Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules.
Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules.
复制标题
DOI：
10.1016/s0021-9258(19)37635-5
复制
发表时间：
1988-10
期刊：
影响因子：
--
通讯作者：
B. Denker;Barbara L. Smith;F. Kuhajda;P. Agre
中科院分区：
文献类型：
--
作者：
B. Denker;Barbara L. Smith;F. Kuhajda;P. Agre
在人类红细胞中鉴定了一种新的Mr 28，000整合膜蛋白（“28 kDa”），并发现其完全与Triton X-100不溶性膜骨架相关。28 kDa的抗体在免疫印迹中与28 kDa和Mr 35，000 - 60，000的弥漫区域（“HMW-28 kDa”）发生强烈反应。膜的选择性蛋白水解酶切证明HMW-28 kDa具有胞外结构域，而28 kDa和HMW-28 kDa都具有胞内结构域。将28 kDa和HMW-28 kDa纯化至均一。定量免疫印迹表明每个红细胞含有120，000 - 160，000个28 kDa拷贝。28 kDa和HMW-28 kDa的二维碘肽图谱几乎相同;纯化的HMW-28 kDa的肽-N-糖苷酶消化证明其为28 kDa的N-糖基化形式。当浓缩时，28 kDa形成一系列在十二烷基硫酸钠中稳定的较大寡聚体。几个nonerythroid组织研究与抗28 kDa的免疫印迹，只有肾脏显示免疫反应28 kDa。通过二维碘肽图谱比较，纯化的大鼠肾28 kDa与大鼠红细胞28 kDa几乎相同。抗28 kDa抗体对人肾的免疫组织化学染色显示近曲小管顶端刷状缘上有明显的染色。从红细胞膜和肾膜中纯化出一种新的膜整合蛋白。这种新的蛋白质可能在连接膜骨架和脂质双层中起作用。
A novel Mr 28,000 integral membrane protein ("28kDa") was identified in human erythrocytes and found entirely associated with the Triton X-100 insoluble membrane skeletons. Antibodies to 28kDa reacted strongly on immunoblots with 28kDa and a diffuse region of Mr 35,000-60,000 ("HMW-28kDa"). Selective proteolytic digestions of membranes demonstrated that HMW-28kDa has an extracellular domain, and both 28kDa and HMW-28kDa have intracellular domains. 28kDa and HMW-28kDa were purified to homogeneity. Quantitative immunoblots indicate that each erythrocyte contains 120,000-160,000 copies of 28kDa. Two-dimensional iodopeptide maps of 28kDa and HMW-28kDa were nearly identical; peptide-N-glycosidase digestion of purified HMW-28kDa demonstrated that it is the N-glycosylated form of 28kDa. When concentrated, 28kDa formed a series of larger oligomers which were stable in sodium dodecyl sulfate. Of several nonerythroid tissues studied with anti-28kDa immunoblots, only kidney displayed immunoreactive 28kDa. Purified rat kidney 28kDa was nearly identical to rat erythrocyte 28kDa when compared by two-dimensional iodopeptide mapping. Immunohistochemical staining of human kidney with anti-28kDa demonstrated prominent staining over the apical brush borders of proximal convoluted tubules. A novel integral membrane protein has been purified from erythrocyte and kidney membranes. This new protein may play a role in linkage of the membrane skeleton to the lipid bilayer.