Subunit affinity chromatography and its application to the isolation of aryl sulfatase A enzymes.
Subunit affinity chromatography and its application to the isolation of aryl sulfatase A enzymes.
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亚基亲和层析及其在芳基硫酸酯酶 A 酶分离中的应用。
DOI：
10.1016/0003-9861(80)90439-7
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发表时间：
1980
影响因子：
3.9
通讯作者：
Waheed,A
中科院分区：
文献类型：
--
作者：
VanEtten,RL;Waheed,A
将兔肝芳基硫酸酯酶A（芳基硫酸酯磺基水解酶，EC 3.1.6.1）的单体形式共价偶联至CNBr活化的琼脂糖凝胶，并检查共价偶联的单体亚基的催化性质。固定化的亚基表现出一个pH最佳接近pH 5.6，这似乎是单体的特征pH最佳。酶-琼脂糖凝胶复合物在pH 5.5时表现出特征性的异常动力学行为，但在pH 4.5时固定化酶没有营业额诱导的失活。检查共价偶联的亚基柱作为亚基亲和色谱介质的能力。发现在pH4.5和pH5.0，I = 0.2时，溶解的芳基硫酸酯酶A从溶液中被除去，并与Sepharose-芳基硫酸酯酶A亲和柱缔合。保留的酶亚基可用0. 2 mTris-HCl（pH7.5）定量洗脱。外源蛋白质，如牛血清白蛋白没有可测量的影响的速率或平衡的酶的共价结合的亚基的协会。发现酶与亲和柱的结合程度强烈依赖于平衡时间和pH。在pH 5.0，I = 0.2下24 h后，约90%的酶被保留。在其他类似条件下，使用Sepharose-6 MB导致比Sepharose-4 B稍快的缔合。在所采用的实验条件下，亲和柱的总容量约为偶联到琼脂糖凝胶上的总芳基硫酸酯酶A的50%。兔肝亚基柱也允许纯化几种其它芳基硫酸酯酶A。因此，亚基亲和柱提供了一个简单，方便，快速的程序，大多数哺乳动物的芳基硫酸酯酶A酶的分离，以及研究种间和种内亚基协会的相互作用。
The monomeric form of rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) was covalently coupled to CNBr-activated Sepharose and the catalytic properties of the covalently coupled monomer subunit were examined. The immobilized subunit showed one pH optimum near pH 5.6 which appears to be the characteristic pH optimum of the monomer. The enzyme-Sepharose complex exhibited the characteristic anomalous kinetic behavior at pH 5.5 but there was no turnover-induced inactivation of the immobilized enzyme at pH 4.5. The covalently coupled subunit column was examined for its ability to act as a subunit affinity chromatography medium. It was found that dissolved aryl sulfatase A was removed from solution at pH 4.5 and pH 5.0,I= 0.2, and became associated with the affinity column of Sepharose-aryl sulfatase A. The retained subunit of the enzyme could subsequently be quantitatively eluted with 0.2mTris-HCl, pH 7.5. Extraneous protein such as bovine serum albumin did not measureably affect the rate or equilibrium for association of the enzyme to the covalently bound subunit. The extent of binding of the enzyme to the affinity column was found to be strongly dependent on the time of equilibration and on the pH. About 90% of the enzyme was retained after 24 h at pH 5.0,I= 0.2. Under otherwise comparable conditions, use of Sepharose-6MB resulted in slightly faster association than did Sepharose-4B. Under the experimental conditions employed, the total capacity of the affinity column was approx 50% of the total aryl sulfatase A coupled to the Sepharose. The rabbit liver subunit column also permits the purification of several other aryl sulfatase A enzymes. Thus, the subunit affinity column provides a simple, convenient, and rapid procedure for the isolation of most mammalian aryl sulfatase A enzymes as well as for studying inter- and intraspecific subunit association interactions.
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共价修饰是芳基硫酸酯酶 A 异常动力学的原因。
影响因子：
3.9
作者：
A. Waheed;R. V. Van Etten
通讯作者：
R. V. Van Etten
脑苷脂 3-硫酸酯作为芳基硫酸酯酶 A 的生理底物。
DOI：
--
发表时间：
1968
期刊：
Biochimica et Biophysica Acta
影响因子：
--
作者：
E. Mehl;H. Jatzkewitz
通讯作者：
H. Jatzkewitz
DOI：
10.1016/s0021-9258(19)41628-1
发表时间：
1975
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
R. Stevens;A. Fluharty;M. Skokut;H. Kihara
通讯作者：
H. Kihara
影响因子：
2.7
作者：
L. W. Nichol;A. Roy
通讯作者：
A. Roy
影响因子：
--
作者：
J. Austin;D. Armstrong;L. Shearer
通讯作者：
L. Shearer