Isolation and characterization of acetylcholine receptor membrane-associated (nonreceptor v2-protein) and soluble electrocyte creatine kinases.
Isolation and characterization of acetylcholine receptor membrane-associated (nonreceptor v2-protein) and soluble electrocyte creatine kinases.
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乙酰胆碱受体膜相关（非受体 v2 蛋白）和可溶性细胞肌酸激酶的分离和表征。
DOI：
10.1016/s0021-9258(18)89467-4
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
A. Zechel
中科院分区：
文献类型：
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作者：
F. Barrantes;A. Braceras;H. Caldironi;G. Mieskes;H. Moser;E. C. Toren;M. E. Roque;T. Wallimann;A. Zechel
肌酸激酶被认为是鱼雷电器官最重要的成分，也是由此获得的乙酰胆碱受体(AChR)膜的少数成分。经低温乙醇提取、镁-酶络合物沉淀和汞-琼脂糖层析纯化，可得到比活性大于550单位/毫克蛋白的可溶性蛋白制剂。离子交换高效液相色谱的保留时间、电泳行为、免疫化学性质、胰酶图谱和氨基酸组成使肌酸激酶同工酶的比较成为可能。优势种的变性亚基具有所谓v2蛋白的特点，pI值为6.3-6.8，mR=40,000-42,000，并显示出与抗BB(“脑”型)肌酸激酶抗体的交叉反应。MM(“肌肉”型)抗原可在总的电细胞中检测到，但在AChR膜中不能检测到；它们的相对分子质量略低，等电点较高。免疫细胞化学证实了BB同工酶的原位膜结合。底物磷酸肌酸的表观Km值为2.2 mM(AChR膜相关酶)和2.5 mM(肌肉形态)。Mg2+-ADP的表观Km值分别为0.54和0.22 mm。因此，由于膜结合，ADP与二元酶-肌酸-P复合体的结合亲和力提高了2倍。
Creatine kinase has been identified as a most prominent component of Torpedo electric organ and a minority constituent of the acetylcholine receptor (AChR) membranes obtained therefrom. Purification by low temperature ethanol extraction, precipitation of the Mg2+-enzyme complex, and mercurial-agarose chromatography yield preparations of soluble kinase with specific activities greater than 550 units/mg protein. Retention times in ion-exchange high performance liquid chromatography, electrophoretic behavior, immunochemical properties, tryptic mapping, and amino acid composition enable the comparison of creatine kinase isoenzymes. The denatured subunits of the predominant species have pI values of 6.3-6.8 and Mr = 40,000-42,000 characteristic of the so-called v2 proteins and show cross-reactivity with antibodies against the BB (“brain” type) creatine kinase. The MM (“muscle” type) antigens could be detected in the total electrocyte, but not in the AChR membranes; they have a slightly lower molecular weight and higher pI. The in situ membrane association of the BB isoenzyme is confirmed by immunocytochemistry. The apparent Km values for the substrate creatine phosphate are 2.2 mM for the AChR membrane-associated enzyme and 2.5 mM for the muscle form. The apparent Km values for Mg2+-ADP are 0.54 and 0.22 mM, respectively. Thus, a 2-fold higher affinity in the binding of ADP to the binary enzyme-creatine-P complex results from membrane association.