Hepatic Na(+)-K(+)-ATPase enzyme activity correlates with polarized beta-subunit expression.
Hepatic Na(+)-K(+)-ATPase enzyme activity correlates with polarized beta-subunit expression.
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肝 Na( )-K( )-ATP 酶活性与极化 β 亚基表达相关。
DOI：
10.1152/ajpcell.1995.269.1.c69
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Sutherland,E
中科院分区：
文献类型：
--
作者：
Simon,FR;Leffert,HL;Ellisman,M;Iwahashi,M;Deerinck,T;Fortune,J;Morales,D;Dahl,R;Sutherland,E
我们研究了肝细胞中Na(+)-K(+)- atp酶的催化亚基在胆小管（顶端）和窦（基底外侧）膜结构域均被发现的潜在原因，而功能活性则优先与窦膜部位相关。在一系列平行研究中，我们通过光镜和电镜确定Na(+)-K(+)- atp酶α -亚基定位于肝细胞的两个膜结构域。使用纯化的肝质膜亚组分，瓦巴因抑制曲线显示出相似的抑制常数（抑制常数为10(-5)M），使用α 1-、α 2-和α 3多克隆和单克隆抗体的免疫印迹显示，在两种膜组分中α 1主要是抗原位点。此外，Northern blot杂交分析仅在肝细胞中发现α 1亚型。与α - 1亚基的双极分布相反，β -亚基仅在单克隆抗体荧光标记的正弦表面被识别。通过Northern blot分析证实了β 1-异构体，并通过多克隆抗体免疫印迹主要存在于正弦域。除了α - 1的双极性分布外，肝质膜亚组分的免疫印迹显示fodrin、锚蛋白、肌动蛋白和E-cadherin在两个结构域对称分布。这些结果表明，功能胜任的α / β复合物形成于正弦域，而只有α - 1亚基存在于顶极。
We have examined underlying causes for observations made in hepatocytes in which catalytic subunits of Na(+)-K(+)-ATPase are found both in bile canalicular (apical) and sinusoidal (basolateral) membrane domains, whereas functional activity is associated preferentially with sinusoidal membrane sites. In a series of parallel studies, we determined by both light and electron microscopy that Na(+)-K(+)-ATPase alpha-subunits were localized to both membrane domains of hepatocytes. With the use of purified liver plasma membrane subfractions, ouabain inhibition curves demonstrated similar inhibition constants (inhibition constant 10(-5) M), and immunoblots using alpha 1-, alpha 2-, and alpha 3-polyclonal and monoclonal antibodies demonstrated antigenic sites predominantly for alpha 1 in both membrane fractions. Also, Northern blot hybridization analysis revealed only the alpha 1-isoform in hepatocytes. In contrast to the bipolar distribution of the alpha 1-subunit, the beta-subunit was identified only at the sinusoidal surface using fluorescence labeling with a monoclonal antibody. The beta 1-isoform was demonstrated by Northern blot analysis and was present predominantly at the sinusoidal domain by immunoblotting with polyclonal antibodies. In addition to the bipolar distribution of alpha 1, immunoblotting of liver plasma membrane subfractions demonstrated a symmetrical distribution of fodrin, ankyrin, actin, and E-cadherin at both domains. These results suggest that functionally competent alpha/beta-complexes form at the sinusoidal domain, whereas only alpha 1-subunits are present at the apical pole.