Identification and isolation of the phosphorylated intermediate of the calcium pump in rat intestinal basolateral membranes.
Identification and isolation of the phosphorylated intermediate of the calcium pump in rat intestinal basolateral membranes.
复制标题
大鼠肠基底外侧膜钙泵磷酸化中间体的鉴定和分离。
DOI：
10.1042/bj2560593
复制
发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Weiser,MM
中科院分区：
文献类型：
--
作者：
Wajsman,R;Walters,JR;Weiser,MM
通过atp依赖的Ca2+泵运输Ca2+已经证明以前在大鼠肠基底侧膜囊。为了鉴定Ca2+泵蛋白，在Ca2+和La3+存在的情况下，用[γ - 32p]ATP磷酸化十二指肠基底外侧膜，在有利于Ca2+泵磷酸化中间体最大形成的条件下。在酸性SDS/聚丙烯酰胺凝胶的射线自显像上可见4条主要的磷酸蛋白条带；磷蛋白（pp）在130 kDa （pp130）下的特性与质膜Ca2+泵的特性一致。该磷酸化蛋白被La3+显著增强，表现出酰基-磷酸键的特征，优先被ATP磷酸化，并被微摩尔浓度的钒酸盐抑制。另一个115 kDa的磷酸化蛋白可能代表内质网Ca2+泵或pp130的片段。其他75和95 kDa的磷酸化蛋白主要表达碱性磷酸酶。与空肠、回肠或其他亚细胞组分相比，十二指肠基底侧膜制剂中pp130的形成最高。在绒毛尖端和隐窝细胞的膜或维生素d缺乏的动物中，Ca2+泵活性与pp130形成之间没有发现类似的相关性。通过钙调蛋白亲和层析分离得到单个磷酸蛋白Pp130。我们得出结论，pp130代表大鼠肠基底侧膜Ca2+泵的磷酸化中间体，可以利用其作为钙调素结合蛋白的特性与其他磷酸化蛋白分离。
Transport of Ca2+ by the ATP-dependent Ca2+ pump has been demonstrated previously in rat intestinal basolateral-membrane vesicles. To identify the Ca2+-pump protein, duodenal basolateral membranes were phosphorylated with [gamma-32P]ATP in the presence of Ca2+ and La3+, under conditions conducive for maximal formation of the phosphorylated intermediate of the Ca2+ pump. Four major phosphoprotein bands were seen on autoradiograms of acidic SDS/polyacrylamide gels; the properties of a phosphoprotein (pp) at 130 kDa (pp130) were consistent with those expected for the plasma-membrane Ca2+ pump. This phosphoprotein was markedly enhanced by La3+, exhibited the characteristics of an acyl-phosphate bond, was preferentially phosphorylated from ATP and inhibited by micromolar concentrations of vanadate. Another phosphoprotein of 115 kDa possibly represented the endoplasmic reticulum Ca2+ pump or a fragment of pp130. Other phosphoproteins of 75 and 95 kDa were predominantly expressions of alkaline phosphatase. Formation of pp130 was highest in duodenal basolateral-membrane preparations when compared with those of jejunum and ileum or other subcellular fractions. A similar correlation between Ca2+-pump activity and pp130 formation was not found in membranes from villus-tip and crypt cells or in vitamin D-deficient animals. pp130 was isolated as a single phosphoprotein by calmodulin-affinity chromatography. We conclude that pp130 represents the phosphorylated intermediate of the rat intestinal basolateral-membrane Ca2+ pump, which can be separated from other phosphoproteins using its properties as a calmodulin-binding protein.