Glyn H, Sleep J
J Physiol. 1985 Aug;365:259-76. doi: 10.1113/jphysiol.1985.sp015770.
The rate of hydrolysis of adenosine triphosphate (ATP) by chemically skinned rabbit muscle fibres was measured as a function of Mg ATP concentration in the range 5 microM to 5 mM. Pyruvate kinase and lactate dehydrogenase were used to link adenosine diphosphate formation to oxidation of nicotinamide adenine dinucleotide which was followed by the change in absorption at 340 nm. The ATPase rate of a fully activated fibre (pCa = 4.5) increased monotonically with Mg ATP concentration in a manner that could be readily fitted by a hyperbola. At 15 degrees C, pH 7 and an ionic strength of 0.2 M the rate at saturating Mg ATP (Vm) was 1.78 +/- 0.2 s-1 per myosin head (mean +/- S.D.; n = 6) and the Mg ATP concentration needed for half the maximal rate (Km) was 16.6 +/- 2 microM. The ATPase of fibres that had been stabilized by cross-linking with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) was also investigated. Cross-linking did not significantly affect the Vm or Km and these fibres proved useful for investigating the adequacy of the pyruvate kinase activity for regenerating hydrolysed ATP. Myofibrils were cross-linked with EDC or glutaraldehyde to prevent shortening. Their ATPase properties were investigated: the values of Vm were 0.85 +/- 0.18 (mean +/- S.D.; n = 14) and 0.82 +/- 0.05 s-1 (n = 6) and of Km were 18.0 +/- 2.8 and 12.4 +/- 2.4 microM respectively. The values of Vm and Km for EDC cross-linked myofibrils were fairly insensitive to ionic strength, the Km decreasing 40% and the Vm increasing 50% for a change from 0.2 to 0.3 M. This slight dependence on ionic strength is considered in relation to the ionic strength dependence of the elementary rate constants of the actomyosin subfragment-1 ATPase cycle.
测定了化学去皮兔肌纤维水解三磷酸腺苷（ATP）的速率与5微摩尔至5毫摩尔范围内的镁ATP浓度的函数关系。使用丙酮酸激酶和乳酸脱氢酶将二磷酸腺苷的形成与烟酰胺腺嘌呤二核苷酸的氧化联系起来，随后监测340纳米处的吸光度变化。完全激活的纤维（pCa = 4.5）的ATP酶速率随镁ATP浓度单调增加，其方式可用双曲线轻松拟合。在15℃、pH 7和离子强度为0.2 M的条件下，饱和镁ATP时的速率（Vm）为每肌球蛋白头部1.78±0.2秒-1（平均值±标准差；n = 6），达到最大速率一半所需的镁ATP浓度（Km）为16.6±2微摩尔。还研究了用1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）交联稳定的纤维的ATP酶。交联对Vm或Km没有显著影响，这些纤维被证明可用于研究丙酮酸激酶活性对再生水解ATP的充足性。肌原纤维用EDC或戊二醛交联以防止缩短。研究了它们的ATP酶特性：Vm值分别为0.85±0.18（平均值±标准差；n = 14）和0.82±0.05秒-1（n = 6），Km值分别为18.0±2.8和12.4±2.4微摩尔。EDC交联肌原纤维的Vm和Km值对离子强度相当不敏感，离子强度从0.2 M变为0.3 M时，Km降低40%，Vm增加50%。结合肌动球蛋白亚片段-1 ATP酶循环基本速率常数对离子强度的依赖性，考虑了这种对离子强度的轻微依赖性。
