Lipskaia T Iu, Rybina I V
Biokhimiia. 1987 Apr;52(4):690-700.
Creatine kinase from pigeon breast muscle was obtained in a homogeneous (as evidenced from polyacrylamide gel SDS electrophoresis) state. The molecular mass of the enzyme monomer is 43,000. Ultracentrifugation in a sucrose density gradient and gel filtration revealed that the enzyme is present in solution as a mixture of two major forms, i.e., octamer and dimer, which differ in their activity. The decrease of ionic strength from 0.25 to 0.02 results in reversible dissociation of the octameric form. A temperature rise from 5 degrees to 20 degrees C or the nature of monovalent anions (e.g., Cl-, CH3COO-, NO3-) and cations (K+, Na+) present in the medium do not influence the distribution of oligomeric forms. At pH 6.0 the major form is represented by the octamer; its dissociation is caused by an increase of pH. The octamer dissociation occurs in a mixture of substrates of the creatine kinase reaction in the presence of Mg2+; no such dissociation is observed in the absence of Mg2+ and in the presence of each of the reaction substrates. The non-interacting pair of substrates--ADP and creatine--causes the dissociation of the octamer in the presence of nitrate ions but not acetate. It is concluded that the dissociating effect of substrates is due to the conformational changes of subunits during catalysis. At physiological concentrations of nucleotide substrates the degree of octamer dissociation depends on the ratio of creatine phosphate and creatine concentrations, as well as on the presence of chlorine and phosphate ions. A qualitative estimation of the rate of pH- and substrate-dependent dissociation of creatine kinase octamer revealed that under the given experimental conditions the pH-dependent dissociation is completed within hours, whereas the substrate-dependent one--within seconds or minutes. According to its properties, mitochondrial creatine kinase from pigeon breast muscle is close to its bovine heart counterpart; the observed differences were found to be quantitative.
从鸽胸肌中提取的肌酸激酶呈均一状态（聚丙烯酰胺凝胶SDS电泳证明）。该酶单体的分子量为43,000。蔗糖密度梯度超速离心和凝胶过滤显示，该酶在溶液中以两种主要形式的混合物存在，即八聚体和二聚体，它们的活性不同。离子强度从0.25降至0.02会导致八聚体形式可逆解离。温度从5℃升至20℃或介质中存在的一价阴离子（如Cl-、CH3COO-、NO3-）和阳离子（K+、Na+）的性质不会影响寡聚体形式的分布。在pH 6.0时，主要形式为八聚体；其解离是由pH升高引起的。八聚体解离发生在肌酸激酶反应底物混合物中且存在Mg2+的情况下；在不存在Mg2+以及存在每种反应底物时未观察到这种解离。非相互作用的底物对——ADP和肌酸——在存在硝酸根离子而非醋酸根离子的情况下会导致八聚体解离。得出的结论是，底物的解离作用是由于催化过程中亚基的构象变化。在核苷酸底物的生理浓度下，八聚体解离程度取决于磷酸肌酸和肌酸浓度的比例，以及氯离子和磷酸根离子的存在。对肌酸激酶八聚体pH和底物依赖性解离速率的定性估计表明，在给定实验条件下，pH依赖性解离在数小时内完成，而底物依赖性解离在数秒或数分钟内完成。根据其性质，鸽胸肌线粒体肌酸激酶与牛心肌线粒体肌酸激酶相近；观察到的差异是定量的。
