PARTIAL PURIFICATION and CHARACTERIZATION of ASPARTATE AMINOTRANSFERASE from the HEPATOPANCREAS of the MUSSEL Mytilus galloprovincialis Lam.

A. Can, N. Akev A. Askhası

EN TR

PARTIAL PURIFICATION and CHARACTERIZATION of ASPARTATE AMINOTRANSFERASE from the HEPATOPANCREAS of the MUSSEL Mytilus galloprovincialis Lam.

Abstract

In this study aspartate and alanine aminotransferase enzymes of Mytilrls gallopro~~incialis Lam., the mussel species specific to the Turkish coast were examined. Because it was found that aspartate aminotransferase activity in the hepatopancreas was superior to alanine aminotransferase while surveying the activities of these enzymes in . the whole body, hepatopancreas and mantle tissues; among these two enzymes, aspartate aminotransferase was partially purified from hepatopancreas and its kinetic properties were studied. Molluscs were collected daily from the Rumelikavagl coast of the Bosphonts and hepatopancreases were homogenized with 0.9% NaCl solution after dissection from the body. The fraction obtained after 35%-65% ammonium sulphate precipitation was applied to hydroxylapatite column. The elution performed by increasing molarity gradient of the phosphate buffer, pH 6.8, resulted in the recovery of the aspartate arninotransferase activity in 100 mM phosphate buffer pool. The 83 fold purified enzyme was applied to polyacrylamide gel electrophoresis and the existence of two activity bands indicated two isoenzymes of the aspartate aminotransferase. Effect of temperature on the activity of the enzyme was examined and it was found that the enzyme exhibited maximum activities at 25 "C and 40 "C; the activity completely dissappeared at 60 "C. Aspartate aminotransferase activity was maximum at I pH 7.6, Km values for aspa$ate,and 2-oksoglutarate were 1.7 mM and 6.6~10-~ mM, Vmax for the same substrates were .I62 U/ml and 0.149 U/ml, respectively.

Keywords

PARTIAL PURIFICATION and CHARACTERIZATION of ASPARTATE AMINOTRANSFERASE from the HEPATOPANCREAS of the MUSSEL Mytilus galloprovincialis Lam.

Abstract

Bu qaligmada, Turkiye sahillerinde bulunan Mytilus gallopi.ovincialis Lam. midye tiirunun aspartat ve alanin aminotransferazlarl incelendi. Tum doku, hepatopankreas ve manto k~simlannda yapllan aragt~rmada, aspartat aminotransferazln hepatopankreastaki aktivitesinin alanin aminotransferaza gore fazla olmasindan dolayl, midye hepatopankreas~ndan aspartat aminotransferaz kismen saflagtlrildi ve bazl kinetik ozellikleri incelendi. Deneyin yapilacagl gun 1stanbul Bogazi Rumelikava@i klyilarindan toplanan midyelerin hepatopankreas k~s~rnlarl qlkanlarak, %0.9 NaCl ile, homojenize edildi. Aspartat aminotransferaz aktivitesi gosteren homojenizatin %35-%65 amonyum sulfat kesiti hidroksilapatit kolona uygulandi. pH's1 6.8 olan ve artan molaritede fosfat tamponu ile yapllan basamaklt elusyon sonucunda, aspartat aminotransferaz 100 mM fosfat tarnponu ile eliie edildi. Bu iglem sonunda, midye hepatopankreaslndan 83 kez safla~tlnlan aspartat aminotransferazln poliakrilamid jel elektroforezinde iki aktivite bandl gostermesi nedeniyle iki izoenzim iqerdiii sonucuna vanldl. Aspartat aminotransferaz iizerine temperaturun etkisi incelendiginde, enzimin 25 "C ve 40 "C'lerde maksimum aktivite gosterdigi ve aktivitenin 60 "C'de tamamen kayboldugu goriildu. Enzimin optimum pH'slnin pH 7.6'da oldugu; aspartat ve 2- oksoglutarata kargi Km degerlerinin slraslyla, 1.7 mM ve 6.6~10-~ mM, aynl substratlara kary Vmax degerlerinin ise 0.162 U/ml ve 0.149 U/ml oldugu bulundu.

Keywords

References

1. Boyd, J.W., Biockenl J., 81,434-441 (1961).
2. Leung, F.Y., Henderson,A.R., Clirl. Cllen~. 2712, 232-238 (1981).
3. Rej, R., CRC Critical Reviebils it1 Clil~. Lab. Sci., 21, 99-186 (1984).
4. Bishop, S.H., Ellis, L.L., Burcham, J.M., "The Mollusca, Metabolic Biochemistry and Molecular Biomechanics" Hochachka, P.W.., Wilbur, K.M., eds. Academic Press Inc., New York, Vol.1, pp.243-327 (1983).- Ref. McCormic, A., Paynter, K.T., Broday, M.M., Bishop, S.H., Conrp. Biocl~enz. Plrysiol., 84 B, 163-166 (1986).
5. Paynter, K.T., Hoffmann, R.J., Ellis, L.L., Bishop, S.H., Tlie Jourtral of Experimental Zoology. 231, 185-197 (1984).
6. Goromosova, S.A., Tamozhnyaya, V.A., Biology Marine, 2,62-68 (1980).
7. Goromosova, S.A., Tamozhnyaya, V.A., Biology Maritre Kiev, 48, 118-122 (1979).
8. Lowry, O.H., Rosenbrough, N.J., Fan; A.L., Randall, R., J. Biol. Clrenr., 193, 265-275 (1951).

9. Warburg, O., Christian, W., Biochen~. Z., 310, 384-421 (1941). Karmen, A., J. Clit~. Invest., 34, 131 -133 (1955).
10. Akev, N., Can, A., Yanardak, R., I.sratib~~l Ecz. Fak. Mec., 31, 1-9 (1995).
11. Rej, R., Bretaudiere, J.-P., Graffunder, B., Clitr. Chen~., 27 (4), 535-542 (1981).
12. Omstein, L., Ann. N.Y. Acad. Sci., 121, 321-349 (1964).
13. Davis, B.J., Ann. N.Y. Acad. Sci., 121,404-427 (1964). . Schwartz, M.K., Nisselbaum, J.S., Bodansky, O., Americarr J. Clitr. Patliology, 40, 103-
14. Tiselius, A., HjertCn, S., Levin, O., A~lr. Biochenr. Bioplrys., 65, 132-155 (1956). Goromosova, S.A., Milovidova, N.Yu., Tamozhnyaya, V.A., Shapiro, A.Z., Girdrobiol ZIr., 23 (I), 61-66 (1987).
15. Regoli, F., Arch. Etr~~irotr. con tun^. Tmicol., 34 (I), 48-63 (1998).
16. Stien, X., Percic, P., Gnassia-Barelli, M., Romeo, M., Lafaurie, M., Etlvirotl. Pollrrr., 99 (3), 339-345 (1998).
17. Guilhermino, L., Barros, P., Silva, M.C., Soares, A.M.B.M., Bionlarkers, 3 (2), 157-163 (1 998).
18. Wong, K.F.,Cossins, E.A., Pll):tocl?enlisrty, 8, 1327-1338 (1969).
19. Reed, R.E., Hess, J.L., J. Biol.Cl~en~., 250,4456-4461 (1975).
20. Oner, N., Can, A., Aydeger, N., Tiitem, E., Acta Pharrnacerctica Trircica. 30,43-48 (1988).
21. Griffith, S.M., Vance, C.P., Plant Plz)isiol., 90, 1622-1629 (1989).
22. Michuda, C.M., Martinez-Carrion, M., Biochemistry, 8, 1095-1 105 (1969).
23. Oner, N., Aydeger, N., Can, A., Acta Pharn~acerrtica Turcica, 31,27-32 (1989).
24. Sauvage, F.X., Romieu, C.G., Flanzy, C., Robin, J.P., Atn. J. E1101.Vitic.. 42,209-218 (1991).
25. Can, A., Akev, N., Acra Pkar~nacerrtica fiircica, 37, 100-104 (1995).
26. Jenkins, W.W.T., Yphantis, D.A., Sizer, LW., J. Biol. Cltem., 234,51-57 (1959).
27. Frankel, S., "Gradwohl's Clinical Laboratory Methods and Diagnosis", Vol.1. p.126.
28. Frankel, S., Reitman, S., Sonnenwirth, A.C. eds. The C.V. Mosby Company, Saint Louis (1970).
29. Goromosova, S.A., Tamozhnyaya, V.A., ZIl. Evol. Biokflinl. Fiziol., 17 (4), 337-341 (1981).
30. Owen, T.G., Hochachka, P.W., Biochenl.J., 143,541-553 (1974).
31. Nisselbaum, J.S., Bodansky O., J. Biol. Cllen~., 241,2661-2664 (1966).
32. Quiroga, C., Busquets, M., Cortes, A., Bozal, J., In[. J. Biochem., 17, 1185-1190 (1985).
33. McCormic, A., Paynter, K.T., Broday, M.M., Bishop, S.H., Conlp. Biochenl. Pllysiol., 84 B, 163-166 (1986).

Details

Primary Language

English

Subjects

Pharmacology and Pharmaceutical Sciences

Journal Section

Research Article

Authors

A. Can, N. Akev A. Askhası This is me

Publication Date

September 8, 2015

Submission Date

September 8, 2015

Acceptance Date

-

Published in Issue

Year 2000 Volume: 33 Number: 0

IZ

https://izlik.org/JA96YX54CF

Cite

RIS / Bibtex

APA

A. Askhası, A. C. N. A. (2015). PARTIAL PURIFICATION and CHARACTERIZATION of ASPARTATE AMINOTRANSFERASE from the HEPATOPANCREAS of the MUSSEL Mytilus galloprovincialis Lam. Journal of Faculty of Pharmacy of Istanbul University, 33, 37-48. https://izlik.org/JA96YX54CF

AMA

1.A. Askhası ACNA. PARTIAL PURIFICATION and CHARACTERIZATION of ASPARTATE AMINOTRANSFERASE from the HEPATOPANCREAS of the MUSSEL Mytilus galloprovincialis Lam. Journal of Faculty of Pharmacy of Istanbul University. 2015;33:37-48. https://izlik.org/JA96YX54CF

Chicago

A. Askhası, A. Can, N. Akev. 2015. “PARTIAL PURIFICATION and CHARACTERIZATION of ASPARTATE AMINOTRANSFERASE from the HEPATOPANCREAS of the MUSSEL Mytilus Galloprovincialis Lam”. Journal of Faculty of Pharmacy of Istanbul University 33 (September): 37-48. https://izlik.org/JA96YX54CF.

EndNote

A. Askhası ACNA (September 1, 2015) PARTIAL PURIFICATION and CHARACTERIZATION of ASPARTATE AMINOTRANSFERASE from the HEPATOPANCREAS of the MUSSEL Mytilus galloprovincialis Lam. Journal of Faculty of Pharmacy of Istanbul University 33 37–48.

IEEE

[1]A. C. N. A. A. Askhası, “PARTIAL PURIFICATION and CHARACTERIZATION of ASPARTATE AMINOTRANSFERASE from the HEPATOPANCREAS of the MUSSEL Mytilus galloprovincialis Lam”., Journal of Faculty of Pharmacy of Istanbul University, vol. 33, pp. 37–48, Sept. 2015, [Online]. Available: https://izlik.org/JA96YX54CF

ISNAD

A. Askhası, A. Can, N. Akev. “PARTIAL PURIFICATION and CHARACTERIZATION of ASPARTATE AMINOTRANSFERASE from the HEPATOPANCREAS of the MUSSEL Mytilus Galloprovincialis Lam”. Journal of Faculty of Pharmacy of Istanbul University 33 (September 1, 2015): 37-48. https://izlik.org/JA96YX54CF.

JAMA

1.A. Askhası ACNA. PARTIAL PURIFICATION and CHARACTERIZATION of ASPARTATE AMINOTRANSFERASE from the HEPATOPANCREAS of the MUSSEL Mytilus galloprovincialis Lam. Journal of Faculty of Pharmacy of Istanbul University. 2015;33:37–48.

MLA

A. Askhası, A. Can, N. Akev. “PARTIAL PURIFICATION and CHARACTERIZATION of ASPARTATE AMINOTRANSFERASE from the HEPATOPANCREAS of the MUSSEL Mytilus Galloprovincialis Lam”. Journal of Faculty of Pharmacy of Istanbul University, vol. 33, Sept. 2015, pp. 37-48, https://izlik.org/JA96YX54CF.

Vancouver

1.A. Can, N. Akev A. Askhası. PARTIAL PURIFICATION and CHARACTERIZATION of ASPARTATE AMINOTRANSFERASE from the HEPATOPANCREAS of the MUSSEL Mytilus galloprovincialis Lam. Journal of Faculty of Pharmacy of Istanbul University [Internet]. 2015 Sep. 1;33:37-48. Available from: https://izlik.org/JA96YX54CF