一种纯化大鼠肝脏酪氨酸转氨酶的简化方法。

A simplified procedure for the purification of rat liver tyrosine aminotransferase.

作者信息

Voigt J, Sekeris C E

出版信息

Hoppe Seylers Z Physiol Chem. 1978 Oct;359(10):1363-9. doi: 10.1515/bchm2.1978.359.2.1363.

DOI:10.1515/bchm2.1978.359.2.1363

Abstract

Rat liver tyrosine aminotransferase was purified by chromatography on CM-Sephadex C-50 and DEAE-cellulose, (NH4)2SO4 fractionation and gel filtration on Sephadex G-200. Livers from 400 rats can be easily worked up by this procedure. Furthermore, this purification method has the advantage that hepatic tryptophan 2,3-dioxygenase, which, like tyrosine aminotransferase, is induced by glucocorticosteroids, can be purified from the same homogenate. Tyrosine aminotransferase purified by this method was shown to be specific for 2-oxoglutarate. Its subunits have a molecular weight of 45 000. The following "apparent" Michaelis constants were determined: L-tyrosine, 1.7 X 10(-3) M; 2-oxoglutarate, 5.9 X 10(-4) M; and pyridoxal 5'-phosphate, 2.1 X 10(-6) M. Tyrosine aminotransferase, depleted of its cofactors, binds 4 molecules of pyridoxal 5'-phosphate per 90 000 daltons with a KA of 2.2 X 10(5) M-1.

摘要

大鼠肝脏酪氨酸转氨酶通过在CM - Sephadex C - 50和DEAE - 纤维素上进行色谱分离、硫酸铵分级分离以及在Sephadex G - 200上进行凝胶过滤来纯化。用这个方法可以轻松处理400只大鼠的肝脏。此外，这种纯化方法的优点是，与酪氨酸转氨酶一样受糖皮质激素诱导的肝脏色氨酸2,3 - 双加氧酶可以从同一匀浆中纯化出来。用这种方法纯化的酪氨酸转氨酶对2 - 氧代戊二酸具有特异性。其亚基的分子量为45000。测定了以下“表观”米氏常数：L - 酪氨酸，1.7×10⁻³ M；2 - 氧代戊二酸，5.9×10⁻⁴ M；以及磷酸吡哆醛，2.1×10⁻⁶ M。去除辅因子的酪氨酸转氨酶每90000道尔顿结合4分子磷酸吡哆醛，结合常数KA为2.2×10⁵ M⁻¹。