Ray T K, Das P K, Nandi J, Banerjee A, Bandopadhyay S
Department of Surgery, SUNY Health Science Center, Syracuse 13210.
Biochemistry. 1988 Dec 13;27(25):8958-68. doi: 10.1021/bi00425a013.
Separation of the gradient-purified gastric microsome into two membrane subfractions of distinct enzymatic and phospholipid composition has been achieved by mild SDS (0.033% w/v) treatment followed by sucrose gradient centrifugation of the pig and rabbit gastric microsomes. While the high-density membranes had all of the (H+,K+)-ATPase and K+-pNPPase activities and revealed a single major 100-kDa band on SDS-PAGE, the low-density membranes contained all of the 5'-nucleotidase and nearly all of the Mg2+-ATPase. In the present study, the low-density subfraction has been characterized to be derived from the apical membranes and the high-density one from the intracellular tubulovesicular membranes of the parietal cells. Such characterization was based primarily on sole dependency of the apical plasma membranes on the endogenous activator for (H+,K+)-ATPase activity, differential sensitivity of the activator (AF)-dependent and -independent (H+,K+)-ATPase on micromolar vanadate and Ca2+, specific vitamin B12 binding ability of the apical plasmalemma, phospholipid and protein profiles of the two membrane subfractions, and other parameters. The AF, mentioned previously, has recently been implicated as a cytosolic regulator of the gastric (H+,K+)-ATPase [Bandopadhyay et al. (1987) J. Biol. Chem. 262, 5664-5670]. Two different forms (i.e., AF-dependent and -independent forms) of the (H+,K+)-ATPase are suggested to be present in the tubulovesicles on the basis of differential vanadate sensitivity while the AF-dependent form alone is present in the apical membranes. The data have been discussed in terms of stimulation-induced membrane transformation characteristic of the H+-secreting epithelia including the acid-secreting cells of the stomach.
通过温和的SDS（0.033% w/v）处理，随后对猪和兔的胃微粒体进行蔗糖梯度离心，已将梯度纯化的胃微粒体分离为具有不同酶和磷脂组成的两个膜亚组分。高密度膜具有所有的（H⁺，K⁺）-ATP酶和K⁺-对硝基苯磷酸酶活性，并且在SDS-PAGE上显示出一条单一的主要100-kDa条带，而低密度膜含有所有的5'-核苷酸酶和几乎所有的Mg²⁺-ATP酶。在本研究中，低密度亚组分已被鉴定为源自顶膜，而高密度亚组分源自壁细胞的细胞内微管泡膜。这种鉴定主要基于顶质膜对（H⁺，K⁺）-ATP酶活性的内源性激活剂的唯一依赖性、激活剂（AF）依赖性和非依赖性（H⁺，K⁺）-ATP酶对微摩尔钒酸盐和Ca²⁺的不同敏感性、顶质膜的特异性维生素B₁₂结合能力、两个膜亚组分的磷脂和蛋白质谱以及其他参数。先前提到的AF最近被认为是胃（H⁺，K⁺）-ATP酶的一种胞质调节剂[班多帕德海伊等人（1987年）《生物化学杂志》262卷，5664 - 5670页]。基于钒酸盐敏感性的差异，提示在微管泡中存在两种不同形式（即AF依赖性和非依赖性形式）的（H⁺，K⁺）-ATP酶，而仅AF依赖性形式存在于顶膜中。已根据包括胃泌酸细胞在内的H⁺分泌上皮细胞的刺激诱导膜转化特征对这些数据进行了讨论。
