Purification of Alkaline Phosphatase from Bovine Milk and Investigation of Inhibitory Effects Of Some Veterinary Drugs on Enzyme Activity

Abstract

In this study, Alkaline Phosphatase EC 3.1.3.1, ALP which has a critical role in phosphate metabolism was purified via bi- ochemical methods and the effects of some veterinary drugs on purified milk ALP were determined on account of the fact that using as a pasteurization indicator in milk. Enzyme was homogenized with different organic solvents, ammonium sulfate precipitation, dialysis and following Sephadex G-100 gel filtration chromatography. The enzyme product yielded bands, app- roximately 85 and 185-190 kDa on SDS-PAGE except casein fractions, while a single band, approximately 170-190 kDa interval. ALP is the most important enzyme in dairy industry as the check for the presence of residual ALP activity in milk is a good indication of proper pasteurization. Heat treatment of milk for adequate pasteurization, which kills pathogenic microorga- nisms, give rise to inactivate ALP. It seems as if milk is pasteurized before not to reach pasteurization temperature, in case of decreasing ALP activity notably by the veterinary drugs. Therefore, in vitro inhibitory effects of some drugs furosemide, atropine sulfate, toldimfos sodium and levamisol/synonim: - - tetramisol HCI were investigated in this study. While atropine sulfate didin’t effect the enzyme activity, the IC50 values of drugs which have inhibitory effect were obtained 45.48 μM, 2.19 and 126.315 mM for tetramisol, furosemide and toldimfos sodium, respectively. When compared to Levamisol, which is a po- tent inhibitor of the enzyme, the inhibitory effects of the drugs on the enzyme activity are weak.

Keywords

• Alkaline phosphatase,purification,Inhibition,veterinary drugs

İnek Sütünden Alkalen Fosfataz Enziminin Saflaştırılması ve Bazı Veteriner İlaçların Enzim Aktivitesi Üzerine İnhibisyon Etkilerinin İncelenmesi

Abstract

Bu çalışmada, fosfat metabolizmasında önemli role sahip olan Alkalen Fosfatazın ALP , sütte pastörizasyon belirteci olarak kullanılması gerekçesiyle sütte bulunan izoformu biyokimyasal yöntemlerle saflaştırılmış ve bazı veteriner ilaçların enzim aktivitesi üzerine etkileri araştırılmıştır. Enzim, çeşitli organik çözücülerle muamele edilerek, amonyum sülfat çöktürme aralıkları belirlenmiş ve elde edilen çökelek diyaliz edilerek, Sephadex G-100 jel filtrasyon kolonuna tatbik edilmiştir. Saflaştırılan enzim SDS poliakrilamid jel elektroforezine uygulanarak, elektroforezde gözlenen kazein fraksiyonları dışında, yaklaşık 85 kDa ve 185-190 kDa molekül ağırlığına sahip bandlar elde edilirken; doğal jel elektroforezinde, yaklaşık 170-190 kDa aralığında tek band gözlenmiştir. Enzim, pastörizasyon sıcaklığına gelindiğinde inaktive olacağından enzim aktivitesinin gözlenmemesi sütün pastörize olduğunu gösterir. Fakat, veteriner ilaçlar tarafından enzimin inaktivasyonu söz konusu olduğunda, pastörizasyon sıcaklığına ulaşılmadan enzim inhibe olacağından doğru bir pastörizasyon gerçekleştirilemeyecektir. Bu sebeple çalışmamızda, bazı ilaçların furosemide, atropine sulfate, toldimfos sodium and levamisol/ - - tetramisol HCI enzim aktivitesi üzerine inhibisyon etkileri incelenmiştir. Atropin sülfat enzim aktivitesini etkilemez iken, enzim aktivitesi üzerinde inhibisyon etki gösteren tetramisol, furosemid ve toldimfos sodyuma ait IC50 değerleri sırasıyla 45.48 μM, 2.19 ve 126.315 mM olarak hesaplanmıştır. ALP’nin bilinen inhibitörü olan Levamisol ile karşılaştırıldığında, enzim aktivitesini azaltan ilaçların zayıf inhibitör etki gösterdiği belirlenmiştir.

Keywords

• Alkalen fosfataz,saflaştırma,inhibisyon,veteriner ilaçlar

References

1. J.L. Millan, Alkaline Phosphatases, Structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes, Review, Purinergic Signalling, 2 (2006) 335-341.
2. J.G. Zalatan, T.D. Fenn and D. Herschlag, Comparative enzymology in the alkaline phosphatase superfamily to determine the catalytic role of an active-site metal Ion, J. Mol. Biol., 384 (2008) 1174–1189.
3. S.C. Bihani, A. Das, K.S. Nilgiriwala, V. Prashar, M. Pirocchi, S.K. Apte, J.-L. Ferrer, M.V. Hosur, X-Ray structure reveals a new class and provides insight into evolution of alkaline phosphatases, Plos One, 6(7) (2011) e22767.
4. J.G. Rola, and M. Sosnowski, Determination of alkaline phosphatase activity in milk and milk products by fluorimetric method, Bull Vet Inst Pulawy, 54 (2010) 537-542.
5. A.V. Vega-Warner, C.-H. Wang, D.M. Smith, and Z. Ustunol, Milk alkaline phosphatase purification and production of polyclonal antibodies, Journal of Food Science, 64(4) (1999) 601-605.
6. P.F. Fox, A.L. Kelly, Indigenous enzymes in milk: overview and historical aspects-Part 2, Review, International Dairy Journal, 16 (2006) 517-532.
7. L. Bjelakovic, G. Kocic, T. Cvetkovic, D. Stojanovic, S. Najman, Z. Pop-Trajkovic, M. Jonovic, B. Bjelakovic, Alkaline phosphatase activity in human milk during the first month of lactation, Acta Fac Med Naiss, 26(1) (2009) 43-47.
8. Y. Kuzuya, Y. Kanamaru and T. Tanahashi, Carbohydrate composition in bovine milk alkaline phosphatase, Jpn. J. Zootech. Sci., 54(7) (1983) 487- 490.

9. J. Fenoll, G. Jourquin, J.M. Kauffmann, Fluorimetric determination of alkaline phosphatase in solid and fluid dairy products, Talanta, 56 (2002) 1021–1026.
10. J.E. Coleman, Structure and Mechanism of Alkaline Phosphatase, Annu. Rev. Biophys. Biomol. Struct., 21 (1992) 441-83.
11. S.A. Rankin, A. Christiansen, W. Lee, Banavara, D. S. and Lopez-Hernandez, A., Invited review: The application of alkaline phosphatase assays for the validation of milk product pasteurization, J. Dairy Sci. 93 (2010) 5538–5551.
12. Alkaline Phosphatase (D-3): sc-137213, Santa Cruz Biotechnology, Inc.
13. R.K. Dhaked, S.I. Alam, A. Dixit, L. Singh, Purification and characterization of thermo-labile alkaline phosphatase from an Antarctic psychrotolerant Bacillus sp. P9, Enzyme and Microbial Technology, 36 (2005) 855–861.
14. J.-Y. Zhu, G.Y. Ye, Q. Fang and C. Hu, Alkaline phosphatase from venom of the endoparasitoid wasp, Pteromalus puparum, Journal of Insect Science 10:14, available online: insectsicence.org/10.14, 2010.
15. B. Serra, M.D. Morales, A.J. Reviejo, E.H. Hall, J.M. Pingarrón, Rapid and highly sensitive electrochemical determination of alkaline phosphatase using a composite tyrosinase biosensor, Analytical biochemistry, 336 (2005) 289-94.
16. A. Aminian, F. Karimian, R. Mirsharifi, A. Alibakhshi, S.M. Hasani, H. Dashti, Y. Jahangiri, H. Ghaderi and A. Meysamie, Correlation of serum alkaline phosphatase with clinicopathological characteristics of patients with oesophageal cancer, Eastern Mediterranean Health Journal, 17(11) (2011) 862-866.
17. M. Abramowitz, P. Muntner, M. Coco, W. Southern, I. Lotwin, T.H. Hostetter and M.L. Melamed, Serum Alkaline Phosphatase and Phosphate and Risk of Mortality and Hospitalization, Clin J Am Soc Nephrol, 5 (2010) 1064–1071.
18. M.W. Eerdekens, E.J. Nouwen, D.E. Pollet, T.W. Briers and M.E. De Broe, Placental Alkaline Phosphatase and Cancer Antigen 125 in Sera of Patients with Benign and Malignant Diseases, Clin. Chem., 31/5 (1985) 687-690.
19. Q.-B. She, J.J. Mukherjee, J.-S. Huang, K.S. Crilly and Z. Kiss, Growth factor-like effects of placental alkaline phosphatase in human fetus and mouse embryo fibroblasts, FEBS Letters, 469 (2000) 163-167.
20. E. Yorganci and E. Akyilmaz, Alkaline phosphatase based amperometric biosensor immobilized by cysteamine-glutaraldehyde modified self-assembled monolayer, Artificial Cells, Blood Substitutes, and Biotechnology, 39 (2011) 317–323.
21. R. Caprita, A. Caprita, Comparative study on milk casein assay methods, Zootehnie si Biotehnologii, Timisoara, 41 (2008) 758-762.
22. M. Inouye, Effect of Acetone on alkaline phosphatase activity, J Biochem, 54 (1963) 138-145.
23. N. Özer, M. Müftüoğlu, D. Ataman, A. Ercan, I.H. Ögüs, Simple, high-yield puriŞcation of xanthine oxidase from bovine milk, J. Biochem. Biophys. Methods, 39 (1999) 153–159.
24. C. O’Fagain, P.F. Cummins and B.F. O’Connor, Gel filtration chromatography, Protein chromatography methods in molecular biology, 681 (2011) 25-33.
25. P. Stanton, Gel filtration chromatography, HPLC of Peptides and Proteins Methods in Molecular Biology™ 251 (2004) 55-73.
26. R. Aschaffenburg, J.E.C. Mullen, A rapid and simple phosphatase test for milk, Journal of Dairy Research, 16 (1949) 58-67.
27. S. Fadıloğlu, O. Erkmen and G. Şekeroğlu, Thermal and carbon dioxide inactivation of alkaline phosphatase in buffer and milk, Food Technol. Biotechnol., 42 (2004) 27-32.
28. M.M. Bradford, A rapid and sensitive method or the quantition of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72 (1976) 248–54.
29. U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227 (1970) 680–5.
30. R.K. Morton, Alkaline phosphatase of milk association of the enzyme with a particulate lipoprotein complex, Department of Biochemistry, University of Cambridge, (1953) 55.
31. H.D. Belitz, W. Grosch, P. Schieberle, Food Chemistry, 4th Revised and Extended Edition, (2009). Hourigan, The use of enzymes for thermal process monitoring: modification of milk alkaline phosphatase heat resistance by means of an immobilization technique, Food Control, 15 (2004) 427–433.
32. G. Linden, C. Alais, Alkaline phosphatase in human, cow and sheep milks: Molecular and catalytic properties and metal ion action, Ann. Biol. anim Bioch. Biophys., 18 (1978) 749-758.
33. Lata Sheo Bachan Upadhyay and Nishant Verma, A three step approach for the purification of alkaline phosphatase from non-pasteurized milk, J Food Sci Technol, 52 (2014) 3140-3146.
34. A.N. Vamvakaki, E. Zoidou, G. Moatsou, M. Bokari, E. Anifantakis, Residual alkaline phosphatase activity after heat treatment of ovine and caprine milk, Small Ruminant Research 65 (2006) 237–241.
35. E.F. Evans and R. Klınke, The Effects of intracochlear and systemic furosemide on the propertıes of single cochlear nerve fibres in the cat, J. Physiol., 331 (1982) 409-427.
36. F.K. Mohammad, F.T. Abachi, A.S. Alias, M.Y. Al-Attar, T.A. Al-Sawah and T.A. Al-Talib, Preparation and Evaluation of Veterinary 0.1% Injectable Solution of Atropine Sulphate, Vet. World, 5 (2012) 145-149.
37. A.Z. Durrani, N. Kamal, A.R. Shakoori, R.M. Younus, Prevalence of post parturient haemoglobinuria in buffalo and therapeutic trials with toldimfos sodium and tea leaves in Pakistan, Turk. J. Vet. Anim. Sci., 34 (2010) 45-51.
38. S. Sadeghi, F. Fathi, J. Abbasifar, Potentiometric sensing of levamisole hydrochloride based on molecularly imprinted polymer, Sensors and Actuators B, 122 (2007) 158–164.
39. P. Jedziniak, M. Olejnik, T. Szprengier-Juszkiewicz, S. Smulski, M. Kaczmarowski and J. Zmudzki, Identification of flunixin glucuronide and depletion of flunixin and its marker residue in bovine milk, J. Vet. Pharmacol. Therap., 36 (2013) 571—575.