Rat liver alcohol dehydrogenase. I. Purification and characterization.
Rat liver alcohol dehydrogenase. I. Purification and characterization.
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大鼠肝脏乙醇脱氢酶。
DOI：
10.1016/0003-2697(83)90095-7
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发表时间：
1983
影响因子：
2.9
通讯作者：
Leffert,HL
中科院分区：
文献类型：
--
作者：
Lad,PJ;Leffert,HL
用差速离心、（NH_4）_2SO_4沉淀、DEAE-Affi-GelBlue、Affi-GelBlue和AMP-琼脂糖层析等方法，在14 h内从Fischer-344大鼠肝脏中纯化出乙醇脱氢酶。在HPLC之后，获得超过240倍的纯化。在变性条件下，该酶在10%十二烷基硫酸钠-聚丙烯酰胺凝胶上迁移为单一蛋白带（Mr为40，000）。在非变性条件下，蛋白质从HPLC I-125柱上洗脱为具有恒定酶比活性的对称峰。当通过分析等电聚焦进行检查时，两个蛋白质和两个酶活性条带在pH 8.8和8.9之间紧密地聚集在一起（宽条带）。纯酶在0.5 mM Tris-HCl、50 mM 2-（N-环己基氨基）乙磺酸（CHES）和50 mM 3-（环己基氨基）-1-丙磺酸（CAPS）缓冲液中的最适pH为8.3 - 8.8，在50 mM甘氨酰-甘氨酸中的最适pH为9.0以上。动力学研究与纯酶，在0.5 m Tris-HCl在不同的pH条件下，揭示了三个特征电离常数的活动：7.4（pK 1），8.0-8.1（pK 2），和9.1（pK 3）。后两者可能代表游离酶中的官能团; pK 1可能代表酶-NAD+复合物中的官能团。纯酶还用于在37°C下在0. 5 mTris-HCl缓冲液（pH 7.4）中测定动力学常数（I = 0.2）。测得Vmax= 2.21 μm/min/mg酶，乙醇Km = 0.156 mm，NAD+ Km = 0.176 mm，NAD+解离常数= 0.306 mm。在pH 7.4和10 mm乙醇条件下，计算得出的速率为2.4 μm/min/g肝脏。
Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr⋍40,000 ) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 m Tris-HCl, 50 mm 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mm 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mm glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 m Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0–8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1may represent a functional group in the enzyme-NAD+complex. Pure enzyme also was used to determine kinetic constants at 37°C in 0.5 m Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax= 2.21 μm/min/mg enzyme, Kmfor ethanol = 0.156 mm, Kmfor NAD+= 0.176 mm, and a dissociation constant for NAD+= 0.306 mm. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mm ethanol, the rate was calculated to be 2.4 μm/min/g liver.
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大鼠肝脏乙醇脱氢酶催化的吡唑抑制和乙醇和视黄醇氧化的动力学研究。
DOI：
10.3891/acta.chem.scand.23-1119
发表时间：
1969
期刊：
Acta chemica Scandinavica
影响因子：
--
作者：
M. Reynier
通讯作者：
M. Reynier
影响因子：
3.9
作者：
N. Mourad;C. Woronick
通讯作者：
C. Woronick
影响因子：
3.5
作者：
H. Jörnvall
通讯作者：
H. Jörnvall
甲状腺激素对肝脏酒精代谢酶活性的影响。
DOI：
10.1016/0006-291x(79)91850-3
发表时间：
1979
影响因子：
3.1
作者：
F. Moreno;R. Teschke;G. Strohmeyer
通讯作者：
G. Strohmeyer
用于抗体测量的 ELISA：与数据表达相关的方面。
影响因子：
2.2
作者：
R. Malvano;A. Boniolo;M. Dovis;M. Zannino
通讯作者：
M. Zannino