Persechini A, White H D, Gansz K J
Department of Physiology, University of Rochester Medical Center, New York 14642, USA.
J Biol Chem. 1996 Jan 5;271(1):62-7. doi: 10.1074/jbc.271.1.62.
We have determined the stoichiometry and rate constants for the dissociation of Ca2+ ion from calmodulin (CaM) complexes with rabbit skeletal muscle myosin light chain kinase (skMLCK), rat brain nitric oxide synthase (nNOS) or with the respective peptides (skPEP and nPEP) representing the CaM-binding domains in these enzymes. Ca2+ dissociation kinetics determined by stopped-flow fluorescence using the Ca2+ chelator quin-2 MF are as follows. 1) Two sites in the CaM-nNOS and CaM-nPEP complexes have a rate constant of 1 s-1. 2) The remaining two sites have a rate constant of 18 s-1 for CaM-nPEP and > 1000 s-1 for CaM-nNOS. 3) Three sites have a rate constant of 1.6 s-1 for CaM-skMLCK and 0.15 s-1 for CaM-skPEP. 4) The remaining site has a rate constant of 2 s-1 for CaM-skPEP and > 1000 s-1 for CaM-skMLCK. Comparison of these rate constants with those determined for complexes between the peptides and tryptic fragments representing the C- or N-terminal lobes of CaM indicate a mechanism for Ca2+ dissociation from CaM-nNOS of 2C slow + 2N fast and from CaM-skMLCK of (2C + 1N) slow + 1N fast. Ca2+ removal inactivates CaM-nNOS and CaM-skMLCK activities with respective rate constants of > 10 s-1 and 1 s-1. CaM-nNOS inactivation is fit by a model in which rapid Ca2+ dissociation from the N-terminal lobe of CaM is coupled to enzyme inactivation and slower Ca2+ dissociation from the C-terminal lobe is coupled to dissociation of the CaM-nNOS complex. CaM-skMLCK inactivation is fit by a model in which the three slowly dissociating Ca(2+)-binding sites are coupled to both dissociation of the complex and enzyme inactivation.
我们已经确定了钙离子（Ca2+）从钙调蛋白（CaM）与兔骨骼肌肌球蛋白轻链激酶（skMLCK）、大鼠脑一氧化氮合酶（nNOS）形成的复合物或与这些酶中代表CaM结合结构域的相应肽段（skPEP和nPEP）解离的化学计量和速率常数。使用Ca2+螯合剂喹啉-2 MF通过停流荧光法测定的Ca2+解离动力学如下：1）CaM-nNOS和CaM-nPEP复合物中的两个位点的速率常数为1 s-1。2）其余两个位点，CaM-nPEP的速率常数为18 s-1，CaM-nNOS的速率常数大于1000 s-1。3）CaM-skMLCK的三个位点的速率常数为1.6 s-1，CaM-skPEP的为0.15 s-1。4）其余位点，CaM-skPEP的速率常数为2 s-1，CaM-skMLCK的大于1000 s-1。将这些速率常数与肽段和代表CaM C端或N端叶的胰蛋白酶片段之间形成的复合物所测定的速率常数进行比较，表明Ca2+从CaM-nNOS解离的机制为2个C端位点慢 + 2个N端位点快，从CaM-skMLCK解离的机制为（2个C端位点 + 1个N端位点）慢 + 1个N端位点快。Ca2+的去除使CaM-nNOS和CaM-skMLCK的活性失活，其各自的速率常数分别大于10 s-1和1 s-1。CaM-nNOS失活符合一个模型，即Ca2+从CaM的N端叶快速解离与酶失活相关联，而从C端叶较慢的Ca2+解离与CaM-nNOS复合物的解离相关联。CaM-skMLCK失活符合一个模型，即三个缓慢解离的Ca(2+)结合位点与复合物的解离和酶失活都相关联。
