Identification of a putative Na(+)-H+ exchanger regulatory cofactor in rabbit renal BBM.
Identification of a putative Na(+)-H+ exchanger regulatory cofactor in rabbit renal BBM.
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兔肾 BBM 中推定的 Na()-H 交换调节辅因子的鉴定。
DOI：
10.1152/ajprenal.1990.259.6.f867
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Weinman,EJ
中科院分区：
文献类型：
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作者：
Morell,G;Steplock,D;Shenolikar,S;Weinman,EJ
先前用洗涤剂溶解兔肾刷边膜（BBM）蛋白进行的体外研究表明，腺苷3′，5′-环单磷酸腺苷（cAMP）依赖性蛋白激酶A （PKA）介导的Na(+)-H+交换器的抑制需要42-kDa辅助因子的存在，而42-kDa辅助因子与交换器本身不同。我们试图确定兔肾BBM囊泡中是否存在一种与42-kDa辅助因子相似的蛋白质。用含有纯化的PKA催化亚基的低渗磷酸化溶液孵育天然BBM囊泡蛋白，导致许多BBM蛋白磷酸化，包括一种表观分子量与从正辛基葡萄糖苷提取的BBM蛋白的阴离子交换柱层析中获得的42 kda辅因子相似但不相同的蛋白。通过磷酸化肽图和放射性碘化肽图确定BBM囊泡蛋白与42-kDa辅因子的同源性。这些结果表明，当PKA和ATP存在于囊泡空间中时，天然BBM囊泡含有许多被PKA磷酸化的蛋白质。其中一种蛋白质似乎与42-kDa蛋白相同，如先前在体外研究中提出的那样，42-kDa蛋白作为调节辅助因子介导PKA对肾Na(+)-H+交换器的抑制作用。
Previous in vitro studies with detergent-solubilized rabbit renal brush-border membrane (BBM) proteins have suggested that adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA)-mediated inhibition of the Na(+)-H+ exchanger requires the presence of 42-kDa cofactor that is distinct from the exchanger itself. We sought to determine whether there was a protein in native rabbit renal BBM vesicles that has characteristics similar to that of the 42-kDa cofactor. Incubation of native BBM vesicle proteins with a hypotonic phosphorylation solution containing purified catalytic subunit of PKA resulted in phosphorylation of a number of BBM proteins, including a protein with an apparent molecular weight that was similar but not identical to that of the 42-kDa cofactor obtained from anion-exchange column chromatography of n-octyl glucoside-extracted BBM proteins. The identity between the BBM vesicle protein and the 42-kDa cofactor was established by phosphopeptide maps and radioiodinated peptide maps. These results indicate that native BBM vesicles contain a number of proteins that are phosphorylated by PKA when the PKA and ATP are present inside the vesicle space. One of these proteins appears to be identical to the 42-kDa protein that, as previously suggested by in vitro studies, acts as a regulatory cofactor mediating the inhibitory effect of PKA on the renal Na(+)-H+ exchanger.