Krause S M, Jacobus W E
Peter Belfer Laboratory for Myocardial Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1992 Feb 5;267(4):2480-6.
The kinetic influence of bound creatine kinase (CK) on the Ca(2+)-activated myosin ATPase was evaluated. ATPase rates were measured from 0.8 microM to 3.2 mM MgATP. Under control conditions, the apparent KmATP was 79.9 +/- 13.3 microM. In contrast, the addition of 12.2 mM phosphocreatine (PCr) decreased the apparent KmATP to a value of 13.6 +/- 1.4 microM. To determine if this reduction was merely the result of an ATP maintenance system, ATP was regenerated using either phosphoenolpyruvate and pyruvate kinase (PEP-PK), or PCr and soluble bovine cardiac CK. Data obtained with PEP + PK indicated an apparent KmATP of 65.5 +/- 7.3 microM. To study the effects of exogenous CK, the endogenous CK was irreversibly inhibited with 1 mM iodoacetamide. The kinetics of the ATPase were then examined by adding soluble CK to the incubation medium. Under these conditions, the KmATP was 56.4 +/- 0.86 microM. Therefore, these two ATP regeneration systems could not duplicate the effects of endogenous CK. The reduction of the apparent KmATP by endogenous CK was not the result of an altered inhibition by MgADP. MgADP inhibition was determined to be non-competitive, with a Ki of 5.0 +/- 0.1 mM. These data suggest that the observed kinetic effects reflect the proximity of the enzymes in the myofibrillar bundle, thus emphasizing the importance of bound CK for the localized regeneration of MgATP utilized by the myosin ATPase.
评估了结合型肌酸激酶（CK）对Ca(2+)激活的肌球蛋白ATP酶的动力学影响。在0.8微摩尔至3.2毫摩尔的MgATP浓度范围内测量ATP酶活性。在对照条件下，表观KmATP为79.9±13.3微摩尔。相比之下，添加12.2毫摩尔磷酸肌酸（PCr）可将表观KmATP降低至13.6±1.4微摩尔。为了确定这种降低是否仅仅是ATP维持系统的结果，使用磷酸烯醇丙酮酸和丙酮酸激酶（PEP-PK）或PCr和可溶性牛心肌CK再生ATP。用PEP + PK获得的数据表明表观KmATP为65.5±7.3微摩尔。为了研究外源性CK的作用，用1毫摩尔碘乙酰胺不可逆地抑制内源性CK。然后通过向孵育培养基中添加可溶性CK来检查ATP酶的动力学。在这些条件下，KmATP为56.4±0.86微摩尔。因此，这两种ATP再生系统不能复制内源性CK的作用。内源性CK导致的表观KmATP降低不是MgADP抑制作用改变的结果。确定MgADP抑制作用为非竞争性，Ki为5.0±0.1毫摩尔。这些数据表明，观察到的动力学效应反映了肌原纤维束中酶的接近程度，从而强调了结合型CK对肌球蛋白ATP酶利用的MgATP局部再生的重要性。
