Kennelly P J, Starovasnik M A, Edelman A M, Krebs E G
Howard Hughes Medical Institute, Seattle, Washington.
J Biol Chem. 1990 Jan 25;265(3):1742-9.
The binding of Ca2+(4).calmodulin (CaM) to rabbit skeletal muscle myosin light chain kinase (MLCK) is required for expression of the enzyme's activity. While both MLCK and CaM were stable at 30 degrees C, their complex was not. The binding of CaM to MLCK resulted in a time- and temperature-dependent inactivation that reflected an intrinsic instability of the complex. Separation of the components of the inactive complex yielded functional CaM, but catalytically inert MLCK, indicating that the site of the inactivating event was confined to MLCK. The behavior of proteolytic fragments further localized this event to the C-terminal 60% of the 603-residue protein. Changes in the tryptophan fluorescence and proteolytic susceptibility of MLCK-CaM indicated that a conformational change accompanied, and thus may have caused, inactivation. Substrates protected against inactivation, as did millimolar concentrations of Mg2+, Mn2+, and Ca2+. These metals appeared to bind to a site on MLCK distinct from that which recognized Mg2+.ATP. A proteolytic fragment of MLCK lacking the ability to bind CaM, C beta 35 (residues 255-584; Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. G. (1985) J. Biol. Chem. 260, 11275-11285), was unstable at 30 degrees C, whereas a similar fragment which does bind CaM, T beta 40 (residues 236-595; Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. (1985) J. Biol. Chem. 260, 11275-11285), was unstable only when CaM was bound.
Ca2+(4)·钙调蛋白（CaM）与兔骨骼肌肌球蛋白轻链激酶（MLCK）的结合是该酶活性表达所必需的。虽然MLCK和CaM在30℃时都很稳定，但它们的复合物却不然。CaM与MLCK的结合导致了一种时间和温度依赖性的失活，这反映了复合物固有的不稳定性。无活性复合物的各组分分离后产生了有功能的CaM，但却是催化惰性的MLCK，这表明失活事件的位点局限于MLCK。蛋白水解片段的行为进一步将该事件定位到603个氨基酸残基的蛋白质的C末端60%区域。MLCK-CaM的色氨酸荧光和蛋白水解敏感性的变化表明，一种构象变化伴随着失活，因此可能导致了失活。底物能防止失活，毫摩尔浓度的Mg2+、Mn2+和Ca2+也能起到同样的作用。这些金属似乎与MLCK上一个不同于识别Mg2+·ATP的位点结合。缺乏结合CaM能力的MLCK蛋白水解片段Cβ35（氨基酸残基255 - 584；埃德尔曼，A.M.，泷雄，K.，布卢门撒尔，D.K.，汉森，R.S.，沃尔什，K.A.，谷仁，K.，和克雷布斯，E.G.（1985年）《生物化学杂志》260，11275 - 11285）在30℃时不稳定，而一个类似的能结合CaM的片段Tβ40（氨基酸残基236 - 595；埃德尔曼，A.M.，泷雄，K.，布卢门撒尔，D.K.，汉森，R.S.，沃尔什，K.A.，谷仁，K.，和克雷布斯，E.（1985年）《生物化学杂志》260，11275 - 11285）只有在结合CaM时才不稳定。
