人红细胞Ca2+泵钙调蛋白结合域的鉴定和初步结构。
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James P, Maeda M, Fischer R, Verma AK, Krebs J, Penniston JT, Carafoli E
人红细胞Ca2+泵钙调蛋白结合域的鉴定和初步结构。
生物化学杂志1988 Feb 25;263(6):2905-10。
- PubMed ID
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2963820 (查看PubMed］
- 摘要
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暴露纯化的Ca2+泵的人红细胞凝乳胰蛋白酶导致钙调蛋白激活的快速损失。在1-2分钟内从atp酶上去除约12 kDa的片段。用125i标记的钙调蛋白进行印迹实验表明，该片段包含钙调蛋白结合区。剩余的atp酶分子被降解为3 ~ 120 kDa的片段;它们都没有结合钙调素。为了分离钙调蛋白结合域，钙调蛋白被耦合到Denny-Jaffe试剂(一种可裂解的放射性光亲和交联剂)上，被允许与Ca2+泵结合。在照明将交联剂耦合到泵上后，可解理键被分裂，钙调素被去除，使泵具有放射性标记。用胰凝乳酶消化，用凝胶渗透色谱分离产物。在反相高压液相色谱法(HPLC)中进一步纯化了唯一的放射性峰(在约12 kDa处迁移)。氨基酸分析显示，该片段的最小分子质量为12.4 kDa，含有一个蛋氨酸。在尝试直接测序肽段失败后。 CNBr digestion was carried out on the labeled ATPase, producing both soluble and insoluble labeled material. After reverse-phase HPLC purification of the soluble material, a single radioactive peak was collected. Its sequence was (Formula: see text). A portion of this peak was passed through a microcalmodulin column; it bound in the presence of Ca2+ and was eluted by EDTA, and by a mixture of EDTA and urea. Staphylococcal V8 protease digestion of the eluted peak produced the same sequence as shown above, but starting at Leu-2 and ending at Glu-32. Structural analysis of this peptide showed that it shares features with the calmodulin binding domains of other enzymes which are regulated by calmodulin.