1916

Plasental alkali fosfatazın saflaştırılması ve kinetik özelliklerinin incelenmesi

Alkali fosfataz insan plasentasından 29.1 kat saflaştırılarak spesifik aktivitesi 6.40 IU/mg olarak bulunmuştur. Saflaştırma işlemleri, pH 8.6 tris tamponu kullanılarak homojenizasyon, butanol ekstarksiyonu, amonyum sülfatla çöktürme, ısı muamelesi ve Sephadex G-200 jel filtrasyonu basamaklarını içermektedir. Kısmen saflaştırılmış enzimin lineweaver-Burk grafiği çizilerek 37°C'de 0. 1M Glisin-1 mM Mg++ tamponunda (pH 10.5) p-NPPsubstratı için Km değeri 0.5 mM olarak saptandı. Enzimin p-NPP substratı için optimum pH'sı 10.6 olarak bulundu. Yapılan ısı ile inaktivasyon çalışmaları sonucunda, enzimin 65°C'de aktivitesinin hemen hemen %100'ünü koruduğu, 78 °C'de ise aktivitesinin tamamını kaybettiği gözlemlenmiştir. Daha sonra enzim üzerine ferröz sülfatın etkileri denenmiş ve ferröz sülfatın enzimi inhibe ettiği ve inhibisyon tipinin un-kompetitif olduğu saptanmıştır.

Purification of placental alkaline phosphatase and investigation of it's kinetic properties

Alkaline phosphatase of human placenta was purified 29.1 fold to a final specific activity of 6.40 U/mg by a procedure involving homogenization with tris buffer pH 8.6, extraction with butanol, ammonium sulphate precipitation, exposure to heat and Sephadex G-200 gel filtration. The apparent Km value of the purified enzyme for p-NPP in 0.1 M glycine-1 mM magnesium buffer, pH 10.5 at 37"C was calculated (estimated) to be 0.50 mM from Lineweaver-Burk plot. The enzyme had a pH optimum 10.6 for p-NPP. Heat inactivation studies showed that the enzyme remained almost 100% stable at 65"C but it was completely inactivated at 78°C. Subsequently, the effects of ferrous sulphate were investigated. The inhibition type for ferrous sulphate was uncompetitive.

PDF

___

1. Solomon, P.: Alkaline phosphatase. Ann Int Med, 67: 183-203, 1967.
2. Sunguroğlu, K., Cökhun, İ.H.: Koyun böbrek alkali ve asit fosfatazlarının kısmen saflaştırılması ve bazı katyonla-rın bu enzimler üzerindeki etkilerinin araştırılması. Optimal Tıp Dergisi, 4: 224-228, 1991.
3. Cari, A.B., Edvvard, R.A.: Tietz textbook of clinical che-mistry. 2 th ed., W.B. Saunders Co., pp. 830-844, 1994.
4. Moss, D.W.: Perspectives in alkaline phosphatase research. Clin Chem, 38: 2486-2492, 1992.
5. Fernley, H.N.: Mammalian alkaline phosphatases. The Enzyme Vol IV. Boyer, D .Ed. Academic Press New York, 3.Ed. 417-447, 1971.
6. Fishman, W.H.: Perspectives on alkaline phosphatase iso-enzymes. Am J Med, 56: 617-50, 1974.
7. Kaplan, M.M.: Alkaline phosphatase: Progress in hepato-logy. Gastroenterology, 62: 452-468, 1972.
8. McComb, R.B., Bowers, G.N., et al,: Alkaline phosphatase. Nevv york Plenum Press, 1979.
9. VVarnes, T.W.: Progress report in alkaline phosphatase. Gut, 13: 926, 1972.
10. Ghosh, N.K., Fishman, W.H.: Purification and properties of molecular-weight variants of human placental alkaline phosphatase. Biochem J, 108: 779-792, 1968.
11. Mabrv, C.C., Bautista, A.: Familial hyperphosphatasia with rnental retardation, seizures, and neurologic deficits. J Pediatr, 74: 74-85, 1970.
12. Roberts, W.M.: Variations in the phosphatase activity of blood in disease. Br J Exp Pathol, 11, 90-95, 1930.
13. Robinson, R.: The possible significance of hexosophospho-ric esters in ossification in vitro. Biochem J., 17: 286-93, 1923.
14. Seetharam, S., Sııssman, N.L., et al.: The mechanism of ele-vated alkaline phosphatase activity after bile duet liga-tion in the rat. Hepatol, 6: 374-80, 1986.
15. Van Floof, V.O., De Broe, M.E.: Interpretation and clinical significance of alkaline phosphatase isoenzyme pat-terns. Crit, Rev, Clin Lab Sci, 31: 197-293, 1994.
16. Van Belle, H.: Alkaline phosphatase I. Kinetics and inhibiti-ons by levamisole of purifiedisoenzyme from humans. Clin Chem, 22: 972-976, 1976.
17. Fishman, W.H., Ghosh, N.K.: Isoenzymes of human alkali-ne phosphatase. Adv Clin Chem, 10: 255-370, 1967.
18. Hulstaert, C.E., Torringa, J.L., et al.: The characteristic dist-ribution of alkaline phosphatase in the full-term pla-centa. Gynecol Invest, 4: 24, 1973.
19. Meyer, R.E., Thompson, S.J., et al.: Maternal serum placen-tal alkaline phosphatase level and risk for preterm de-livery. Am J Obstet Gynecol, 173, 181-186, 1995.
20. Lovvry, O.H., Rosebrough, J., et al.: Protein measurement vvith the folinphenolreagent. J Biol Chem, 193: 265-275, 1951.
21. Bessey, O.A., Lovvry, O.H., et al.: ) Biol Chem, 164: 321, 1946.
22. Barman, T.E., Gutfreund, H.: The catalytic activity and kine-tic properties of bovine milk alkaline phosphatase
23. Harada, M., Hiroaka, K., et al.: Purification and properties of bovine dental-pulp alkaline phosphatase. Arch Oral Biol, 27: 69-74, 1982.
24. Kilpatrick, D.C., Crofton, P.M.: Alkaline phosphatase from human thyroid. Clin Chim Açta, 117: 307-315, 1981.
25. Latner, A.L., Hodson, A.W.: Human liver alkaline phospha-tase purified by affinity ehromatography, ultracentrifu-gation and polyacrylamide-gel eleetrophoresis. Bioc-hem J, 159: 697-705, 1976.
26. Doellgast, G., Fishman, W.H.: Purification of human pla-cental alkaline phosphatase: salt effects in affinity ehromatography. Biochem J, 141: 103-112, 1974.
27. Ghosh, N.K., Fishman, W.H.: Purification and properties of molecular-weight variants of human placental alkaline phosphatase. Biochem J, 108: 779-792, 1968.
28. Harkness, D.R.: Studies on human placental alkaline phosp-hatase 1 .Purification and crystallization. Arch Biochim Biophys, 126: 503-512, 1968.
29. Ghosh, N.K.: Purification and molecular properties of pla-cental and intestinal alkaline phosphatase. Ann NY Acad Sci, 166: 604-640, 1969.
30. Shameem, G.M.M., Quadri, F.: Isolation and purification of placental type alkaline phosphatase from seminoma. Clin Chem, 33: 248-52, 1987.
31. Fernley, H.N.: Mammalian alkaline phosphatases. The Enzyme Vol IV. Boyer, D .Ed. Academic Press Nevv York, 3.Ed. 417-447, 1971.
32. Fleisch, H., Bisaz, S.: Isolation from urine of pyrophospha-te, a calcifcation inhibitor. Amer J Physiol, 203: 671, 1962.