Lukas T J, Collinge M, Haiech J, Watterson D M
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, IL 60611-3008.
Biochim Biophys Acta. 1994 Sep 29;1223(3):341-7. doi: 10.1016/0167-4889(94)90093-0.
Yeast calmodulin binds only three calcium ions in the presence of millimolar concentrations of magnesium due to a defective calcium-binding sequence in its carboxyl terminal domain. Yeast calmodulin's diminished calcium-binding activity can be restored to that of other calmodulins by the use of site-directed mutagenesis to substitute its fourth calcium-binding domain with that of a vertebrate calmodulin sequence. However, the repair of yeast calmodulin's calcium-binding activity is not sufficient to repair quantitatively yeast calmodulin's defective protein kinase activator activity. Yeast calmodulin's activator activity with smooth muscle and skeletal muscle myosin light chain kinases and brain calmodulin-dependent protein kinase II can be progressively repaired by additional substitutions of vertebrate calmodulin sequences, provided that the four calcium-binding sites remain intact. An unexpected result obtained during the course of these studies was the observation that myosin light chain kinases from smooth and skeletal muscle tissues can respond differently to mutations in calmodulin. These and previous results indicate that the binding of four calcium ions by calmodulin is necessary but not sufficient to bring about quantitative activation of protein kinases, and are consistent with the conformational selection/restriction model of the dynamic equilibrium among calcium, calmodulin and each calmodulin regulated enzyme.
在存在毫摩尔浓度镁的情况下，酵母钙调蛋白仅结合三个钙离子，这是由于其羧基末端结构域中钙结合序列存在缺陷。通过定点诱变将酵母钙调蛋白的第四个钙结合结构域替换为脊椎动物钙调蛋白序列，可使其降低的钙结合活性恢复到其他钙调蛋白的水平。然而，修复酵母钙调蛋白的钙结合活性并不足以定量修复其有缺陷的蛋白激酶激活活性。只要四个钙结合位点保持完整，通过进一步替换脊椎动物钙调蛋白序列，酵母钙调蛋白对平滑肌和骨骼肌肌球蛋白轻链激酶以及脑钙调蛋白依赖性蛋白激酶II的激活活性可逐步得到修复。在这些研究过程中获得的一个意外结果是观察到平滑肌和骨骼肌组织中的肌球蛋白轻链激酶对钙调蛋白突变的反应可能不同。这些结果以及之前的结果表明，钙调蛋白结合四个钙离子是蛋白激酶定量激活所必需的，但并不充分，这与钙、钙调蛋白和每种钙调蛋白调节酶之间动态平衡的构象选择/限制模型一致。
