Ibragimova S.A., Riger N.G., Karabalin A.B., Kulbaeva G.A.,  Gaiypbayeva A.N.

Purified antibodies as sensible test for evolutionary young plant enzyme complex MDh GOAD.

 M.A. Aytkhozhin`s institute of Molecular Biology and Biochemistry, 050012 Kazakhstan, Almaty, Dosmukhamedova str 86. baltakay@mail.ru

The appearance of the new proteins in the course of evolution, gives to individual organism the new significant adaptive advantage in changing environmental and climatic conditions (1,2). Namely this is the material basis of the emergence of new taxonomic groups during the evolution.  The investigations, of the evolutionary new proteins have importance for evolution theory, for genomics and genetic, for biochemistry, for physiology and for ecology.

Until now, is considered that the main enzyme of the glutamate metabolism is the glutamate dehydrogenase (GDh). One of the negative property of the reaction of GDh is the allocation of toxic ammonia. It is well known that ammonia able to destroy of biomembranes. For example, Helicobacter pylori during its life liberates ammonia which destroys of the biomembranes of the gastric wall (3). This process is specially very dangerous when plants are in the stress conditions.

In laboratory of the enzymes structure and regulation it was discovered  in new enzyme complex (EC) (4,5). It was shown that EC consists of two enzymes:  malate dehydrogenase (MDh) and glutamateoxaloacetate aminotranferase (GOAT).  EC catalyses the irreversible reaction of the cleavage of the glutamate without allocation of toxic ammonia. But this reaction is impossible when this reaction are catalyzed by MDh and GOAT separately. It means that EC is not a simple sum of the two enzymes, but it is the new enzyme complex in which enzymes have acquired new evolutionary changes. Due to these changes EC works like one enzyme of the irreversible cleavage of glutamate without allocation of ammonia. As it was shown by as (6) the EC is absent in:  in animals, in microorganisms, in algae, in mushrooms, in lower plants. Also EC is absent in Gymnospermae plants and in ancient floral plants such as Magnoliesea, Liliales plants. Thus EC is evolutionary young protein complex. The most reliable method for detection of the new proteins is the receiving the specific antibodies to new protein. In this reason the task of our study is the receiving Purified antibodies as sensible test for evolutionary young plant EC MDh GOAD.

            The materials and methods the seeds of winter wheat (Triticum aestivum L.) “Steklovidnaya -24” cultivar .  Also we used the guinea pigs (Cavia porcellus). 

            The reaction mixture for spectrophotometeric determination of EC activity contents: 1.1 mM NAD, sodium malate 12mM, 87 mM sodium glutamate, 0,05 M tris – phosphate  buffer, pH 8.0 till total volume - 2 ml. Also the quantity of the proteins of the measured by Bradford methods(7).  In the study we used the spectrophotometer Ultrospec 1100 Bioscience, UC. Column chromatography performed using the instrument control elution UA-6 UV / VIS Detector (Teledyne Isco, USA).

Results and discussion

1.The purification of the EC.

It was shown that the maximum of activity of EC has on third day of the seed germination. In this reason the for purification of EC which used three days germinated seeds.   This seeds were homogenized in the chilled porcelain mortar with 0.05 M tris – phosphate buffer, pH 7, 5.  Than the homogenized was centrifuged at 10000xg during 15 minutes. The supernatant was used for purification EC. The first supernatant was purified by gel-chromatography on Sephacryl S-300 column. The results of purification are presented at figure 1. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


Figure 1 - gel - chromatography of EC on Sephacryl S-300 column.

The fraction with activity of EC was collected. This fraction was eluted in volume which corresponds to volume of protein with molecular mass 110 kilo Dalton. Then collected fraction was purified by ion-exchange chromatography on DE-52 type DEAE-cellulose column. The fraction with EC activity was eluted by 0,15 M KCl in 0.05 M tris – phosphate buffer, pH 7,5.  The purified EC has molecule masse 110 kilo Dalton.  The purified EC was separated by SDS electrophoreses (4). The result is presented at figure 2.

SDS electrophoreses of EC

kDA

94.0

 

 

67.0

 

 

60.0

50.0

43.0

 

 

 

 

 

 

 

 

30.0

 

 

 

 

 

 

 

 

 

 

 

20.0

 

 

 

 

 

 

 

 

14.0

 

 

 

 

 

 

 

 

 

      1              2               3

 

Figure 2.  1-marker proteins; 2-EC before purification; 3-purofeid EC.

Thus EC have molecule masse 110 kDA an consists of two proteins with molecule masse 50 and 60 kDA.

2. Purification of antibodies to EC.

For purification antibodies to EC we carried out immunization of adult female guinea pigs with purified EC. Immunization was performed three times every day. After 45 days the blood was taken from guinea pig heart. The obtained blood was centrifuged for receiving of the plasma. Immunoglobulin fraction obtained by fractional precipitation with ammonium sulfate in the range of 20-50% of saturation. Then obtained immunoglobulins were purified by affinity chromatography on protein - G Sepharose. This sorbent are used for purification of antibodies.

Protein G sepharose was designed for easy, one-step purification of classes of immunoglobulin’s which have Fc region.

In bases of this massed is phenomena of Fc region which involved in antibody binding. Protein G-Sepharose beads are prepared by covalently coupling recombinant Protein G to 6% cross-linked Sepharose beads. Affinity chromatography of antibodies to EC was performed according to the practical recommendation 1999 Roche Company (France). The immunoglobulin’s fraction after ammonia sulfate precipitation was purified by affinity chromatography on protein - G Sepharose. The column was washed by start buffer 20 mM nutria phosphates, pH7, 0. For removing the ballast proteins the column was washed by start buffer with addition 150mM NaCl and 2 mM EDTA. Antibodies to EC were eluted by 0,1M glycine –HCl buffer pH 2, 7. Thus we are obtained highly purified antibodies to FC. They can be successfully used to identify of EC in the different taxonomic group of the floral plant.

References

1.      Evolutionarily Young Protein Helps Ancient RNA Get Into Shape. http://news.yale.edu/2010/10/13/evolutionarily-young-protein-helps-ancient-rna-get-shape

2.      Evolutionary change during experimental ocean acidification. http://www.ncbi.nlm.nih.gov/pubmed/23569232

3.      Molecular and cellular activities of Helicobacter pylori pathogenic factors. http://www.ncbi.nlm.nih.gov/pubmed/10376670

4.      Gilmanov M.K., Kudiyarova Zh.S., Rakhmetova Zh.K., Omirbekova N.Zh., Bekbaeva L.K., Kurmanov B.K. The structure and functions of evolutionary young enzyme complex MDh- GOAT // Nitrogen 2007 An international symposium on the nitrogen nutrition of plants, Lancaster University- UK, 27- 31 July, 2007

5.      The structure and functions of evolutionary young enzyme complex MDh- GOAT Nitrogen 2007 An international symposium on the nitrogen nutrition of plants, Lancaster University- UK, 27- 31 July, 2007. Kudiyarova Zh.S., Rakhmetova Zh.K., Omirbekova N.Zh., Bekbaeva L.K., Kurmanov B.K.

6.      Gilmanov M.K., Rakhmetova Zh.K., Sartbaeva I.A.,  “The study of the activity, physico-chemical properties and regulation of the MDh-GOAT enzyme complex in the evolutionary different plants and organisms”. In the materials of the international conference “Actual problems of the biological sciences” Almaty 2005 pp.423-428. (in kazakh)

7.     Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248-254. 1976.