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Malate Dehydrogenase Isoenzymes in wheat Seeds Imbibed in Saline Medium
Biochem. Physiol. Pflanzen (BPP), Bd. 1G3, S. GOS-G10 (1972)
Short Commuuication Division of Bioehemistry, Indian Agricultural Research Institute, New Delhi -
12, India
Malate Dehydrogenase Isoenzymes in wheat Seeds Imbibed in Saline Medium By H. S. NAIl\'AWATEEI) and N. B. DAS
t
With one figure (Received lYray 21, 1972)
Summary The presence of salts in the imbibition medium showed a considerable eUeet upon malate dehydrogenase isoenzymes in wheat. The intensity of l\IDH band with Rf 0.50 increased with increase in sodium chloride concentration, when' as the same isoenzyme band disappeared in the presence of sodium sulphate.
Introduction
The effect of salts like sodium chloride and sodium sulphate on malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) activity have been studied by many workers (PORATH and MAYBER 1964; EYANS and SORGER 1966 and KAHANE and MAYBER 1968). Increasing concentrations of sodium chloride and sodium sulphate in growth medium induced a progressive decrease in specific activity of MDH from pea root mitochondria (PORATH and MAYBER 1964). Contrary to this, the formation of a new MDH isoenzyme at lower sodium chloride concentration was observed by PORATH and MAYBER (1969). Therefore, in present study the effect of sodium chloride and sodium sulphate was studied on MDH isoenzymes at 5, 10 and 15 atmosphere during imbibition of wheat seeds. Experimental
Seeds of Triticum aestivum Linn. variety Kalyan sana were imbibed in deionized water, and 5, 10 and 15 atmosphere concentrations of sodium chloride and sodium sulphate in petri plates, under sterilized conditions for 48 hours in a germinating chamber at 20°C. 1) Present address: Department of Chemistry and Biochemistry, Haryana Agricultural University, Hissar (llaryana) India.
:\Ialate Dehydrogenase Isoenzymes in wheat Seeds Imbided in Saline Medium
609
Enzyme extraction was done by grinding seed in 0.05 M Tris-Hel buffer (pH 7.4) containing 5 mM 2-mercaptoethanol and 5 mM EDTA. The slurry was centrifuged at 10,000 xg for 30 minutes. The suitable aliquot of supernatent (200,ug protein) was layered on 7.5 % polyacrylamide gel and electrophoresis was done using anionic system (DAVIS 1964). Isoenzyme of MDH were detected by zymogram staining technique using nitro blue tetrazolium and phenazine methosulphate (GOLDBERG 1963). Proteins were estimated using Folin-phenol reagent (LOWRY et al. 1951). Result and Discussion
Results presented in the table and figure show the effect of sodium chloride and sodium sulphate on MDH isoenzymes during imbition of wheat seeds. Seeds soaked in 5 atmosphere sodium chloride and 5 atmosphere sodium sulphate showed all the three isoenzymes having Rf 0.40, 0.44 and 0.50, which were similar to the isoenzyme bands of seeds imbibed in deionized water. At higher concentration of sodium chloride (10 atmosphere) the intensity of MDH band having Rf 0.50 increased and that of 0.44 decreased. At 15 atmosphere sodium chloride concentration the intensity of MDH band with Rf 0.50 showed still higher intensity whereas that of the band having Rf 0.44 decreased. Table 1
MDH isoenzymes in wheat seeds imbided in saline medium MDH Isoenzyme Rf
0.40 0.44 0.50
+
Sodium sulphate
Sodium chloride 5 atm
10 atm
15atm
5atm
10atm
15atm
++ ++ +++
++ + +++
++ + ++++
++ ++ +++
++ + ++
++ +
indicates relative intensity of colour of isoenzyme band.
Seeds imbibed in sodium sulphate medium gave contrasting results to that of seeds imbibed in sodium chloride medium. There was a decrease in the intensity of MDH band having Rf 0.50 at 10 atmosphere sodium sulphate concentration and at 15 atmosphere concentration it disappeared. The other isoenzyme band having Rf 0.44 also showed a decrease in intensity, whereas the isoenzyme band having Rf 0.40 remained almost same. MDH is a salt sensitive enzyme and it appears that there is a differential effect of sodium chloride and sodium sulphate upon its isoenzymes. In pea, stimulation of one of the isoenzyme is known (WEIMBERG 1968), whereas, the increasing concentrations of sodium chloride and sodium sulphate have been found to result in the decrease in mitochondrial MDH activity in pea roots (PORATH and MAYBER 1964). 41
Biochern. Physiol. Pflanzen Bd. 163
H. S. NAINA"'ATEE and N. B. DAS
610
Rr
Rr
0 ••0
0••0
O.H
O.H
0.:;0
0.:;0
z
J
s
Fig.l. Polyacrylamide gels showing malate dehydrogenase isoenzyme patterns in wheat seeds imbided in (1) 5 atm NaCl (2) 10 atm NaCl (3) 15 atm NaCI (4) 5 atmNa 2 S0 4 (5) 10 atm Na 2 S0 4 and (6) 15 atm Na 2 S04 solutions.
6
In the present study, the MDH-isoenzyme with high electrophoretic mobility was differentially influenced by sodium chloride and sodium sulphate. This isoenzyme is a soluble MDH and the other one with Rf 0.40, which showed immunity to salts is a chloroplast malate dehydrogenase isoenzyme (NAINAWATEE, 1971). The effect of salinity conditions on soluble MDH isoenzymes of wheat could possibily due to the differential regulation of synthesis of malate dehydrogenase proteins. Details of which need a careful investigation. Acknowledgement One of us (H. S. N.) is grateful to the Indian Council of Agricultural Research for awarding a senior research fellowship.
Literature DAVIS, 13. J., Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci., 121, 404-427 (1964). EVANS, H. J., and SORGER, G. J., Role of mineral elements with emphasis on the univalent cations. A Rev. PI. Physiol. 17, 47 -76 (1966). GOLDBERG, E., LDH and MDH in human spermatazoa. Science, N. Y., 139, 602-603 (1963). KAHANE, I., and MAYBER, A. P., Effect of substrate salinity on the ability for protein synthesis in pea roots. PI. Physiol. Lancaster, 43,1115-1119 (1968). LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J., Protein measurement with the Folin-phenol reagent. J. bioI. Chern., 193, 265-275 (1951). NAINAW_\TEE, H. S., Studies on soluble proteins and isoenzymes of malate dehydrogenase of wheat. Ph. D. thesis, I. A. R. I., New Delhi-12 (1971). PORATH, E. H., and MAYBER, A. P., Effect of salinity on metabolic pathways in pea root tips. Israel J. Bot. 13, 115-121 (1964). PORATH, E. H. and MAYBER, A. P., The effect of salinity on the malate dehydrogenase of pea roots. PI. Physiol. Lancaster, 44, 1031-1034 (1969). WEIMBERG, R., Effect of sodium chloride on the activity of a soluble ilJIDH from pea seeds. J. bioI. Chern., 242, 3000-3006 (1968). Authors' address: Prof. Dr. H. S. NAINAWATEE, Department of Chemistry and Biochemistry, Haryana Agricultural University, Hissar, Haryana (India).
Short Commuuication Division of Bioehemistry, Indian Agricultural Research Institute, New Delhi -
12, India
Malate Dehydrogenase Isoenzymes in wheat Seeds Imbibed in Saline Medium By H. S. NAIl\'AWATEEI) and N. B. DAS
t
With one figure (Received lYray 21, 1972)
Summary The presence of salts in the imbibition medium showed a considerable eUeet upon malate dehydrogenase isoenzymes in wheat. The intensity of l\IDH band with Rf 0.50 increased with increase in sodium chloride concentration, when' as the same isoenzyme band disappeared in the presence of sodium sulphate.
Introduction
The effect of salts like sodium chloride and sodium sulphate on malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) activity have been studied by many workers (PORATH and MAYBER 1964; EYANS and SORGER 1966 and KAHANE and MAYBER 1968). Increasing concentrations of sodium chloride and sodium sulphate in growth medium induced a progressive decrease in specific activity of MDH from pea root mitochondria (PORATH and MAYBER 1964). Contrary to this, the formation of a new MDH isoenzyme at lower sodium chloride concentration was observed by PORATH and MAYBER (1969). Therefore, in present study the effect of sodium chloride and sodium sulphate was studied on MDH isoenzymes at 5, 10 and 15 atmosphere during imbibition of wheat seeds. Experimental
Seeds of Triticum aestivum Linn. variety Kalyan sana were imbibed in deionized water, and 5, 10 and 15 atmosphere concentrations of sodium chloride and sodium sulphate in petri plates, under sterilized conditions for 48 hours in a germinating chamber at 20°C. 1) Present address: Department of Chemistry and Biochemistry, Haryana Agricultural University, Hissar (llaryana) India.
:\Ialate Dehydrogenase Isoenzymes in wheat Seeds Imbided in Saline Medium
609
Enzyme extraction was done by grinding seed in 0.05 M Tris-Hel buffer (pH 7.4) containing 5 mM 2-mercaptoethanol and 5 mM EDTA. The slurry was centrifuged at 10,000 xg for 30 minutes. The suitable aliquot of supernatent (200,ug protein) was layered on 7.5 % polyacrylamide gel and electrophoresis was done using anionic system (DAVIS 1964). Isoenzyme of MDH were detected by zymogram staining technique using nitro blue tetrazolium and phenazine methosulphate (GOLDBERG 1963). Proteins were estimated using Folin-phenol reagent (LOWRY et al. 1951). Result and Discussion
Results presented in the table and figure show the effect of sodium chloride and sodium sulphate on MDH isoenzymes during imbition of wheat seeds. Seeds soaked in 5 atmosphere sodium chloride and 5 atmosphere sodium sulphate showed all the three isoenzymes having Rf 0.40, 0.44 and 0.50, which were similar to the isoenzyme bands of seeds imbibed in deionized water. At higher concentration of sodium chloride (10 atmosphere) the intensity of MDH band having Rf 0.50 increased and that of 0.44 decreased. At 15 atmosphere sodium chloride concentration the intensity of MDH band with Rf 0.50 showed still higher intensity whereas that of the band having Rf 0.44 decreased. Table 1
MDH isoenzymes in wheat seeds imbided in saline medium MDH Isoenzyme Rf
0.40 0.44 0.50
+
Sodium sulphate
Sodium chloride 5 atm
10 atm
15atm
5atm
10atm
15atm
++ ++ +++
++ + +++
++ + ++++
++ ++ +++
++ + ++
++ +
indicates relative intensity of colour of isoenzyme band.
Seeds imbibed in sodium sulphate medium gave contrasting results to that of seeds imbibed in sodium chloride medium. There was a decrease in the intensity of MDH band having Rf 0.50 at 10 atmosphere sodium sulphate concentration and at 15 atmosphere concentration it disappeared. The other isoenzyme band having Rf 0.44 also showed a decrease in intensity, whereas the isoenzyme band having Rf 0.40 remained almost same. MDH is a salt sensitive enzyme and it appears that there is a differential effect of sodium chloride and sodium sulphate upon its isoenzymes. In pea, stimulation of one of the isoenzyme is known (WEIMBERG 1968), whereas, the increasing concentrations of sodium chloride and sodium sulphate have been found to result in the decrease in mitochondrial MDH activity in pea roots (PORATH and MAYBER 1964). 41
Biochern. Physiol. Pflanzen Bd. 163
H. S. NAINA"'ATEE and N. B. DAS
610
Rr
Rr
0 ••0
0••0
O.H
O.H
0.:;0
0.:;0
z
J
s
Fig.l. Polyacrylamide gels showing malate dehydrogenase isoenzyme patterns in wheat seeds imbided in (1) 5 atm NaCl (2) 10 atm NaCl (3) 15 atm NaCI (4) 5 atmNa 2 S0 4 (5) 10 atm Na 2 S0 4 and (6) 15 atm Na 2 S04 solutions.
6
In the present study, the MDH-isoenzyme with high electrophoretic mobility was differentially influenced by sodium chloride and sodium sulphate. This isoenzyme is a soluble MDH and the other one with Rf 0.40, which showed immunity to salts is a chloroplast malate dehydrogenase isoenzyme (NAINAWATEE, 1971). The effect of salinity conditions on soluble MDH isoenzymes of wheat could possibily due to the differential regulation of synthesis of malate dehydrogenase proteins. Details of which need a careful investigation. Acknowledgement One of us (H. S. N.) is grateful to the Indian Council of Agricultural Research for awarding a senior research fellowship.
Literature DAVIS, 13. J., Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci., 121, 404-427 (1964). EVANS, H. J., and SORGER, G. J., Role of mineral elements with emphasis on the univalent cations. A Rev. PI. Physiol. 17, 47 -76 (1966). GOLDBERG, E., LDH and MDH in human spermatazoa. Science, N. Y., 139, 602-603 (1963). KAHANE, I., and MAYBER, A. P., Effect of substrate salinity on the ability for protein synthesis in pea roots. PI. Physiol. Lancaster, 43,1115-1119 (1968). LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J., Protein measurement with the Folin-phenol reagent. J. bioI. Chern., 193, 265-275 (1951). NAINAW_\TEE, H. S., Studies on soluble proteins and isoenzymes of malate dehydrogenase of wheat. Ph. D. thesis, I. A. R. I., New Delhi-12 (1971). PORATH, E. H., and MAYBER, A. P., Effect of salinity on metabolic pathways in pea root tips. Israel J. Bot. 13, 115-121 (1964). PORATH, E. H. and MAYBER, A. P., The effect of salinity on the malate dehydrogenase of pea roots. PI. Physiol. Lancaster, 44, 1031-1034 (1969). WEIMBERG, R., Effect of sodium chloride on the activity of a soluble ilJIDH from pea seeds. J. bioI. Chern., 242, 3000-3006 (1968). Authors' address: Prof. Dr. H. S. NAINAWATEE, Department of Chemistry and Biochemistry, Haryana Agricultural University, Hissar, Haryana (India).