Kanoh S, Ito M, Niwa E, Kawano Y, Hartshorne D J
Faculty of Bioresources, Mie University, Tsu, Japan.
Biochemistry. 1993 Aug 31;32(34):8902-7. doi: 10.1021/bi00085a023.
The objective of this study was to localize the actin-binding site in the smooth muscle myosin light chain kinase. Limited proteolysis by thermolysin indicated that hydrolysis of the kinase at the N-terminal end of the molecule resulted in loss of actin-binding ability. Various methods of cleavage were investigated for the generation of a discrete actin-binding peptide. The method chosen was cleavage at the cysteine residues by the 5,5'-dithiobis(2-nitrobenzoic acid)-cyanide complex. This procedure yielded an actin-binding peptide of approximate M(r) 17,000. The peptide was purified and shown to possess the actin-binding properties of the native myosin light chain kinase. The binding constant of the isolated peptide and parent enzyme to actin was estimated as 7.5 x 10(4) M-1. From the amino acid composition of the peptide and comparison with the sequence of gizzard myosin light chain kinase, it was suggested that the actin-binding site is located within the N-terminal sequence 1-114. Comparison with other actin-binding proteins shows some similarities to gizzard alpha-actinin and caldesmon.
本研究的目的是定位平滑肌肌球蛋白轻链激酶中的肌动蛋白结合位点。嗜热菌蛋白酶的有限蛋白水解表明，在分子的N末端对激酶进行水解会导致肌动蛋白结合能力丧失。研究了各种切割方法以产生离散的肌动蛋白结合肽。所选择的方法是用5,5'-二硫代双（2-硝基苯甲酸）-氰化物复合物在半胱氨酸残基处进行切割。该方法产生了一个分子量约为17,000的肌动蛋白结合肽。该肽被纯化并显示具有天然肌球蛋白轻链激酶的肌动蛋白结合特性。分离的肽和亲本酶与肌动蛋白的结合常数估计为7.5×10⁴ M⁻¹。根据该肽的氨基酸组成并与砂囊肌球蛋白轻链激酶的序列进行比较，表明肌动蛋白结合位点位于N末端序列1-114内。与其他肌动蛋白结合蛋白的比较显示与砂囊α-辅肌动蛋白和钙调蛋白有一些相似之处。
