
I:·'-j
~ r---~~~-t-~-7.~.~ I/
0····· .... ~
~/.:.....
2
... 0
......
0
... .. 0 .....
/···1 ... ,0'
....
""...
0'"
f
10
~'" •. ··#/#0,.
o ... o
20
TI!1E (min)
25
JO
Fig. 1. Tillle course of glutamine syllthetase activity of Yigna 1IIUligO. With ('omplete assay mixture in cell -free extraets of leaves (0- -0); in frozen/thawed ( •....• ); in toluene-treated (A--A) leaf slices. GHA, glntamylhydroxamate.
360
M. H.
SIDDIQUI
et a1.
Table 1. Apparent Km of glutamine synthetase for different substrate. The biosynthetie glutalllyl hydroxalllate assay was used in these experiments Substrate
Cell-tree extraet
Frozenjtha wed
Toluene -treated
L-giutalllate
4.00 IllM 0.40 mM 0.40 mM
:2.GG mM 0.:25m1\[ 0.40 Ill;\I
:2.:2:2mM 0.:20 Illl1 0.40 Illl1
ATP Hydroxylamille
Table:2. Glutall/illc synthetase activity in Yigna mungo (a), total, containing the normal leaf slice preparation; (b), resichml, !'ontaining the slices of normal preparation + freshly added buffer; (c), supernat
Fraetions
Frozen/Thawed (a) Total (b) Residual (!') Supernatant
1) :\Iean
10.24 10.1:2 4.84
± 0.7;)1) ± 0.97 ± 0.34
± Standard deviation (:'{ =
Toluene treated 19.0G 7.77 11.29
± 0.8:2 ± 0.90 ± 0.41
:l). GHA, glutamyl hydroxamate
Increase in freezing time from 30 min upto 3 h steadily increased the activity in the leaves. No further increase in the activity could be observed in leaves frozen for durations longer than 3 h. Thus 3 h proved the optimum freezing time. Likewise, increasing concentration of toluene from 0.01 to 0.05 ml in 1 ml of leaf slice suspension increased the enzymp activity in them (leaf slices). Any further increase in its concentration, however, had an inhibitory effect.
Properties of glutctmine synthetase in cell-free extracts and in whole-tissue systems In establishing a techniqup it is important to compare the properties of the enzyme as obtained by the use of the new tpchnique with those obtained from measurements, in cell-free extracts. While the pH optima showed only a slight variation (7.8 for the cell-free extracts and 7.6 for the whole-tissue system), apparent Km value for glutamate and ATP varied grpatly from one system of assay to another (Table 1). By contrast, apparent Km value for hydroxylamine remained same for all the three systems of assay (Table 1).
In vivo assa,y The question whether the conditioning of the leaves resulted in the leakage of the enzyme or allowed its in vivo assay needed verification. For this, the enzyme activity was measured in three kinds of preparations as follows: (i) total preparation - normally
Glutamine Synthetase Vigna mungo
361
treated suspension of leaf slices, (ii) residual fraction - treated leaf slices, resuspended in fresh buffer, and (iii) supernatant fraction - the buffer part only of the treated leaf slice suspension. The results are summarized in Table 2. Both toluene and the freezing treatments caused some leakage of the enzyme in the buffer, the leakage was greater from toluene-treated leaves than from frozen/thawed ones. The leakage study showed that enzyme came in contact with other assay constituents in its native form. That is why we got more activity in these two in vivo systems than in cell-free extracts where enzyme gets disturbed during extraction. Discussion
The results presented here clearly demonstrate that glutamine synthetase can be conveniently measured in leaves permeabilised by either solvent treatments or by freezing in a deep-freezer (-14°C). They also show that much higher activity can be obtained with both the whole tissue systems than in measurements with cell-free extracts. The solvent treatment, however, not only proved marginally superior to the freeze/thaw procedure, but also proved more convenient, for it cuts on the pre-assay conditioning time. Further, since the technique eliminates the need for any freezing equipment, it can be useful for field or, when suitably developed, even for in situ assays. The three systems of assay differed markedly from each other from the stand-point of such properties of the enzyme as apparent Km values for glutamate and ATP, which varied considerably from one method of assay to another. RHODES and STEWART (1974), who used freeze/thaw technique for the assay of glutamine synthetase provide different Km for hydroxylamine in cell-free extract and in vivo system. This kind of relatively reduced Km values in the whole-cell assays were, however, observed by MUKERJI and MORRIS (1976) during their study of the enzymes RuBPCase and PEPCase. They suggested that the enzyme in whole-cells, though altered to an extent by the treatment given, approximates more closely to its natural state than in cell-free extracts. Although neither of the two whole-tissue methods examined have proved a completely in vivo system, similar explanation as above can be advanced for the reduced Km values observ~ ed. For it is possible that freezing or solvent treatment may cause release of the enzyme from the plant tissue much less drastically than is obtainable with the usual methods of extraction. Thus while both the whole tissue methods could prove useful, the use of the toluene treated leaves should provide a convenient and a more efficient alternative for the assay of glutamine synthetase in plant tissues. Acknowledgment
xl.
H. Siddiqui is grateful to the authorities of the CSIR, New Delhi, for financial assistance.
References D. P., and XIrLLS, S. K: A technique for the assay of enzymes in intaet plant cell in presence of dimethylsulfoxide. Plant Physiol. 44, 153-l55 (l969).
DELMER,
362
M. H. SIDDIQUI et aI., Glutamine Synthetase Vigna mungo
KANAMORI, T., and MATSUMOTO, H.: Glutamine synthetase from rice plant roots. Arch. Biochem. Biophys. 120, 404-412 (1972). KLEPPER, L., FLESHER, D., and H.\GEMAN, R. H.: Generation of reduced nicotinamide adenine dinurleotide for nitrate reduction in green leaves. Plant Physiol. 48, 580-590 (1971). l\IrKEHJI, D., and MORRIS, I.: Measurement of Carboxylases (RuDPCase and PEPCase) in cell suspension of Phaeodactylu1l1 tricorlluturI! treated with organic solvents. Z. Pflanzenphysiol. 90, 96-99 (1978). MUKERJI, D., and MORRIS, I.: Photosynthetic Carboxylating Enzymes in Phaeodactylum tricornutttln: Assay Methods and Properties. Mar. BioI. 36, 199-206 (1976). RIIODER, D., and STE\HRT, G. R.: A procedure for the in vivo determination of enzyme activity in higher plant tissue. Planta 118, 133-144 (1974). SRlY.\ST.\Y.\, R. C., MUKERJI, D., and MATHUR, S. N.: Freeze/thaw technique for estimation of nitrogenase activity in detached nodules of Yigna mungo. Ann. App. BioI. 96, 235-241 (1980)
Received October 2, 1981 Authors' addresses: M. H. SIDDIQUI, ANJALI MATHUR, S. N. MATHUR, Plant Physiology Research Laboratory, Department of Botany, University of Gorakhpur, Gorakhpur - 273001 (India), D. MuKERJI, Department of Biology, Zambia University, P. O. Box 32379, Lusaka, Zambia.
in,
357-362 (1982)
Assay of Glutamine Synthetase Activity in Leaf Tissue of Vigna mungo M. H. SIDDIQUI, ANJALI MATHUR, D. MUKERJI and S. N. MATHURl) Plant Physiology Research Laboratory, Department of Botany, University of Gorakhpur, India Key Term Index: glutamine synthetase, enzyme activity determination in vivo and in vitro; Vigna mungo
Summary Freezing at -14°C in a deep freezer followed by thawing or toluene treatment of the leaf slices of l'igna mungo in Tris buffer allowed the measurement of glutamine synthetase (EC 6.3.1.2) without the need of cell-free extracts. Both the whole-tissue systems showed more than two-fold higher activity than was obtainable with cell-free extracts. Also, much reduced apparent Km values for glutamate and ATP were obtained with the whole tissue systems. Relatively, in terms of absolute activity and Km values, the toluene treated leaves proved superior to and more efficient than the freeze/thaw system. It is thus suggested that the use of toluene-treated leaves should provide a convenient and efficient alternative for the measurement of glutamine synthetase in plant tissues.
Introduction
Although measurement of enzyme activity in cell-free extracts continues to be by far the most common practice in biochemical studies, many workers have successfully measured activity of single enzymes within whole cells of numerous organism (see MUKERJI and MORRIS 1978). In whole cell assay accessibility of the enzyme to substrate and other ingredients of the assay is achieved by permeabilisation of the cell membrane either through the treatment of the cell with organic solvents or through their subjection to cycles of freeze/thaw. For accession into higher plant tissues, vacuum infiltration or treatment with dimethylsulfoxide used to be employed occasionally (KLEPPER et al. 1971; DELMER and MILLS 1969), until RHODES and STEWART (1974) made use of freeze/thaw technique for the assay of different plant enzymes, including glutamine synthetase, in leaves of aquatic plants, mainly Lemna minor and some others. The procedure included subjection of leaf slices to cycles of freezing (in liquid N2) and thawing. Alternatively permcabilisation of higher plants tissues through freezing in a deep-freeze (-14°C) or through treatment with organic solvents, if proved effective, should provide a convenient method for in vivo enzyme assays. Recently we have described a method for the assay of nitrogenase in detached root nodules of Vigna mungo (SRIVASTAVA et al. 1980). In this the permeability barrier of the nodule tissue (including bacteroids) was overcome by freezing them at -14°C in 1) To whom request for reprints be made.
358
1\1. H. SIDDIQUI et a1.
the presence of 0.05 ml of toluene/100 mg nodule. In the present investigation this possibility has been examined with respect to the assay of glutamine synthetase in leaves of a terrestrial crop plant, Vigna mungo.
Material and Methods Plant malerial Seeds of Black-gram (rigna II1UlIgO (L.) HEPPEH, cv. Type-9) were obtained locally, Culture of Rhizobium legulI1inosarum from Division of Microbiology, Indian Agricultural Research Institute, New Delhi and the chemicals from B.D. H., V.l'. Chest Institllte, New Delhi, and Sigma Chemical Co. St. Louis, U.R.A. Seeds were disinfected with 95 % ethanol for 10 min, washed several times with distilled water and were inoculated with Rhizobium by keeping them dipped in yeast extract mannitol broth (YEMA medium) for 12 h. The inoculated seeds were sown in pots containing add washed neutral sand and grown in natural ("onditions of light and temperature, with nutrient supplied on every fourth day. The leaves of 8 to 10 day old plants were taken for enzyme assay.
Enzyme assay Following KANAMORI and MATSUMOTO (1972), glutamine synthetase activity was measured by the biosynthetic assay based on glutamyl hydroxamate synthesis. The enzyme was assayed in cell-free extracts as well as in variously treated leaf tissues. For the former, freshly uprooted Vigna mungo plants were washed thoroughly with water followed by further washing of detached leaves in distilled water. One gram washed leaves were chilled for 1 h and then crushed in chilled mortar and pestle with cold 3 ml of 00 mM Tris buffer, pH 7.8 containing 1 mM mercaptoethanol, 2 mM EDTA and 2% polyvinylpyrrolidone. The homogenate was filtered through muslin cloth, the filter ate was made to 5 ml, and was then centrifuged at 20,000 rpm for 20 min at 4°C. The clear supernatant was used as the cell-free extracts of the enzyme. For whole tissue assay, the leaves were sliced into 1 to 3 mm segments. Two hundred milligram samples were place in 10 ml vaccine vial in 1 ml Tris buffer, pH 7.6. The leaf sliee suspensions so prepared were either frozen for 3 h at -14°C in a deep-freeze or were treated with 0.05 ml toluence for 80 min at 10°C followed by thawing in case of frozen samples, and were then used for enzyme assay. Cell-free extract assay was initiated by adding 0.5 ml of the enzyme extract to 1. 7 ml of the assay mixture, which was initially pre-incubated at 30°C for 2 min. In case of leaf tissue assay, 1.2 ml of the pre-incubated assay mixture was added to leaf slice preparations. The vials containing reaction mixture were allowed to incubate at 30°C in a constant temperature incubator. The assay mixture contained 50 mM Tris buffer, pH 7.6, 70 mM sodium glutamate, 2.5 mM ATP, 45 mM MgS0 4 , 4.5 mM cysteine, and 20 mM hydroxylamine (pH 7.0, freshly prepared) in a final volume of 2.2 ml. Appropriate blanks were run with cell-free extract and differentally conditioned leaf slices and they were incubated without substrate for desired time. At desired time, the reaction was terminated by transferring 1.5 ml samples from the reaction mixture to cold ferric chloride reagent (equal volumes of 5 % FeCl 3 in 0.1 N HCI, 12 % TCA and 25 % HCI were mixed together). Precipitated protein was removed by centrifugation and the intensity of the brown colour produced was read at 540 nm in a Carl Zeiss Spectrocolorimeter model Spekol 10. For single point measurements, 1.5 ml samples were transferred to 0.0 ml ferric chloride reagent after 20 min of incubation. For time course experiments (for which the total volume was adequately raised without altering relative concentration of the ingredients) 1.5 ml samples were transferred first immediately after initiation and then after every 0 min upto 30 min. For determination of Km, the rate was estimated for different substrate at different concentrations (starting from the lower level i.e. around the a.pparent Km values to optimum concentration) in all the three assay conditions and
359
UJ utumine Synthetase l'igna mungo
values l/S and l/V were plotted, and Km values were calculated by linear regression. The enzyme activity is expressed as the amount of glutamyl hydroxamate (GHA) formed per unit fresh weight of leaves. The amount of GHA produeed was calculated from the standard curve prepared with its authentic: sample. All experiments were done in triplieates and were repeated three times.
Results
Glutamine synthetase activity in differently conditioned leaf slices and in cell-free extracts Fig. 1 shows glutamine synthetase activity in cell-free extracts and in differently conditioned leaf slices of Vigna mungo. Pre-assay conditioning involved either freezing (for 3 h) of leaf slices (in buffer) at -14 DC in a deep-freezer followed by thawing at room temperature (25-30 DC) or their treatment with 0.05 ml of toluene in presence of 1 ml buffer for 80 min. It can be seen from Fig. 1 that the activity was linear with time in cell-free extracts as well as when measured in frozen/thawed or toluene treated leaf tissues. It was also found (not shown) that when the three assay were done without substrate (L-glutamate) formation of some GHA was observed, which became constant after 20 min. This formation of GHA without L-glutamate showed utilization of endogenous L-glutamate by the enzyme. Relatively, the highest activity was obtained with the toluene treated leaves while there was not much difference between the toluene treated and frozen/thawed leaves, both the whole tissue systems showed more than two times as high activity as was obtained with the cell-free extracts. J1 moles OHA . g-l fr. wI.
81
--,------------,
""
5
r----------+------------+-------A
I:·'-j
~ r---~~~-t-~-7.~.~ I/
0····· .... ~
~/.:.....
2
... 0
......
0
... .. 0 .....
/···1 ... ,0'
....
""...
0'"
f
10
~'" •. ··#/#0,.
o ... o
20
TI!1E (min)
25
JO
Fig. 1. Tillle course of glutamine syllthetase activity of Yigna 1IIUligO. With ('omplete assay mixture in cell -free extraets of leaves (0- -0); in frozen/thawed ( •....• ); in toluene-treated (A--A) leaf slices. GHA, glntamylhydroxamate.
360
M. H.
SIDDIQUI
et a1.
Table 1. Apparent Km of glutamine synthetase for different substrate. The biosynthetie glutalllyl hydroxalllate assay was used in these experiments Substrate
Cell-tree extraet
Frozenjtha wed
Toluene -treated
L-giutalllate
4.00 IllM 0.40 mM 0.40 mM
:2.GG mM 0.:25m1\[ 0.40 Ill;\I
:2.:2:2mM 0.:20 Illl1 0.40 Illl1
ATP Hydroxylamille
Table:2. Glutall/illc synthetase activity in Yigna mungo (a), total, containing the normal leaf slice preparation; (b), resichml, !'ontaining the slices of normal preparation + freshly added buffer; (c), supernat
Fraetions
Frozen/Thawed (a) Total (b) Residual (!') Supernatant
1) :\Iean
10.24 10.1:2 4.84
± 0.7;)1) ± 0.97 ± 0.34
± Standard deviation (:'{ =
Toluene treated 19.0G 7.77 11.29
± 0.8:2 ± 0.90 ± 0.41
:l). GHA, glutamyl hydroxamate
Increase in freezing time from 30 min upto 3 h steadily increased the activity in the leaves. No further increase in the activity could be observed in leaves frozen for durations longer than 3 h. Thus 3 h proved the optimum freezing time. Likewise, increasing concentration of toluene from 0.01 to 0.05 ml in 1 ml of leaf slice suspension increased the enzymp activity in them (leaf slices). Any further increase in its concentration, however, had an inhibitory effect.
Properties of glutctmine synthetase in cell-free extracts and in whole-tissue systems In establishing a techniqup it is important to compare the properties of the enzyme as obtained by the use of the new tpchnique with those obtained from measurements, in cell-free extracts. While the pH optima showed only a slight variation (7.8 for the cell-free extracts and 7.6 for the whole-tissue system), apparent Km value for glutamate and ATP varied grpatly from one system of assay to another (Table 1). By contrast, apparent Km value for hydroxylamine remained same for all the three systems of assay (Table 1).
In vivo assa,y The question whether the conditioning of the leaves resulted in the leakage of the enzyme or allowed its in vivo assay needed verification. For this, the enzyme activity was measured in three kinds of preparations as follows: (i) total preparation - normally
Glutamine Synthetase Vigna mungo
361
treated suspension of leaf slices, (ii) residual fraction - treated leaf slices, resuspended in fresh buffer, and (iii) supernatant fraction - the buffer part only of the treated leaf slice suspension. The results are summarized in Table 2. Both toluene and the freezing treatments caused some leakage of the enzyme in the buffer, the leakage was greater from toluene-treated leaves than from frozen/thawed ones. The leakage study showed that enzyme came in contact with other assay constituents in its native form. That is why we got more activity in these two in vivo systems than in cell-free extracts where enzyme gets disturbed during extraction. Discussion
The results presented here clearly demonstrate that glutamine synthetase can be conveniently measured in leaves permeabilised by either solvent treatments or by freezing in a deep-freezer (-14°C). They also show that much higher activity can be obtained with both the whole tissue systems than in measurements with cell-free extracts. The solvent treatment, however, not only proved marginally superior to the freeze/thaw procedure, but also proved more convenient, for it cuts on the pre-assay conditioning time. Further, since the technique eliminates the need for any freezing equipment, it can be useful for field or, when suitably developed, even for in situ assays. The three systems of assay differed markedly from each other from the stand-point of such properties of the enzyme as apparent Km values for glutamate and ATP, which varied considerably from one method of assay to another. RHODES and STEWART (1974), who used freeze/thaw technique for the assay of glutamine synthetase provide different Km for hydroxylamine in cell-free extract and in vivo system. This kind of relatively reduced Km values in the whole-cell assays were, however, observed by MUKERJI and MORRIS (1976) during their study of the enzymes RuBPCase and PEPCase. They suggested that the enzyme in whole-cells, though altered to an extent by the treatment given, approximates more closely to its natural state than in cell-free extracts. Although neither of the two whole-tissue methods examined have proved a completely in vivo system, similar explanation as above can be advanced for the reduced Km values observ~ ed. For it is possible that freezing or solvent treatment may cause release of the enzyme from the plant tissue much less drastically than is obtainable with the usual methods of extraction. Thus while both the whole tissue methods could prove useful, the use of the toluene treated leaves should provide a convenient and a more efficient alternative for the assay of glutamine synthetase in plant tissues. Acknowledgment
xl.
H. Siddiqui is grateful to the authorities of the CSIR, New Delhi, for financial assistance.
References D. P., and XIrLLS, S. K: A technique for the assay of enzymes in intaet plant cell in presence of dimethylsulfoxide. Plant Physiol. 44, 153-l55 (l969).
DELMER,
362
M. H. SIDDIQUI et aI., Glutamine Synthetase Vigna mungo
KANAMORI, T., and MATSUMOTO, H.: Glutamine synthetase from rice plant roots. Arch. Biochem. Biophys. 120, 404-412 (1972). KLEPPER, L., FLESHER, D., and H.\GEMAN, R. H.: Generation of reduced nicotinamide adenine dinurleotide for nitrate reduction in green leaves. Plant Physiol. 48, 580-590 (1971). l\IrKEHJI, D., and MORRIS, I.: Measurement of Carboxylases (RuDPCase and PEPCase) in cell suspension of Phaeodactylu1l1 tricorlluturI! treated with organic solvents. Z. Pflanzenphysiol. 90, 96-99 (1978). MUKERJI, D., and MORRIS, I.: Photosynthetic Carboxylating Enzymes in Phaeodactylum tricornutttln: Assay Methods and Properties. Mar. BioI. 36, 199-206 (1976). RIIODER, D., and STE\HRT, G. R.: A procedure for the in vivo determination of enzyme activity in higher plant tissue. Planta 118, 133-144 (1974). SRlY.\ST.\Y.\, R. C., MUKERJI, D., and MATHUR, S. N.: Freeze/thaw technique for estimation of nitrogenase activity in detached nodules of Yigna mungo. Ann. App. BioI. 96, 235-241 (1980)
Received October 2, 1981 Authors' addresses: M. H. SIDDIQUI, ANJALI MATHUR, S. N. MATHUR, Plant Physiology Research Laboratory, Department of Botany, University of Gorakhpur, Gorakhpur - 273001 (India), D. MuKERJI, Department of Biology, Zambia University, P. O. Box 32379, Lusaka, Zambia.