Functional expression and segmental localization of rat colonic K-adenosine triphosphatase.
Functional expression and segmental localization of rat colonic K-adenosine triphosphatase.
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大鼠结肠 K-腺苷三磷酸酶的功能表达和节段定位。
DOI：
10.1172/jci118247
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Binder,HJ
中科院分区：
文献类型：
--
作者：
Lee,J;Rajendran,VM;Mann,AS;Kashgarian,M;Binder,HJ
最近克隆了结肠K-ATP酶的推定cDNA（Crowson，MS，和G. E. Shull 1992. J.Biol.Chem.267：13740-13748）。大量证据表明，在大鼠远端结肠中存在两种K-ATP酶和活性钾吸收过程：一种对哇巴因敏感，另一种对哇巴因不敏感。本研究利用杆状病毒表达系统在草地贪夜蛾（Spodoptera frugiperda，Sf 9）细胞中表达K-ATP酶活性，并以K-ATP酶cDNA 5'端编码区327个核苷酸的片段为抗原，制备多克隆抗体（M-1），鉴定K-ATP酶蛋白的特异性定位。K-ATP酶活性（28.7 +/- 1.2 nmol无机磷酸盐/mg蛋白/min）在从用含有具有推定的K-ATP酶cDNA的重组DNA的杆状病毒感染的Sf 9细胞分离的质膜中表达。K-ATP酶的Km为1.2 mM。哇巴因（1 mM）不抑制表达的K-ATP酶活性;而钒酸盐抑制的Ki为8.3 μ M。用M-1抗体的Western印迹分析确定了从远端而不是近端大鼠结肠制备的顶端膜中的100 kD蛋白。用M-1抗体进行的免疫组化研究仅定位于大鼠远端结肠表面细胞的顶膜，而针对Na，K-ATP酶的mAb（c464.6）定位于大鼠远端结肠表面细胞和隐窝细胞的基底外侧膜。总之，推测的K-ATP酶cDNA编码一种哇巴因不敏感的K-ATP酶，仅存在于大鼠远端结肠表面细胞的顶膜中。图片
A putative cDNA for the colonic K-ATPase has recently been cloned (Crowson, M.S., and G. E. Shull. 1992. J. Biol. Chem. 267:13740-13748). Considerable evidence exists that there are two K-ATPases and active K absorptive processes in the rat distal colon: one that is ouabain sensitive and the other ouabain insensitive. The present study used the baculovirus expression system to express K-ATPase activity in insect Spodoptera frugiperda (Sf 9) cells and a polyclonal antibody (M-1), developed against a fusion protein produced from the 327 nucleotide fragment from 5' coding region of the putative K-ATPase cDNA, to identify the specific localization of the K-ATPase protein. K-ATPase activity (28.7 +/- 1.2 nmol inorganic phosphate/mg protein min) was expressed in plasma membranes isolated from Sf 9 cells infected with baculovirus containing recombinant DNA with the putative K-ATPase cDNA. Km for K for the K-ATPase was 1.2 mM. The expressed K-ATPase activity was not inhibited by ouabain (1 mM); while the Ki for vanadate inhibition was 8.3 microM. Western blot analysis with the M-1 antibody identified a 100-kD protein in apical membranes prepared from distal, but not proximal, rat colon. Immunohistochemical studies with M-1 antibody localized K-ATPase only in the apical membrane of surface cells, while an mAb (c464.6) against Na,K-ATPase localized basolateral membranes of both surface and crypt cells of rat distal colon. In conclusion, the putative K-ATPase cDNA encodes an ouabain-insensitive K-ATPase that is present only in the apical membrane of surface cells of rat distal colon.Images