Gray K A, Grossman S H, Summers D D
Comp Biochem Physiol B. 1986;83(3):613-20. doi: 10.1016/0305-0491(86)90305-6.
Creatine kinase from nurse shark brain and muscle has been purified to apparent homogeneity. In contrast to creatine kinases from most other vertebrate species, the muscle isozyme and the brain isozyme from nurse shark migrate closely in electrophoresis and, unusually, the muscle isozyme is anodal to the brain isozyme. The isoelectric points are 5.3 and 6.2 for the muscle and brain isozymes, respectively. The purified brain preparation also contains a second active protein with pI 6.0. The amino acid content of the muscle isozyme is compared with other isozymes of creatine kinase using the Metzger Difference Index as an estimation of compositional relatedness. All comparisons show a high degree of compositional similarity including arginine kinase from lobster muscle. The muscle isozyme is marginally more resistant to temperature inactivation than the brain isozyme; the muscle protein does not exhibit unusual stability towards high concentrations of urea. Kinetic analysis of the muscle isozyme reveals Michaelis constants of 1.6 mM MgATP, 12 mM creatine, 1.2 mM MgADP and 50 mM creatine phosphate. Dissociation constants for the same substrate from the binary and ternary enzyme-substrate complex do not differ significantly, indicating limited cooperatively in substrate binding. Enzyme activity is inhibited by small planar anions, most severely by nitrate. Shark muscle creatine kinase hybridizes in vitro with rabbit muscle or monkey brain creatine kinase; shark brain isozyme hybridizes with monkey brain or rabbit brain creatine kinase. Shark muscle and shark brain isozymes, under a wide range of conditions, failed to produce a detectable hybrid.
护士鲨大脑和肌肉中的肌酸激酶已被纯化至表观均一性。与大多数其他脊椎动物物种的肌酸激酶不同,护士鲨的肌肉同工酶和大脑同工酶在电泳中迁移距离相近,而且不同寻常的是,肌肉同工酶在大脑同工酶的阳极位置。肌肉同工酶和大脑同工酶的等电点分别为5.3和6.2。纯化后的大脑制剂还含有另一种活性蛋白,其等电点为6.0。使用梅茨格差异指数来估计组成相关性,将肌肉同工酶的氨基酸含量与其他肌酸激酶同工酶进行比较。所有比较都显示出高度的组成相似性,包括龙虾肌肉中的精氨酸激酶。肌肉同工酶对温度失活的耐受性略高于大脑同工酶;肌肉蛋白对高浓度尿素没有表现出异常的稳定性。对肌肉同工酶的动力学分析显示,其米氏常数分别为:MgATP 1.6 mM、肌酸12 mM、MgADP 1.2 mM和磷酸肌酸50 mM。二元和三元酶 - 底物复合物中相同底物的解离常数没有显著差异,表明底物结合中的协同作用有限。酶活性受到小的平面阴离子抑制,其中硝酸盐的抑制作用最为严重。鲨鱼肌肉肌酸激酶在体外与兔肌肉或猴脑肌酸激酶杂交;鲨鱼脑同工酶与猴脑或兔脑肌酸激酶杂交。在广泛的条件下,鲨鱼肌肉和鲨鱼脑同工酶未能产生可检测到的杂交产物。