İnsan Kanserli Kolon Dokusundan CA İzoenzimlerinin Saflaştırılması ve Enzim Aktivitesi Üzerine Bazı Analjezik İlaçların İnhibitör Etkisi

Year 2012, Volume: 40 Issue: 2, 127 - 132, 01.04.2012

Ramazan Demirdağ Emrah Yerlikaya Ömer İrfan Küfrevioğlu Cemal Gündoğdu

Abstract

K arbonik anhidraz CA karbon dioksitin karbonik aside hidrasyonundan sorumlu bir enzimdir ve aynı zamanda canlı organizmalarda birçok biyolojik proseslerde yer almaktadır. Bu çalışmada, CA izoenzimleri CA II ve CA IX birlikte sepharose 4B-L-tirozin sülfanilamid afinite kromatografisi kullanılarak 106.67 spesifik aktivite ile % 54.86 verimle 78.4 kat saflaştırıldı. SDS-PAGE’de CA II ve CA IX izoenzimlerin molekül ağırlıkları sırası ile 29 kDa ve 56 kDa olarak hesaplandı. Ayrıca saflaştırılan total enzim üzerine bazı analjezik ilaçların inhibisyon etkisi araştırıldı. Deksketoprofen, pethidin, pheniramidol, tramadol’ün IC50 değerleri sırasıyla 0.0077, 0.025, 0.011, ve 0.04 mM olarak bulundu

Keywords

Saflaştırma, Kanser, Kolon, Karbonik Anhidraz, İnhibitör

Purification of Ca Isoenzymes from Human Cancerous Colon Tissue and Inhibitory Effects of Some Analgesics on Enzyme Activity

Abstract

Carbonic Anhydrase CA is an enzyme which is responsible for the hydration of carbon dioxide to carbonic acid and it also takes places in many biological processes in the living organisms. In this study, CA isoenzymes CA II and CA IX together were purified 78.4 fold with a yield of 54.86 and specific activity of 106.67 by using Sepharose 4B-L-tyrosine sulfanilamide affinity chromatography. In SDS-PAGE molecular weights of CA II and CA IX were calculated as 29 kDa and 56 kDa respectively. Besides inhibitory effects of some analgesics on purified total enzyme was investigated. IC50 values were found as 0.0077, 0.025, 0.011 and 0.04 mM for dexketoprofen, pethidine, phenyramidol and tramadol respectively.

Keywords

purification, Cancer, Colon, Carbonic anhydrase, Inhibitor

References

• 1. C.T. Supuran, A. Scozzafava, Carbonic Anhydrase Inhibitors, Current Medic. Chem., 1 ( 2001) 61.
• 2. J.B. Feldstein, D.N. Silverman, Purification and characterization of carbonicanhydrase from the saliva of the rat, J. Biol. Chem., 259 (1984) 5447.
• 3. S.R. Krungkrai, N. Suraveratum, S. Rochanakij, J. Krungkrai, Characterization of carbonic anhydrase in Plasmodium falciparum. International Journal of Parasitology, 31, (2001) 661.
• 4. N. Demir, Y. Demir, H. Nadaroglu, Carbonic anhydrase from bovine bone, Prep. Biochem. & Biotech., 31 (2001) 33.
• 5. S. Beydemir, M. Çiftçi, Ö. Küfrevioglu, Effects of gentamicine sulfate on enzyme activities of carbonic anhydrase from human erytrocytes in vitro and from rat erytrocytes in vivo, Biol. & Pharmac. Bull., 25 (2002) 966.
• 6. S. Beydemir, İ. Gülçin, Effects of melatonin on carbonic anhydrase from human erytrocyte in vitro and from rat erytrocytes in vivo, J. Enzyme Inhibition & Medic. Chem., 19 (2004) 193.
• 7. W.R. Chegwidden, S.J. Dodgson, I. M. Spencer, In the Carbonic Anhydrase-New Horizons, Birkhauser Verlag, Basel, ( 2000) 343.
• 8. W.R. Chegwidden, Y. Edwards, N. Carter, The Carbonic Anhydrases-New Horizons, Mol. Bases of Inher. Disease (Scriver, C. R., Beaudet, A. L.,Sly,W. S., and Valle, D., eds) 8th Ed., pp. (2000), 2165, McGraw-Hill, Inc.,NewYork.
• 9. C.T. Supuran, A. Scozzafava, Carbonic anhydrase inhibitors: Part 94. 1,3,4-thiadiazole-2-sulfonamide derivatives as antitumor agents?, Eur. J. Med. Chem, 35 (2000) 867.
• 10. C.T. Supuran, Indisulam: An anticancer sulfonamide in clinical development, Expert Opin Investig Drugs., 12 (2003) 283.
• 11. C.T. Supuran, A. Innocenti, A. Mastrolorenzo, A. Scozzafava, Antiviral sulfonamide derivatives, Mini. Rev. Med. Chem., 4 (2004) 189.
• 12. I. Nishimoiri, Carbonic anhydrase relatedproteins, in Carbonic Anhydrase, Its Inhibitors and ActiVators (Supuran, C. T., Scozzafava, A., and Conway, J., Eds.), Acatalytic CAs, CRC Press, Boca Raton, FL, (2004) 24.
• 13. M. Hilvo, M. Tolvanen, A. Clark, B.R. Shen, G.N. Shah, A. Waheed, P. Halmi, M. Hanninen, J.M. Hamalainen, M. Vihinen, WS. Sly, S. Parkkıla, Characterızatıon of CA XV, a new GPI-Anchored form of carbonıc anhydrase, Biochem. J., 392 (2005) 83, Part 1.
• 14. S.V. Ivanov, I. Kuzmin, M.H. Wei, S. Pack, B.E. Johnson, E.J. Stanbridge, M.I. Lerman, Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type Von Hippel-Lindau transgenes, Pros. Natl. Acad. Sci., USA, 95 (1998) 12596.
• 15. J. Pastorek, S. Pastrekova, I. Callebaut, J.P. Mornon, V. Zelnik, R. Opavský, M. Zat’ovicová, S. Liao, D. Portetelle, E.J. Stanbridge, Cloning and characterization of M.N, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment, Oncogene, 9 (1994) 2877.
• 16. S. Ivanov, SY. Liao, A. Ivanova, A. DanilkovitchMiagkova, N. Tarasova, G. Weirich, M.J. Merrill, M.A. Proescholdt, E.H. Oldfield, J. Lee, J. Zavada, A. Waheed, W. Sly, MI. Lerman, E.J. Stanbridge, Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer, Am. J. Pathol., 158 (2001) 905.
• 17. C.C. Wykoff, N.J. Beasley, P.H. Watson, K.J. Turner, J. Pastorek, A. Sibtain, G.D. Wilson, H. Turley, K.L. Talks, P.H. Maxwell, C.W. Pugh, P.J. Ratcliffe, A.L. Harris, Hypoxia-inducible expression of tumor-associated carbonic anhydrases, Cancer Res., 60 (2000) 7075.
• 18. A. Giatromanolaki, M.I. Koukourakis, E. Sivridis, J. Pastorek, C.C. Wykoff, K.C. Gatter, A.L. Harris, Expression of hypoxia-inducible carbonic anhydrase-9 relates to angiogenic pathways and independently to poor outcome in non-small cell lung cancer, Cancer Res., 61 (2001) 7992.
• 19. S.K. Chia, C.C. Wykoff, P.H. Watson, C. Han, R.D. Leek, J. Pastorek, K.C. Gatter, P. Ratcliffe, A.L. Harris, Prognostic significance of a novel hypoxia-regulated marker, carbonic anhydrase IX, in invasive breast carcinoma, J. Clinic. Onco., 19 (2001) 3660.
• 20. N. Robertson, C. Potter, A.L. Harris, Role of carbonic anhydrase IX in human tumor cell growth, survival and invasion, Cancer Res., 64 (2004) 6160.
• 21. http://www.pharmaceutical-drug-manufacturers. com/pharmaceutical-drugs/analgesics. html
• 22. http://en.wikipedia.org/wiki/
• 23. K.M. Wilbur, N.G. Anderson, Electrometric and colorimetric determination of carbonic anhydrase, J. Biol. Chem., 176 (1948) 147.
• 24. J.A. Verpoorte, S. Mehta, J.T. Edsall, Esterase Activities of Human Carbonic Anhydrases B and C, J. Biol. Chem., 242 (1967) 4221.
• 25. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248.
• 26. D.K. Laemmli, Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4, Nature, 227 (1970) 680.
• 27. H. Lineweaver, D. Burk, The determination of enzyme dissociation constants, J. Am. Chem. Soc., 57 (1934) 685.
• 28. D.E. Swinson, J.L. Jones, D. Richardson, C. Wykoff, H. Turley, J. Pastorek, Carbonic anhydrase IX expression, a novel surrogate marker of tumor hypoxia, is associated with a poor prognosis in non-small-cell lung cancer, J. Clin. Oncol., 21 (2003) 473.
• 29. P. Vermylen, C. Roufosse, A. Burny, A. Verhest, T. Bosschaerts, S. Pastorekova, V. Ninane, JP. Sculier, Carbonic anhydrase IX antigen differentiates between preneoplastic malignant lesions in non-small cell lung carcinoma, Eur. Respir. J., 14 (1999) 806.
• 30. S. Rasheed, P.J. McDonald, M. John, J. Northover, T. Guenther, Angiogenesis and hypoxic factors in colorectal cancer, Path. Res. & Pract., 204 (2008) 501.
• 31. D. Schottenfield, Epidemiology of Colorectal Cancer, McGraw-Hill Inc., (1995) 11-23.
• 32. J. Saarnio, S. Parkkila, A.K. Parkkila, K. Haukipuro, S. Pastoreková, J. Pastorek, M.I. Kairaluoma, T.J. Karttunen, Immunohistochemical Study of Colorectal Tumors for Expression of a Novel Transmembrane Carbonic Anhydrase, MN/CA IX, with Potential Value as a Marker of Cell Proliferation, Am. J. Pathol. 153 (1998) 279.
• 33. N. Robertson, C. Potter, A.L. Harris, Role of carbonic anhydrase IX in human tumor cell growth, survival, and invasion, Cancer Res., 64 (2004) 6160.