Renal membrane-bound carbonic anhydrase. Purification and properties.
Renal membrane-bound carbonic anhydrase. Purification and properties.
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肾膜结合碳酸酐酶。
作者：
P. Wistrand;K. Knuuttila
用蔗糖差速离心法分离人供肾微粒体，彻底洗涤后用非离子洗涤剂-β--D-葡萄糖苷增溶。该增溶材料首先被应用于乙酰唑胺-环氧乙烷-SepharoseR-CL-4B亲和层析柱上，以去除污染的细胞质碳酸酐酶同工酶CA I和CA II，然后加入到与CM Bio-Gel AR偶联的对氨基甲基苯磺酰胺亲和层析柱上，以纯化膜结合的碳酸酐酶活性。这就产生了50%的纯酶。然后在阴离子交换柱上浓缩和分级，并通过凝胶过滤脱盐和纯化至均一(SDS-PAGE和等电聚焦)。该酶从可提取的膜蛋白中纯化了411倍。经凝胶过滤和SDS-PAGE分析，其相对分子质量为34.4 kDa，氨基酸分析为36.7 kDa。其氨基酸组成不同于细胞质同工酶CA I、II和III。兔抗纯化的SDS处理的酶产生的抗血清与天然的非变性膜酶蛋白反应，但与CA II反应较弱。该酶在动力学上与CA II的水合酶和酯酶活性相似，但不受各种磺胺类药物的抑制。综合考虑，这些数据表明，人类肾脏含有一种膜结合的碳酸氢酶蛋白，它不同于唾液中的细胞质同工酶CA I、II和III以及分泌型(CAVI)。
Microsomes from perfused human donor kidneys were separated by differential centrifugation in sucrose, and thoroughly washed before solubilization by the nonionic detergent nonyl-beta-D-glucoside. The solubilized material was first applied onto an affinity chromatographic column of acetazolamide-oxirane-SepharoseR-CL-4B to remove contaminating cytoplasmic carbonic anhydrase isozymes CA I and CA II. It was then added onto an affinity column of p-aminomethylbenzene sulfonamide coupled to CM Bio-gel AR to purify the membrane-bound carbonic anhydrase activity. This resulted in a 50% pure enzyme. It was then concentrated and fractionated on an anion-exchange column, and desalted and purified to homogeneity (SDS-PAGE and isoelectric focusing) by gel filtration. The enzyme was now purified 411-fold from extractable membrane protein. Its molecular weight was 34.4 kDa from gel filtration and SDS-PAGE, and 36.7 kDa from amino acid analysis. The amino acid composition differed from that of the cytoplasmic isozymes CA I, II, and III. Antisera, produced in rabbits against the purified SDS-treated enzyme, reacted with native nondenatured membrane enzyme protein but only weakly with CA II. Kinetically the enzyme was similar to CA II with respect to hydrase and esterase activities and to inhibition by various sulfonamides. Considered together, the data suggest that the human kidney contains a membrane-bound carbonic anhydrase protein that differs from the cytoplasmic isozymes CA I, II, and III and the secretory form (CA VI) in the saliva.
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DOI：
10.1016/s0021-9258(19)75797-4
发表时间：
1987-01
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
H. Murakami;W. Sly
通讯作者：
H. Murakami;W. Sly
DOI：
--
发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Whitney,PL;Briggle,TV
通讯作者：
Briggle,TV
影响因子：
2.9
作者：
ROSEVEAR, P;VANAKEN, T;FERGUSONMILLER, S
通讯作者：
FERGUSONMILLER, S
犬肾皮质膜结合碳酸酐酶的动力学和抑制。
DOI：
10.1016/0005-2744(81)90136-4
发表时间：
1981
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Sanyal,G;Pessah,NI;Maren,TH
通讯作者：
Maren,TH
通过选择性抑制定义近端小管碳酸酐酶的功能。
DOI：
10.1152/ajprenal.1983.245.4.f443
发表时间：
1983
期刊：
The American journal of physiology
影响因子：
--
作者：
Lucci,MS;Tinker,JP;Weiner,IM;DuBoseJr,TD
通讯作者：
DuBoseJr,TD