Maruyama K, Nonomura Y
J Biochem. 1984 Sep;96(3):859-70. doi: 10.1093/oxfordjournals.jbchem.a134904.
In the microsome of scallop adductor striated muscle, 30K, 55K, 90K, and 360K proteins were detected as calcium binding proteins by 45Ca autoradiography on the transferred nitrocellulose membrane after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The 360K protein was directly extracted with Triton X-100 from the whole homogenate of striated portion of scallop adductor muscle and purified through DEAE cellulose and hydroxyapatite column chromatography. This purified scallop high molecular weight calcium binding protein (SHCBP) showed a faster mobility in SDS PAGE in the presence of Ca2+ than in its absence. The decrease of tryptophan fluorescence had a half maximum near pCa 7 and was slightly co-operative with Mg2+. UV absorbance was slightly increased with Ca2+. The CD spectrum also changed with Mg2+ and Ca2+. These results reflect that this SHCBP binds calcium ions under near physiological conditions. SHCBP-like high molecular weight calcium binding proteins were also detected in the smooth muscle portion of adductor muscle and branchiae of scallop by 45Ca autoradiography, but not in liver. The adductor muscle of clam had a high molecular weight calcium binding protein whose molecular weight was a little smaller than that of SHCBP. The foot of turban shell had the same molecular weight calcium binding protein as SHCBP. Stains-all, a cationic carbocyanine dye, which has been reported to stain calcium binding proteins blue, stained SHCBP blue. The spectrum of SHCBP stained with Stains-all was very similar to that of calsequestrin. Although the function of SHCBP is still unknown, it might be expected to correspond to calsequestrin of vertebrate skeletal muscle, a calcium sequestering protein, in the sarcoplasmic reticulum.
在扇贝闭壳肌的微粒体中，经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS - PAGE）后，通过对转移至硝酸纤维素膜上的样品进行45Ca放射自显影，检测到30K、55K、90K和360K的蛋白质为钙结合蛋白。360K的蛋白质可通过Triton X - 100直接从扇贝闭壳肌横纹肌部分的全匀浆中提取，并经DEAE纤维素和羟基磷灰石柱色谱法纯化。这种纯化的扇贝高分子量钙结合蛋白（SHCBP）在SDS - PAGE中，Ca2+存在时比不存在时迁移速度更快。色氨酸荧光的降低在pCa 7附近达到最大降幅的一半，并且与Mg2+有轻微协同作用。紫外吸光度随Ca2+略有增加。圆二色光谱也随Mg2+和Ca2+发生变化。这些结果表明，这种SHCBP在接近生理条件下能结合钙离子。通过45Ca放射自显影，在扇贝闭壳肌的平滑肌部分和鳃中也检测到了类似SHCBP的高分子量钙结合蛋白，但在肝脏中未检测到。蛤的闭壳肌有一种高分子量钙结合蛋白，其分子量比SHCBP略小。笠贝的足部有与SHCBP分子量相同的钙结合蛋白。据报道，阳离子花青染料“全染剂”能将钙结合蛋白染成蓝色，它将SHCBP染成了蓝色。用全染剂染色后的SHCBP光谱与肌集钙蛋白的光谱非常相似。尽管SHCBP的功能仍不清楚，但可能预期它与脊椎动物骨骼肌肌浆网中的钙螯合蛋白肌集钙蛋白相对应。
