Absence of the alpha subunit of (Na+, K+)ATPase on luminal cell membranes.
Absence of the alpha subunit of (Na+, K+)ATPase on luminal cell membranes.
复制标题
管腔细胞膜上不存在 (Na , K )ATP 酶的 α 亚基。
DOI：
--
复制
发表时间：
1988
影响因子：
3.2
通讯作者：
A. Yamamoto
中科院分区：
文献类型：
--
作者：
Y. Tashiro;K. Omori;A. Yamamoto
最近，我们在该杂志上发表了两篇关于（Na+，K+）ATPasC在大鼠胰腺外分泌细胞（1）和大鼠腮腺（2）中的定量免疫电镜定位的报告。这些研究中使用的抗体是针对全（NatK）ATP酶的，该酶已通过Takemura等人（3）的方法从大鼠肾脏中纯化，如Akayama等人（4）所述。免疫印迹分析结果表明，抗体选择性结合α和3亚基，并显著抑制（Na，K）ATP酶活性（4）。(Na用此抗体和包埋后蛋白A-gold技术对大鼠胰腺外膜细胞和腮腺内膜细胞的~（+，K ~+）ATP酶进行了定位。在胰腺外分泌细胞的腺泡和导管细胞中，金颗粒与管腔和基底外侧表面膜结合，但管腔表面的颗粒密度较高（1）。在腮腺腺泡细胞中，在管腔质膜上检测到少量但数量可观的金颗粒，而在导管细胞中，金颗粒仅与基底外侧膜结合（2）。这些结果似乎与我们实验室先前的发现一致，即犬肝细胞的管腔（胆小管）表面膜上存在大量的（Na+，K+）ATP酶（3）。最近，Sztul等人批评了最后一份报告的结果（5）。根据他们的研究，特异于α亚基胞内域的单克隆抗体仅将该亚基定位于肝细胞的基底外侧表面，并且未检测到小管质膜的标记。他们使用针对（NatK）ATP酶α亚基的多克隆抗体进行的蛋白质印迹显示，肝细胞的基底外侧域基本上具有细胞的所有估计的（NaF，K+）ATP酶催化活性。他们认为，我们针对流浪汉（Na+，K+）ATP酶产生的多克隆抗体识别可能与（Na，K）ATP酶共享某些抗原决定簇的蛋白质，如Ca 2或Mg 2 ATP酶，或者我们最初的（Na '，K）ATP酶制剂含有高度抗原性的污染蛋白质。我们重新运行我们以前的实验，使用单特异性抗体对大鼠肾脏（Na+，K+）ATP酶的α亚基。大鼠流浪汉（Na，K）ATP酶，按别处所述方法纯化（4），用制备性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）将其分离成α和β亚基，然后将亚基电泳转移到硝基纤维素片上。与抗体孵育后，切下对应于a亚基的条带，从其中洗脱与a亚基结合的抗体，并用于a亚基定位的免疫电镜测定。我们的结果总结如下（表1）。大鼠肝细胞、胰腺外分泌腺泡细胞或腮腺腺泡细胞的腔表面膜没有金颗粒标记，而这些细胞基底外侧表面膜上的颗粒密度与先前报道的相似（1，2，3）。这些结果表明，在这些细胞的腔表面膜上没有可检测到的量的（Na+，K+）ATP酶的a亚基。因此，我们提出的大鼠胰腺外分泌腺泡细胞（1）和腮腺腺泡细胞（2）的腔表面上存在大量（Na+，K）ATP酶的建议没有得到实验支持。目前尚不清楚当使用抗hobo（Na，K）ATP酶抗体时腔表面膜被金颗粒染色的原因。一种可能性是，我们最初的（Na，K）ATP酶制备物，尽管通过SDS-PAGE判断是高度纯化的，但可能含有小但高度抗原性的污染蛋白，如Sztul等人所建议的（5）。另一
Recently we published two reports in this journal on quantitative immunoelectron microscopic localization of (Na+,K+)ATPasC in rat exocrine pancreatic cells (1) and rat parotid gland (2). The antibody used in those studies was against holo(NatK )ATPase which had been purified from rat kidney by the procedure ofTakemura et al. (3), as described by Akayama et al. (4). Results of immunoblot analyses showed that the antibody bound selectively to the a and 3 subunits and that it markedly inhibited (Na ,K ) ATPase activity (4). (Na+,K+)ATPase was localized in rat exocnine pancreatic cells and parotid gland by use ofthe antibody and the post-embedding protein A-gold technique. In acinar and duct cells ofexocrine pancreatic cells, gold partides bound to both the luminal and the basolateral surface membranes, but with higher particle density on the luminal surface (1). In the acinar cells ofparotid gland, a small but significant number ofgold particles were detected on the luminal plasma membranes, whereas in duct cells gold particles bound exclusively to the basolatenal membranes (2). These results appeared to us to be consistent with the previous finding from our laboratory that an appreciable amount of(Na+,K+)ATPase is present on the luminal (bile canalicular) surface membrane in canine hepatocytes (3). Recently, the results of this last report have been criticiied by Sztul et al. (5). According to them, a monocbonal antibody specific for the endodomain of the a subunit localized this subunit exclusively on the basolateral surfaces of hepatocytes, and no labeling of the canalicular plasma membrane was detected. Their Western blots that used a polyclonal antibody against the (NatK )ATPase a subunit showed that the basolateral domain of a hepatocyte has essentially all the cell’s estimated (NaF,K+)ATPase catalytic activity. They suggested that either our polycbonal antibodies produced against hobo(Na+,K+)ATPase recognized proteins such as Ca2 or Mg2 ATPase that may share certain antigenic determinants with (Na ,K ) ATPase or that our original (Na’,K )ATPase preparation contained a highly antigenic contaminating protein. We have re-run our previous experiments using antibody monospecific against the a subunit of rat kidney (Na+,K+)ATPase. Rat hobo(Na ,K ) ATPase, purified as described elsewhere (4), was separated into its a and l subunits by preparative sodium dodecyl sulfate-polyacrylamide gel dcctrophoresis (SDS-PAGE), after which the subunits were transferred dcctrophoretically to a nitrocellubose sheet. After incubation with the antibody, the band corresponding to the a subunit was cut out, and the antibody bound to the a subunit was eluted from it and used for immunoelectron microscopic determination of localization of the a subunit. Our results are summarized as follows (Table 1). There was no labeling ofthe luminal surface membranes ofrat hepatocytes, exocrine pancreatic acinar cells, or parotid gland acinar cells with gold particles, whereas the particle densities on the basolateral surface membranes ofthese cells were similar to those reported previously (1,2,3). These results indicate that there is no detectable amount of the a subunit of (Na+,K+)ATPase on the luminal surface membranes of these cells. Therefore, our proposal that an appreciable amount of(Na+,K )ATPase is present on the luminal surfaces of rat exocrine pancreatic acinar cells (1) and on those of parotid gland acinar cells (2) is not supported experimentally. It is not yet clear why the luminal surface membranes were stained with gold particles when anti-hobo(Na ,K )ATPase antibody was used. One possibility is that our original (Na ,K )ATPase preparation, although highly purified as judged by SDS-PAGE, may have contained a small but highly antigenic contaminating protein, as suggested by Sztul et al. (5). Another