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

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.

Anahtar Kelimeler:

Mussel, Mytil~~s galloproviizcialis Lam., aspartate aminotransferase, alanine aminotransferase, transaminase

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

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:

Mussel, Mytil~~s galloproviizcialis Lam., aspartate aminotransferase, alanine aminotransferase, transaminase,

PDF

___

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).

ISSN: 2548-0731
Yayın Aralığı: Yılda 3 Sayı
Başlangıç: 1965
Yayıncı: İstanbul Üniversitesi

Arşiv

Sayıdaki Diğer Makaleler

CHEMICAL CONSTITUENTS OF CENTA UREA AIMANICOLA

E. İşik S. ÖKSÜZ

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

A. Can, N. Akev A. ASKHASI

THE EFFECT OF PARSLEY LEAVES AND SEED EXTRACTS ON BLOOD GLUCOSE LEVELS IN RABBITS

R. Yanardağ Ö.ÖZSOY

COMPOSITION OF THE ESSENTIAL OILS OF IAJULA VISCOSA, I.GRAVEOLENS AND I. HELENIUM SUBSP. TURCORACEMOSA

C. Karamenderes*, U. ZEYBEK*

DRUG RELEASE FROM PEG SUPPOSITORY BASES AND FROM THEIR COMBINATION WITH POLYMERS

1 M. Akhtar, 2 N. Akhtar, 3 M. Ahmad 4 A.S. ARIF, 5 PERVAIZ A. SHAH

LOCAL NAMES OF SOME PLANTS FROM KARAMAN PROVINCE

S. Koçak N. ÖZHATAY