Urushidani T, Hanzel D K, Forte J G
Department of Physiology-Anatomy, University of California, Berkeley 94720.
Am J Physiol. 1989 Jun;256(6 Pt 1):G1070-81. doi: 10.1152/ajpgi.1989.256.6.G1070.
When isolated rabbit gastric glands were stimulated with histamine plus isobutylmethylxanthine, a redistribution of H+-K+-ATPase, from microsomes to a low-speed pellet, occurred in association with the phosphorylation of an 80-kDa protein (80K) in the apical membrane-rich fraction purified from the low-speed pellet. Histamine alone or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), but not carbachol, also stimulated both the redistribution of H+-K+-ATPase and phosphorylation of 80K. Under stimulated conditions, 80K copurified in the apical membrane fraction along with H+-K+-ATPase and actin; whereas purified microsomes from resting stomach were highly enriched in H+-K+-ATPase but contained neither 80K nor actin. Treatment of the apical membranes with detergents, salts, sonication, and so on, led us to conclude that 80K is a membrane protein, unlike actin; however, the mode of association of 80K with membrane differed from H+-K+-ATPase, an integral membrane protein. Isoelectric focusing and peptide mapping revealed that 80K consists of six isomers of slightly differing pI, with 32P occurring only in the three most acidic isomers and exclusively on serine residues. Moreover, stimulation elicited a shift in the amount of 80K isomers, from basic to acidic, as well as phosphorylation. We conclude that 80K is an apical membrane protein in the parietal cell and an important substrate for cAMP-dependent, but not calcium-dependent, pathway of acid secretion.
当用组胺加异丁基甲基黄嘌呤刺激分离的兔胃腺时，H⁺-K⁺-ATP酶会从微粒体重新分布到低速沉淀中，这与从低速沉淀中纯化的富含顶端膜的部分中一个80kDa蛋白（80K）的磷酸化有关。单独使用组胺或二丁酰腺苷3',5'-环磷酸（DBcAMP），而非卡巴胆碱，也会刺激H⁺-K⁺-ATP酶的重新分布和80K的磷酸化。在刺激条件下，80K与H⁺-K⁺-ATP酶和肌动蛋白一起在顶端膜部分共纯化；而来自静息胃的纯化微粒体中H⁺-K⁺-ATP酶高度富集，但既不含有80K也不含有肌动蛋白。用去污剂、盐、超声处理等对顶端膜进行处理后，我们得出结论，与肌动蛋白不同，80K是一种膜蛋白；然而，80K与膜的结合方式不同于整合膜蛋白H⁺-K⁺-ATP酶。等电聚焦和肽图分析表明，80K由六个等电点略有不同的异构体组成，³²P仅出现在三个酸性最强的异构体中，且仅在丝氨酸残基上。此外，刺激引起80K异构体数量从碱性向酸性的转变以及磷酸化。我们得出结论，80K是壁细胞中的一种顶端膜蛋白，是酸分泌的cAMP依赖性而非钙依赖性途径的重要底物。
