James P, Maeda M, Fischer R, Verma A K, Krebs J, Penniston J T, Carafoli E
Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich.
J Biol Chem. 1988 Feb 25;263(6):2905-10.
Exposure of the purified Ca2+ pump of human erythrocytes to chymotrypsin led to the rapid loss of calmodulin activation. A fragment of about 12 kDa was removed from the ATPase in 1-2 min. Blotting experiments with 125I-labeled calmodulin showed that this fragment contains the calmodulin binding region. The remainder of the ATPase molecule was degraded to a number of fragments ranging from 3 to 120 kDa; none of them bound calmodulin. To isolate the calmodulin binding domain, calmodulin which had been coupled to the Denny-Jaffe reagent (a cleavable radioactive photoaffinity cross-linker) was allowed to bind to the Ca2+ pump. After illumination to couple the cross-linker to the pump, the cleavable bond was split and the calmodulin removed, leaving the pump radioactively labeled. This pump was digested with chymotrypsin, and the products were separated by gel permeation chromatography. The only radioactive peak (migrating at about 12 kDa) was further purified on reverse-phase high pressure liquid chromatography (HPLC). Amino acid analysis showed the fragment to have a minimal molecular mass of 12.4 kDa and to contain a single methionine. After attempts to sequence the peptide directly failed. 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.
将人红细胞纯化的Ca2+泵暴露于胰凝乳蛋白酶会导致钙调蛋白激活迅速丧失。在1 - 2分钟内，ATP酶上一个约12 kDa的片段被切除。用125I标记的钙调蛋白进行的印迹实验表明，该片段包含钙调蛋白结合区域。ATP酶分子的其余部分被降解为多个大小从3到120 kDa不等的片段；它们都不与钙调蛋白结合。为了分离钙调蛋白结合结构域，让与丹尼 - 贾菲试剂（一种可裂解的放射性光亲和交联剂）偶联的钙调蛋白与Ca2+泵结合。光照使交联剂与泵偶联后，裂解可裂解键并去除钙调蛋白，使泵带有放射性标记。用胰凝乳蛋白酶消化该泵，产物通过凝胶渗透色谱分离。唯一的放射性峰（迁移率约为12 kDa）在反相高压液相色谱（HPLC）上进一步纯化。氨基酸分析表明该片段的最小分子量为12.4 kDa，且含有一个甲硫氨酸。直接对该肽进行测序的尝试失败后，对标记的ATP酶进行了CNBr消化，产生了可溶性和不溶性标记物质。对可溶性物质进行反相HPLC纯化后，收集到一个单一的放射性峰。其序列为（公式：见文本）。该峰的一部分通过微钙调蛋白柱；它在Ca2+存在下结合，并被EDTA以及EDTA和尿素的混合物洗脱。对洗脱峰进行葡萄球菌V8蛋白酶消化产生与上述相同的序列，但从Leu - 2开始到Glu - 32结束。对该肽的结构分析表明，它与其他受钙调蛋白调节的酶的钙调蛋白结合结构域具有共同特征。
