Purification and properties of hog liver 4-hydroxyphenylpyruvate dioxygenase.
Purification and properties of hog liver 4-hydroxyphenylpyruvate dioxygenase.
复制标题
猪肝4-羟苯基丙酮酸双加氧酶的纯化及性质。
DOI：
10.1016/0003-9861(82)90188-6
复制
发表时间：
1982
影响因子：
3.9
通讯作者：
Hamilton,GA
中科院分区：
文献类型：
--
作者：
Roche,PA;Moorehead,TJ;Hamilton,GA
The purification of hog liver 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27), and the determination of some of its characteristics, are reported. The enzyme was purified 330-fold in 22% yield from an acetone powder extract by ammonium sulfate fractionation, chromatography twice using sulfopropyl Sephadex under carefully controlled pH conditions (once at pH 5.36 and a second time with a pH gradient from 5.25 to 5.80), and a final chromatography on DEAE-cellulose. The purified enzyme was found to be homogeneous by several standard criteria, but activity measurements indicated that a small amount (less than 5%) of a carboxylesterase (EC 3.1.1.1) isoenzyme is present as a minor impurity. On long-term storage at −20 °C the enzyme forms polymers but this can be reversed with thiols. The molecular weight of the freshly prepared or depolymerized enzyme was estimated to be 89,000 ± 2000 by equilibrium ultracentrifugation, and 50,000 to 54,000 by gel filtration. Sodium dodecyl sulfate-gel electrophoresis experiments, performed in the presence and absence of mercaptoethanol, indicate that the enzyme is composed of two nonidentical subunits with similar molecular weights (44,000 ± 2000). The enzyme gives a typical protein ultraviolet absorption spectrum with no noticeable peaks above 300 nm, it has no detectable carbohydrate content, and it contains 0.9 atom iron and 0.4 atom copper/89,000 daltons. Added iron and copper salts activate the enzyme to some extent but by less than a factor of 2. The enzymatic reaction has a large temperature coefficient (the rate increases ca. fivefold for each 10 °C rise) and is markedly stimulated (up to sixfold) by the presence of some organic solvents in concentrations up to 10% of the medium. These results suggest that a protein conformation change, possibly aided by binding of the organic solvent, is involved in the rate-determining step of the reaction. The similarities and differences of this 4-hydroxyphenylpyruvate dioxygenase to those from other sources, and to prolyl hydroxylase, are discussed.
