Wajsman R, Walters J R, Weiser M M
Division of Gastroenterology and Nutrition, State University of New York, Buffalo General Hospital 14203.
Biochem J. 1988 Dec 1;256(2):593-8. doi: 10.1042/bj2560593.
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.
先前已证实在大鼠小肠基底外侧膜囊泡中存在由ATP依赖性钙泵介导的钙离子转运。为了鉴定钙泵蛋白，在钙离子和镧离子存在的情况下，在有利于钙泵磷酸化中间体最大程度形成的条件下，用[γ-32P]ATP对十二指肠基底外侧膜进行磷酸化。在酸性SDS/聚丙烯酰胺凝胶的放射自显影片上可见四条主要的磷蛋白带；130 kDa的磷蛋白（pp130）的特性与质膜钙泵预期的特性一致。这种磷蛋白被镧离子显著增强，表现出酰基磷酸键的特征，优先从ATP进行磷酸化，并受到微摩尔浓度钒酸盐的抑制。另一种115 kDa的磷蛋白可能代表内质网钙泵或pp130的片段。75 kDa和95 kDa的其他磷蛋白主要是碱性磷酸酶的表达。与空肠、回肠或其他亚细胞组分的制备物相比，十二指肠基底外侧膜制备物中pp130的形成最高。在绒毛顶端和隐窝细胞的膜或维生素D缺乏的动物中，未发现钙泵活性与pp130形成之间存在类似的相关性。通过钙调蛋白亲和层析将pp130分离为单一磷蛋白。我们得出结论，pp130代表大鼠小肠基底外侧膜钙泵的磷酸化中间体，利用其作为钙调蛋白结合蛋白的特性可将其与其他磷蛋白分离。
