Miller C J, Wong W W, Bobkova E, Rubenstein P A, Reisler E
Department of Chemistry and Biochemistry, University of California, Los Angeles 90095, USA.
Biochemistry. 1996 Dec 24;35(51):16557-65. doi: 10.1021/bi962388+.
Yeast actin mutants with acidic residues at the N terminus either neutralized (DNEQ) or deleted (delta-DSE) were used to assess the role of N-terminal acidic residues in the interactions of actin with myosin in the contractile cycle. Cosedimentation experiments revealed an approximately 3-fold decrease in the binding constant for DNEQ and delta-DSE actins to myosin subfragment-1 (S1) relative to that of wild type actin both in the presence of MgATP and in the absence of nucleotides (strong binding). DNEQ and delta-DSE actins protected S1 from tryptic digestion as well as the wild type and rabbit actins. The activation of S1 ATPase by DNEQ and delta-DSE actins (up to 50 microM) was very low but increased greatly after cross-linking these mutant actins to S1 by dimethyl suberimidate. Thus, the increased dissociation of mutant actins from S1 in the presence of ATP is the main cause for the low acto-S1 ATPase activities. At low-ionic strength conditions and in the presence of methylcellulose, the DNEQ and delta-DSE actins moved in the in vitro motility assays at a mean velocity similar to that of wild type actin (3.0 microns/s). Yet, the sliding velocity of the N-terminal and D24A/D25A and E99A/E100A mutant actins decreased relative to that of the wild type at all levels of external load introduced into the assay and at low densities of heavy meromyosin (HMM) on the cover slip. This indicates a lower relative force generation with the mutant actins. In contrast, the force generated under the same conditions with the 4Ac mutant actin (with four acidic charges at the N terminus) was higher than with wild type actin. At higher-ionic strength conditions (I = 150 mM), the sliding of the DNEQ and delta-DSE as well as that of the D24A/D25A and E99A/E100A actins ceased even in the presence of methylcellulose, while I341A actin (deficient in strong binding to myosin) still moved. These results indicate the importance of electrostatic actomyosin interactions under physiological salt conditions and show functionally distinct roles for the different myosin binding sites on actin.
将N端带有酸性残基的酵母肌动蛋白突变体进行中和（DNEQ）或缺失（delta-DSE）处理，以评估N端酸性残基在收缩循环中肌动蛋白与肌球蛋白相互作用中的作用。共沉降实验表明，在存在MgATP和不存在核苷酸（强结合）的情况下，相对于野生型肌动蛋白，DNEQ和delta-DSE肌动蛋白与肌球蛋白亚片段-1（S1）的结合常数降低了约3倍。DNEQ和delta-DSE肌动蛋白对S1的胰蛋白酶消化的保护作用与野生型和兔肌动蛋白相同。DNEQ和delta-DSE肌动蛋白（浓度高达50 microM）对S1 ATP酶的激活作用非常低，但在用辛二酸二甲酯将这些突变肌动蛋白与S1交联后，激活作用大大增加。因此，在ATP存在下突变肌动蛋白与S1解离增加是肌动蛋白-S1 ATP酶活性低的主要原因。在低离子强度条件下且存在甲基纤维素时，DNEQ和delta-DSE肌动蛋白在体外运动分析中的平均移动速度与野生型肌动蛋白相似（3.0微米/秒）。然而，在引入分析的所有外部负载水平以及盖玻片上重酶解肌球蛋白（HMM）低密度的情况下，N端以及D24A/D25A和E99A/E100A突变肌动蛋白的滑动速度相对于野生型有所降低。这表明突变肌动蛋白产生的相对力较低。相比之下，在相同条件下，4Ac突变肌动蛋白（N端带有四个酸性电荷）产生的力高于野生型肌动蛋白。在较高离子强度条件下（I = 150 mM），即使存在甲基纤维素，DNEQ和delta-DSE以及D24A/D25A和E99A/E100A肌动蛋白的滑动也会停止，而I341A肌动蛋白（与肌球蛋白的强结合缺陷）仍会移动。这些结果表明生理盐条件下静电肌动球蛋白相互作用的重要性，并显示了肌动蛋白上不同肌球蛋白结合位点的功能不同作用。
