Detergent solubilization, functional reconstitution, and partial purification of epithelial amiloride-binding protein.
Detergent solubilization, functional reconstitution, and partial purification of epithelial amiloride-binding protein.
复制标题
上皮阿米洛利结合蛋白的洗涤剂溶解、功能重建和部分纯化。
DOI：
10.1021/bi00364a028
复制
发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Benos,DJ
中科院分区：
文献类型：
--
作者：
Sariban-Sohraby,S;Benos,DJ
阿拉巴马大学伯明翰分校生理学和生物物理系，1986年1月31日收到；修订稿收到1986年4月7日。摘要：从培养的蟾蜍肾细胞(A6)中提取的阿米洛利结合蛋白(A6)在3-[(3-胆酰胺丙基)二甲基氨基]-1-丙烷磺酸盐(CHAPS)中溶解，功能重组为脂质体，并部分纯化。在完整的A6上皮细胞、A6细胞匀浆(H)、顶端质膜囊泡(V)和CHAPS增溶的V)以及CHAPS溶解质膜囊泡与琼脂糖固定的小麦胚凝集素(WGA)亲和层析后获得的材料上，测定了[~3H]甲基溴氨利([~3H]CH3BrA)的特异性结合。H、VH和WGA材料与[~3H]CH3BrA的结合在10min后达到平衡。[~3H]CH3BrA与V！结合的Scatchard分析WGA材料显示了一类均一的结合位点，ITD分别为130和128 nm。这些Kd值与阿米洛利抑制完整A6上皮和VV的22Na+内流所测得的表观抑制解离常数相似。Vi蛋白的总结合位点数为4pmol/mg，是H蛋白的10倍，WGA材料的66.6pmol/mg，是H蛋白的148倍。根据[~3H]CH_3BrA与V_1结合的缔合/置换动力学研究，得到缔合速率常数(Ka)和解离速率常数(Kd)分别为3.6×10~5M~(-1)S~(-1)和49.5×10~(-3)S~(-1)。这些值产生的平衡离解常数为138 nm。在溶解的V3蛋白中，结合活性比H增加约20倍，并显著依赖于洗涤剂和磷脂的相对浓度。CHAPS对Vt的增溶使蛋白质平均回收率为44%，保留了90%的[~3H]CH3BrA结合位点。经WGA层析后，回收了2.7%的蛋白和46%的特异性结合位点。将溶解的Vi蛋白重组为磷脂酰胆碱小泡后，检测到对阿米洛利敏感的22Na+摄取，表明转运功能保持不变。以牛肾乳头为起始材料进行了初步的平行研究。在表现出高跨上皮电阻性的Na+转运组织中发现了对阿米洛利敏感的Na+通道，并参与了许多生理过程，包括盐和水的平衡。该通道的电生理学已被广泛研究(Macknight等人，1980；林德曼，1984；Sariban-Sohraby&Beños，1986)，但其潜在的分子机制以及该通道的一级结构尚不清楚。一种调查这些问题的潜在方法已经变得显而易见，因为有了特定的探头
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294 Received January 31, 1986; Revised Manuscript Received April 7, 1986 abstract: The amiloride-binding protein from cultured toad kidney cells (A6) was solubilized in 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), functionally reconstituted into liposomes, and partially purified. The specific binding of [3H] methylbromoamiloride ([3H] CH3BrA) was measured in intact A6 epithelia, A6 cell homogenate (H), apical plasma membrane vesicle (V)), and CHAPS-solubilized V) and on material obtained after affinity chromatography of CHAPS-solubilized plasma membrane vesicles on agarose-immobilized wheat germ agglutinin (WGA). Specific [3H] CH3BrA binding to H, Vh and WGA material reached equilibrium after 10 min. Scatchard analysis of [3H] CH3BrA binding to V! and WGA material revealed a homogeneous class of binding sites with iTD’s of 130 and 128 nM, respectively. These KD values were similar to the apparent inhibitory dissociation constant determined from amiloride inhibition of 22Na+ influx in both intact A6 epithelia and Vv The total number of specific binding sites was 4 pmol/mg of Vi protein, which represented a 10-fold enrichment compared to H, and 66.6 pmol/mg of WGA material (a 148-fold enrichment). From association/displacement kinetic studies of specific [3H] CH3BrA binding to V,, the rate constants of association (ka) and dissociation (kd) were calculated to be 3.6 X 105 M~‘s-1 and 49.5 X 10-3 s-1, respectively. These values yield an equilibrium dissociation constant of 138 nM. In solubilized V3 protein, binding activity was enriched approximately 20-fold over H and was markedly dependent upon the relative concentrations of detergent and phospholipid. CHAPS solubilization of Vt resulted in an average 44% recovery of protein with 90% retention of the total number of specific [3H] CH3BrA binding sites. After WGA chromatography 2.7% of the applied protein and 46% of the specific binding sites were recovered. Amiloride-sensitive 22Na+ uptake was measured after reconstitution of the solubilized Vi protein into phosphatidylcholine vesicles, indicating preservation of transport function. Initial parallel studies were performed with bovine kidney papilla as starting material. Comparable results were obtained.-^ Amiloride-sensitive Na+ channels are found in Na+-transporting tissues displaying high transepithelial electrical re-sistance and are involved in many physiological processes, including salt and water balance. The electrophysiology of the channel has been widely studied (Macknight et al., 1980; Lindemann, 1984; Sariban-Sohraby & Beños, 1986), but the molecular mechanisms underlying the Na+ transport function as well as the primary structure of the channel are unknown. A potential approach to investigating these problems has been made apparent by the availability of specific probes having
登录
查看更多内容
水、尿素、钠、氯和钾在体外分离的灌注乳头集合管中的转运。
影响因子：
19.6
作者：
Antonino S. Rocha;Lúcia H. Kudo
通讯作者：
Lúcia H. Kudo
来自电鱼电板的溶解的河豚毒素结合成分。
影响因子：
2.9
作者：
W. Agnew;M. Raftery
通讯作者：
M. Raftery
兔膀胱钠通道的周转、膜插入和降解。
DOI：
10.1126/science.6612343
发表时间：
1983
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Loo,DD;Lewis,SA;Ifshin,MS;Diamond,JM
通讯作者：
Diamond,JM
DOI：
10.1016/s0021-9258(17)43460-0
发表时间：
1984-02
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
R. Hartshorne;W. Catterall
通讯作者：
R. Hartshorne;W. Catterall
甲基化增加钠转运至 A6 顶膜囊泡：醛固酮作用的可能模式。
影响因子：
56.9
作者：
S. Sariban;M. Burg;W. Wiesmann;P. Chiang;J. Johnson
通讯作者：
J. Johnson