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دراسة مقارنة على إنزيم جلوتاميت-5-فوسفوترانسفريز

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Ahlam Saleh

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Transcript of دراسة مقارنة على إنزيم جلوتاميت-5-فوسفوترانسفريز

Comparative Study on L-glutamate-5- phosphotransferase in Some C3 Plants Under Stress Conditions

Prof. Dr. Hamed M. El-Shora

Prof. of Plant Physiology
Botany Department,
Faculty of Science,
Mansoura University
Prof. Dr. Mohammed E. El-Naggar

Prof. of Phycology
Botany Department,
Faculty of Science,
Mansoura University

Prof. Dr. Hamed M. El-Shora

Prof. of Plant Physiology
Botany Department,
Faculty of Science,
Mansoura University

Prof. Dr. Mahmoud Abo El-Yazeed

Prof. of Plant Physiology
Botany Department,
Faculty of Science,
Tanta University

Prof. Dr. Samia El-Sayed Saafan

Prof. of Phycology
Botany Department,
Faculty of Science,
Zagazig University


L-glutamate-5-phosphotransferase

Glutamate kinase, the enzyme that catalyzes the first step in the pathway from glutamate to proline.

Plants evolved a variety of mechanisms for adapting to environmental stresses, one of which is accumulation of proline.



proline plays an important role as a compatible solute.


Plants have two proline biosynthetic pathways: 1- The glutamate pathway (predominant role under osmotic stress)
2- The ornithine pathway




Referees
Introduction
Materials and Methods
L-Glutamate+ ATP Mg2+ glutamyl phosphate + ADP
GK

Glutamyl phosphate + NADPH glutamate semialdehyde+NADP++Pi


Glutamate semialdehyde 1-pyrolline-5-carboxylate

1-pyrolline-5-carboxylate+NADPH L-proline + NADP+

Aim of the work

Supervisors
1) Study the induction of glutamate kinase (GK) by various growth regulators.

2) Isolate and investigate the activity level of glutamate kinase in marrow leaves.

3) Study the heavy metal stresses such Ni2+ and Hg2+ on the GK activity level in the leaves.

4) Study the various possible methods for alleviation of the stress.

5) Study the activities of antioxidant enzymes such as super oxide dismutase, glutathione reductase (GR) and ascorbate peroxidase (APX) under heavy metal stress.


In the glutamate pathway, glutamate is converted by glutamyl kinase to glutamyl phosphate, which is reduced by glutamyl phosphate reductase to glutamyl semialdehyde.

This product cyclizes spontaneously to pyrroline-5-carboxylate, which is reduced by NADPH to proline by pyrroline-5-carboxylate reductase.
6) Study lipid peroxidation and protein carbonylation under stress conditions.

7) Determine the contents of ascorbate and reduced glutathione (GSH) under stress condition.

8) Purify glutamate kinase from stressed and non-stressed plants and comparing the specific activities of the enzyme from both plants.

9) Compare the characteristics of the GK from stressed and non-stressed plants including:
a) Enzyme constants.
b) Effect of reducing agents.
c) Effect of thiol binding and tryptophan reagents.
d) Thermostability of GK in presence and absence of polyols

Seeds were germinated according to
El-Shora (2001)

Crude enzyme extraction
(El-Shora, 2001).

Glutamate kinase activity was assayed by
Hayzer and Leisinger(1980)

Determination of antioxidant enzymes

Activity of ascorbate peroxidase (APx) was measured according to
Cakmak (1994)

Total ascorbate was determined according to
Cakmak and Marschner (1992)

APX activity was determined according to the method of (Nakano & Asada, 1981)

APX activity was determined according to the method of (Nakano & Asada, 1981)

Glutathione reductase was assayed using the method described by Cakmak (1994).

Glutathione reductase was assayed using the method described by Cakmak (1994)

Glutathione reductase was assayed using the method described by Cakmak (1994)

Estimation of Reduced Glutathione by the method of Anderson (1985)
Levels of GK activity in C3 plants
Effect of JA on GK activity
Effect of GA3 on GK activity
Results
Conclusion
The algal extracts, ALA, HA and coronatine succeeded in partial alleviation of the toxicity of both Ni2+ and Hg2+. This is of great importance in agricultural purposes if we need to ameliorate the toxicity of heavy metals.

The polyols particularly sorbitol expressed the highest protection of the enzyme against thermostability. This is of great importance to keep enzyme at high temperature.

algae are found in large quantities.

Also, the enzyme from stressed plants was more robust and more resisGK from marrow leaves were induced by algal extracts, HA, ALA and coronatine. In fact the induction by algal extracts is of great importance if we need to produce GK on industrial scale, since the marine tant to the higher temperatures over the optimal one.

Effect of benzyl adenine on the enzyme activity
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