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首页> 美国卫生研究院文献>Biochemical Journal >Simultaneous presence of two distinct endoplasmic-reticulum-type calcium-pump isoforms in human cells. Characterization by radio-immunoblotting and inhibition by 25-di-(t-butyl)-14-benzohydroquinone.
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Simultaneous presence of two distinct endoplasmic-reticulum-type calcium-pump isoforms in human cells. Characterization by radio-immunoblotting and inhibition by 25-di-(t-butyl)-14-benzohydroquinone.

机译：在人类细胞中同时存在两种不同的内质网型钙泵同工型。通过放射免疫印迹进行表征并通过25-二-（叔丁基）-14-苯并氢醌进行抑制。

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Phosphorylation, immunoblotting, limited proteolysis and drug-sensitivity analysis were used to characterize the sarcoendoplasmic-reticulum Ca2+ ATPases in a variety of human cell types. In platelets, several megakaryoblastoid and lymphoblastoid cell lines two distinct autophosphorylated forms of these ATPases with molecular mass of 100 and 97 kDa could be observed, whereas in several other cell types the 97 kDa form was absent. On immunoblots the 97 kDa species was specifically recognized by an inhibitory monoclonal antibody raised against the Ca2+ pump of platelet internal membranes, yielded on trypsinolysis a major fragment of 80 kDa, exhibited a distinct electrophoretic migration pattern as compared with the skeletal-, cardiac- and smooth-muscle Ca2+ pumps, and its autophosphorylation was strongly inhibited by the Ca(2+)-mobilizing agent 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ). The 100 kDa species reacted with an antibody specific for the cardiac- and smooth-muscle Ca2+ pumps, yielded on trypsinolysis fragments of 55 and 35 kDa, and its autophosphorylation was much less sensitive to tBHQ inhibition. These findings indicate the simultaneous presence of two different endoplasmic-reticulum Ca2+ pumps in a variety of human cell types, and may explain the previously observed differences in the Ca(2+)-handling characteristics of different intracellular Ca2+ pools and cell types.
机译：磷酸化，免疫印迹，有限的蛋白水解和药物敏感性分析被用来表征各种人类细胞类型中的肌内质网Ca2 + ATPase。在血小板中，可以观察到几种巨核细胞和淋巴母细胞系，这些ATP酶的两种不同的自磷酸化形式，分子量分别为100和97 kDa，而在其他几种细胞类型中则没有97 kDa形式。在免疫印迹中，针对血小板内膜的Ca2 +泵产生的抑制性单克隆抗体可特异性识别97 kDa物种，在胰蛋白酶消化后产生80 kDa的主要片段，与骨骼，心脏和骨骼肌相比，表现出独特的电泳迁移模式平滑肌Ca2 +泵，并通过Ca（2+）动员剂2,5-二-（叔丁基）-1,4-苯并氢醌（tBHQ）强烈抑制其自磷酸化。 100 kDa物种与针对心脏和平滑肌Ca2 +泵的特异性抗体反应，产生55 kDa和35 kDa的胰蛋白酶裂解片段，并且其自身磷酸化对tBHQ抑制的敏感性大大降低。这些发现表明在两种人类细胞类型中同时存在两种不同的内质网Ca2 +泵，并且可以解释先前观察到的不同细胞内Ca2 +池和细胞类型在Ca（2+）处理特性上的差异。

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