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

Yıl 2000, Cilt: 33 , 37 - 48, 08.09.2015

A. Can, N. Akev A. Askhası

Öz

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.

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.

Öz

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

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