Filipek A, Wojda U
Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Biochem J. 1996 Dec 1;320 ( Pt 2)(Pt 2):585-7. doi: 10.1042/bj3200585.
A novel protein target of mouse calcyclin (S100A6) was detected by a gel overlay method with 125I-labelled calcyclin. Interaction of calcyclin with its 30 kDa target protein (p30) present in Ehrlich ascites tumour (EAT) cells depended on the presence of Ca2+ ions. The binding of p30, evidenced by the reaction with 125I-labelled calcyclin, was found to be of higher affinity than the binding between mouse calcyclin and annexin II or glyceraldehyde-3-phosphate dehydrogenase. Examination of tissue extracts by the gel overlay method has shown that p30 is present not only in the EAT cells but also in mouse brain and spleen. This novel target protein of mouse calcyclin was purified to homogeneity from EAT cells by means of Phenyl-Sepharose chromatography, affinity chromatography and CM-cellulose chromatography. Purified p30 was digested with alpha-chymotrypsin and a partial amino acid sequence of one of the resulting peptides was established. A database search analysis revealed that the sequence is unique, with a similarity of less than 55% to any other known protein sequence.
采用¹²⁵I标记的钙周期蛋白通过凝胶覆盖法检测到小鼠钙周期蛋白（S100A6）的一种新型蛋白质靶点。钙周期蛋白与其存在于艾氏腹水瘤（EAT）细胞中的30 kDa靶蛋白（p30）的相互作用依赖于Ca²⁺离子的存在。通过与¹²⁵I标记的钙周期蛋白反应证明，p30的结合亲和力高于小鼠钙周期蛋白与膜联蛋白II或甘油醛-3-磷酸脱氢酶之间的结合。通过凝胶覆盖法对组织提取物的检测表明，p30不仅存在于EAT细胞中，也存在于小鼠脑和脾脏中。通过苯基-琼脂糖凝胶色谱、亲和色谱和CM-纤维素色谱法从EAT细胞中纯化出这种小鼠钙周期蛋白的新型靶蛋白，使其达到均一性。用α-胰凝乳蛋白酶消化纯化的p30，并确定所得一种肽的部分氨基酸序列。数据库搜索分析表明该序列是独特的，与任何其他已知蛋白质序列的相似性小于55%。
