Seval YILMAZ, İbrahim Hanifi ÖZERCAN, Temizer Sema OZAN

Pirüvat kinazın normal ve tömür meme dokusundan saflaştırılması ve karakterizasyonu

İnsan normal ve tümör meme doku pirüvat kinazı saflaştırılarak moleküler ağırlığı ve kinetik özellikleri araştırılmış, normal meme dokusu ile tümör meme dokusu pirüvat kinaz aktivite düzeyleri karşılaştırılmıştır. DEAE Sefadeks A-SO kromatografi ile meme dokusunda pirüvat kinazın iki farklı formunun varlığı gösterilmiştir. SDS-PAGE ile alt birimlerinin moleküler ağırlığı normal dokuda 1. pikte 30.000 Da, II. pikte 63.000 Da, tümör dokusunda ise 1. pikte 16.500 Da, II. pikte 60.000 Da olarak saptanmıştır. Pirüvat kinaz aktivitesi tümör meme dokusunda normal meme dokusuna göre 5,2 kat fazla bulunmuştur. Pirüvat kinaz enzimi normal meme dokusunda 1. pik 1591 kat, II. pik 636,4 kat, tümör dokusunda I. pik 219 kat, II. pik ise 318 kat saflaştırılabilmiştir. Reaksiyon hız eğrisi normal ve tümör meme dokusu pirüvat kinazının 1. pikinde hiperbolikti ve FDP tarafından aktive edilmemiştir. II. pikte reaksiyon hız eğrisi tümör meme dokusunda hiperbolik, normal meme dokusunda ise sigmoidaldır ve FDP tarafından aktive edilmiştir. Hill katsayısı PEP için enzimde en az iki bağlanma bölgesinin varlığını göstermiştir. İnsan meme dokusundan izole edilen pirüvat kinaz izoenzimleri diğer doku pirüvat kinaz izoenzimleri ile karşılaştırıldığında bunların M, ve M2 izoenzim olduğu ve normal meme dokusundaki M2 izoenzimin tümör meme dokusunda K izoenzime dönüşebileceği düşünülebilir.

Prufication and characterization of pyruvate kinase from normal and tumor breast tissues

The molecular weight and kinetic properties of pyruvate kinase purified from human normal and tumor breast tissues were studied and the activity levels of pyruvate kinase from normal and tumor breast tissues were compared. The presence of 2 forms of pyruvate kinase in human breast tissue was demonstrated by DEAE Sephadex A-50 chromatography. The molecular weight of subunits estimated by SDS-PAGE in forms I. and II. in normal breast tissue and in forms I. and II. in tumor breast tissue were 30,000 and 63,000 Da and 16,500 and 60,000 Da, respectively. It was found that the pyruvate kinase activity in tumor tissue was 5.2 times higher than that in normal tissue. Peaks I and II of pyruvate kinase were able to be purified about 1591-fold and 636.4-fold in normal breast tissue, and 219-fold and 318-fold in tumor breast tissue, respectively. The reaction rate curve was hyperbolic in the first peaks of both normal and tumor breast tissue pyruvate kinase and was not activated by fructose-1, 6-diphosphate (FDP). Reaction rate curves in the second peaks of tumor breast tissue and normal breast tissue pyruvate kinase were hyperbolic and sigmoidal, respectively, and were activated by FDP. The Hill coefficient showed that there were at least 2 binding regions in the enzyme for PEP. When compared with other tissue pyruvate kinase isozymes, it seems likely that the isoenzymes of pyruvate kinase isolated from human breast tissue were M, and M2 isozymes and that the M2 isozyme of pyruvate kinase from normal breast tissue changed into K isoenzyme in tumor breast tissue.

PDF

___

1. Onat, T., Emerk, K.: Temel Biyokimya. Saray Medikal Yayıncılık, İzmir. 1997:661-662.
2. Muirhead, H.: Isoenzymes of pyruvate kinase. Biochem. Soc. Trans., 1990; 18: 193-196.
3. Cuenllas, E., Gaitan, S., Bueren, J.A., Tejero, C: Kinetic studies of pyruvate kinase during in vitro differentiation of GM-CFC haemopoietic precursor and bone marrow cells in mice. Biosci. Rep., 1990; 10: 141-154.
4. Eigenbrodt, E., Reinacher, M., Scheefers-Borchel, U., Scheefers, H., Friis, R.: Double role for pyruvate kinase type M2 in the expansion of phosphometabolite pool found in tumor cells. Crit. Rev. Oncog., 1992:3:91-115.
5. Heinrichs, M., Jacobasch, G., Scheiner-Bobis, K., Bertram, S., Eigenbrodt, E., Reinacher, M.: Human erythrocyte pyruvate kinase (L7R-PK): Production and characterization of a monoclonal antibody. Biomed. Biochim. Acta., 1987; 46: 223-228.
6. Kiffmeyer, W.R., Farrar, W.W.: Purification and properties of pig heart pyruvate kinase. J. Protein Chem., 1991; 10: 585-591.
7. Ibsen, K.H.: Interrelationships and functions of the pyruvate kinase isozymes and their variant forms: A Review. Cancer Res., 1977:37:341-353.
8. Kaloyianni-Dimitriades, M.G., Beis, I.D.: Purification, catalytic and regulatory properties of Rana ridibunda erythrocyte pyruvate kinase. Comp. Biochem. Physiol., 1984; 7B: 245-250.
9. Elbers, J.R., Unnik, J.A., Rijksen, G., Oirschot, B.A., Roholl, P.J., Ostihg, J., Staal, G.E.: Pyruvate kinase activity and isozyme composition in normal fibrous tissue and fibroblastic proliferations. Cancer, 1991; 67: 2552-2559.
10. Mazurek, S., Grimm, H., Oehmke, M., Weisse, G., Teigelkamp, S., Eigenbrodt, E.: Tumor M2-PK and glutaminolytic enzymes in the metabolic shift of tumor cells. Anticancer Res., 2000; 20: 5151-5154.
11. Terlecki, G.: Purification and properties of pyruvate kinase type Mt from bovine brain. Int. J. Biochem., 1989; 21: 1053-1060.
12. Weernink, P.A.O., Rijksen, G., Staal, G.E.J.: Phosphorylation of pyruvate kinase and glycolytic metabolism in three human glioma cell lines. Tumour Biol., 1991; 12: 339-352.
13. Helmy, E., Unnik, A.M., Rijksen, G., Smite, G., Staal, J.: Cellular expression of K-type pyruvate kinase in normal and neoplastic human tissues. Cancer, 1991; 68: 2595-2601.
14. Arai, T., Ogino, T., Gunji, M.. Washizu, T., Komdfi, S., Washizu, M.: Changes in glucose transport activities in mammary adenocarcinoma of dogs. Res. Vet. Sci., 1997; 62: 85-86.
15. Kwiatkowska, D., Dawiskiba, J., Kwiatkowska, J.: Alterations in erythrocyte enzymes in cancer. Neoplasma,-1980; 27: 415-422.
16. Yılmaz, S., Ozan, S., Özercan, I.H.: Comparison of pyruvate kinase variants from breast tumor and normal breast. ArctvMed. Res., 2003:34:315-324.
17. Cardenas, J.M., Dyson, R.D., Strandholm, J.J.: Bovine pyruvate kinases. I. purification and characterization of the skeletal muscle isozyme. J. Biol. Chem., 1973; 248: 6931-6937.
18. Beutler, E., Blume, K.G., Kaplan, J.C., Löhr, G.W., Ramot, B., Valentine, W.N.: International committee for standardization in haematology: Recommended methods for red-cell enzyme analysis. Br. J. Haematol., 1977; 35: 311-340.
19. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the folin phenol reagent. J. Biol. Chem., 1951; 193: 265-275.
20. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophag T4. Nature, 1970; 227: 680-685.
21. Imamura, K., Tanaka, T.: Pyruvate kinase isozymes from rat, In: Wood, M. A. Eds. In Methods in Enzymology. Academic Press, New York, 1982; 150-165.
22. Hilf, R.. Wittliff, J.L., Rector, W.D., Savlov, E.D., Hall, T.C., Orlando, R.A.: Studies on certain cytoplasmic enzymes and specific estrogen receptors in human breast cancer and in non- malignant diseases of the breast. Cancer Res., 1973; 33: 2054-2062.
23. Pedersen, N.: The glycolytic enzyme activity of the human cervix uteri. Cancer, 1975; 35: 469-474.
24. Ibsen, K.H., Orlando, R.A., Garratt, K.N., Hernandez, A.M., Giorlando, S., Nungaray, G.: Expression of multimolecular forms of pyruvate kinase in normal benign and malignant human breast tissue. Cancer Res., 1982; 42: 888-892.
25. Larner, E.H.: Rutherford, C L: Application of a microchemical technique to the elucidation of enzyme activity profiles within single human mammary tumors. Cancer, 1978; 41: 1863-1870. 26. Hoopmann, M., Warm, M., Mallmann, P., Thpmas, A., Gohring, U.J., Schondorf, T.: Tumor M2 pyruvate kinas'e-determination in breast cancer patients receiving trastuzumab "therapy. Cancer Lett., 2002; 187:223-228. 27. Luftner, D., Mesterhanm, J., Âkrivakis, C, Geppert, R., Petrides, P.E., Wernecke, K.D., Possinger, K.: Tumor type M2 pyruvate kinase expression in advanced breast cancer. Anticancer Res., 2000; 20: 5077-5082.
28. Farina, F.A., Shatton, J.D., Morris, H.P., Weinhouse, S.: Isozymes of pyruvate kinase in liver and hepatomas of the rat. Cancer Res., 1974; 34: 1439-1446.
29. Tolle, S.W., Dyson, R.D., Neburgh, R.W., Cardenas, J.M.: Pyruvate kinase isozymes in neurons, glia, neuroblastoma and glioblastoma. J. Neurochem., 1976; 27: 1355-1360.
30. Noda, S., Horn, F., Under, D., Schoner, W.: Purified pyruvate kinases type M2 from unfertilized hen's egg substrates of protein kinase C. Eur. J. Biochem., 1986; 155: 643-651.
31. Yılmaz, S.: însan eritrosit ve karaciğer pirüvat kinazının kinetik özellikleri, Doktora Tezi. Sağlık Bilimleri Enstitüsü, Elazığ. 1997.
32. Leon, O., Moran, A., Gonzalez, R.: Purification and characterization of pyruvate kinase from muscle of the sea mollusc, Concholepas concholepas. Com. Biochem. Physiol., 1982; 72B: 65-69.