Mustafa ERAT, Ahmet ADIGÜZEL, Mehmet ÇİFTÇİ, Fikrettin ŞAHİN

Bacillus sp.(BA-142) bakterisinden glukoz 6-fosfat dehidrogenaz enziminin kısmen saflaştırılması

Glukoz 6-fosfat dehidrogenaz enzimi (G6PD) Bacillus sp.(BA-I42) bakterisinden homojenatın hazırlanması ve amonyum sülfat çöktürmesi vasıtasıyla kısmi olarak saflaştırıldı ve bazı kinetik özellikleri araştırıldı. Saflaştırma prosedürü iki basamaktan oluştu; homojenatın hazırlanması ve amonyum sülfat çöktürmesi. Bu iki basamak sonucunda 0,23 EÜ/mg protein spesifik aktivitesine sahip enzim % 79 verimle 82 kat saflaştırıldı. Enzim için optimum pH, stabil pH, optimum sıcaklık değerleri bulundu. Ayrıca, NADP+ ve glukoz 6-fosfat (G6P) için KM ve VITMX değerleri tespit edildi.

Partially prification of glucose 6-phoshate dhydrogenase from bacillus sp. (BA-142) and investigation of some kinetic properties

Glucose 6-phosphate dehydrogenase (G6PD) was purified from Bacillus sp.(BA-142) bacterium cells, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation, and ammonium sulfate precipitation. After two consecutive steps, the enzyme, specific activity was 0.23 EU/mg protein, was purified with a yield of 79% and 82 fold purification. Optimal pH, stable pH, optimal temperature, KM and V,^ values for NADP+ and glucose 6-phosphate (G 6 P) were also determined for the enzyme.

PDF

___

Beutler E., 1971. Red cell metabolism manual of biochemical methods, p. 19,68. Academic Press, London.
Bradford M.M., 1976. A rapid and sensitive method for the quantition of microgram quantities of protein utilizing l:he principle of protein-dye binding. Anal. Biochem., 72: 248-251.
Çakmakçı, R., Kantar, F., Şahin, F., 2001. Effect of N2-fixing bacterial inoculations on yield of sugar beet and barley. J. Plant Nutr. Soil ScL, 164:527-531.
Çiftçi, M., Beydemir, Ş., Yılmaz, H., Altıkat, S., 2003. Purification of glucose 6-phosphate dehydrogenase from Bufalo (Bubalus bubalis) erythrocytes and investigation of some kinetic properties. Protein Expression and Purification. 29: 304-310.
Delgado C, Tejedor MJ, Luque J., 1990. Partial purification of glucose 6-phosphate dehydrogenase and phosphofructokinase from rat erythrocyte haemolysate by partitioning in aqueous two-phase systems. Journal of Chromatograpy, 498:159-168.
Deutsch J., 1983. Glucose-6-phosphate dehydrogenase. In Methods of enzymatic analysis, Edited by Bergmeyer HU and Bergmeyer J, 3:190-196.
Heise, N., Opperdoes, F.R., 1999. Purification, localisation and characterisation of glucose-6-phosphate dehydrogenase of Trypanosoma brucei. Molecular and Biochemical Parasitology, 99:21-32.
Keha EE, Küfrevioğlu Öt., 1997. Biyokimya, Şafak Yayınevi, Erzurum. Lehninger A.L., Nelson, D.L., Cox, M.M., 1993. Principles of Biochemistry. Worth Publishers, New York.
Morelli, A., Benatti, U., Gaetani, G.F., De Flora, A., 1978. Biochemical mechanisms of glucose 6-phosphate dehydrogenase deficiency. ProcNatl Acad Sci., 75: 4-9
Niehaus, W.G., Mallett, T.C., 1994. Purification and characterization of glucose-6-phosphate dehydrogenase from Crytococcus neoformans: Identification as " Nothing dehydrogenase".Archives of Biochemistry and Biophysics, 313(2): 304-309.
Ninfali, P., Orsenigo, T., Barociani, L., Kapa, S., 1990. Rapid purification of glucose 6-phosphate dehydrogenase from mammal's erythrocytes. Preparative Biochemistry, 20:297-309.
Okuno, H., Nagata, K., Nakajima, H., 1985. Purification and properties of glucose-6-phosphate dehydrogenase from Bacillus stearothermophilus. Journal of Applied Biochemistry, 7: 192-201.
Reuter, R., Naumann, P., Metz, P., Kopperschlager, G., 1990. Purification :and characterization of glucose-6-phosphate dehydrogenase from Pseudomonas W6. Biomed. Biochim. Acta 49(7): 539-546.
Scopes, P.K., Testolin, V., Stoter, A., Griffiths-Smith, K., Algar, E.M.., 1985. Simultaneous purification and characterization of glucose-6-phosphate dehydrogenase from Zymomonas mobilis. Biochem. J., 228:627-634.
Shreve, D.S., Levy, H.R.. 1997. On the molecular weight of human glucose 6-phosphate dehydrogenase. Biochemical and Biophysical Research Communications, 78:1369-1375.
Weksler, B.B., Moore, A., Tepler, J., 1990. Hematology. In Cecil essentials of medicine, Edited by Andreoli TE, Carpenter CCJ, Plum F, Smith LH., Jr., 2nd edn, Philadelphia: WB Saunders Compainy, 341-363.
Yılmaz, H., Çiftçi, M., Beydemir., Bakan Küfrevioğlu, 2003., Purification and properties of glucose 6-phosphate dehydrogenase from turkey erythrocytes. Indian Journal of Biochemistry and Biophysics, 40: 62-65.
Yılmaz H., Çiftçi, M., Beydemir, Ş. Bakan E., 2002 Purification of glucose 6-phosphate dehydrogenase from chicken erythrocytes and investigation of some kinetic properties. Preparative Biochemistry and Biotechnology. 32 (3): 287-301.
Yoshida, A., Huang, I.Y., 1986. Structure of human G6PD. Academic Press Inc Ltd, London.
Yüreğir, T.G., Aksoy, K., Dikmen, N., Ünlükurt, L, 1988. Glukoz 6-fosfat dehidrogenaz enziminin insan eritrositlerinden kısmi saflaştırılması ve enzimolojik 'özelliğinin incelenmesi. IX.Ulusal Biyoloji Kongresi, Genel Biyoloji, Nümerik Taksonami ve Kantitatif Ekoloji Paneli bildirileri, 1:125-130.