Blumenthal D K, Takio K, Edelman A M, Charbonneau H, Titani K, Walsh K A, Krebs E G
Proc Natl Acad Sci U S A. 1985 May;82(10):3187-91. doi: 10.1073/pnas.82.10.3187.
In the course of determining the primary structure of rabbit skeletal muscle myosin light chain kinase (MLCK; ATP:protein phosphotransferase, EC 2.7.1.37) a peptide fragment was obtained that appears to represent the calmodulin-binding domain of this enzyme. Low concentrations of the peptide inhibited calmodulin activation of MLCK (Ki congruent to 1 nM). The peptide was not associated with a catalytically active, calmodulin-independent form of MLCK that was obtained by limited proteolysis. The peptide is 27 residues in length and represents the carboxyl terminus of MLCK. The sequence of the peptide shows no significant homology with any known protein sequence. The peptide contains one tryptophanyl residue and a high percentage of basic and hydrophobic residues, but no acidic or prolyl residues. Much of the sequence has a high probability of forming alpha helix. A chemically synthesized peptide has been prepared to study the interactions of the peptide and calmodulin in more detail. The intrinsic tryptophan fluorescence of the synthetic peptide shows a significant enhancement (approximately equal to 45%) in the presence of Ca2+ and calmodulin; fluorescence enhancement is maximal at a peptide:calmodulin stoichiometry of 1:1. Calmodulin-Sepharose affinity chromatography in the presence of 2 M urea indicates that the interaction of peptide and calmodulin is Ca2+-dependent. The results of these studies indicate that the catalytic and calmodulin-binding domains of MLCK represent distinct and separable regions of the protein. In addition, the results provide a basis for future studies of the molecular and evolutionary details of calmodulin-dependent enzyme regulation.
在确定兔骨骼肌肌球蛋白轻链激酶（MLCK；ATP：蛋白质磷酸转移酶，EC 2.7.1.37）一级结构的过程中，获得了一个肽片段，它似乎代表了该酶的钙调蛋白结合结构域。低浓度的该肽抑制MLCK的钙调蛋白激活作用（Ki约为1 nM）。该肽与通过有限蛋白酶解获得的无钙调蛋白时具有催化活性的MLCK形式无关。该肽长度为27个残基，代表MLCK的羧基末端。该肽的序列与任何已知蛋白质序列均无明显同源性。该肽含有一个色氨酸残基，碱性和疏水残基比例较高，但没有酸性或脯氨酸残基。该序列的大部分很可能形成α螺旋。已制备了一种化学合成肽，以更详细地研究该肽与钙调蛋白的相互作用。合成肽的固有色氨酸荧光在Ca2+和钙调蛋白存在下显示出显著增强（约45%）；在肽与钙调蛋白的化学计量比为1:1时荧光增强最大。在2 M尿素存在下进行的钙调蛋白-琼脂糖亲和层析表明，肽与钙调蛋白的相互作用是Ca2+依赖性的。这些研究结果表明，MLCK的催化结构域和钙调蛋白结合结构域代表了该蛋白质不同且可分离的区域。此外，这些结果为未来研究钙调蛋白依赖性酶调控的分子和进化细节提供了基础。
