Coan C R, Inesi G
J Biol Chem. 1977 May 10;252(9):3044-9.
Vesicular fragments of sarcoplasmic reticulum (SR) were labeled with the --SH-directed spin label 2,2,6,6-tetra-methyl,4-amino(N-iodoacetamide). Colorimetric titrations of the remaining --SH residues and determinations of unbound spin label indicated that primarily 3 residues/enzyme molecule were labeled under saturating conditions. This labeling was accompanied by minimal losses in activity, providing precautions were taken to prevent sulfhydryl oxidation during the labeling process. Additions of ATP produced a new "highly constrained" component in the ESR spectrum of the labeled SR, an effect not noted in previous studies. It is demonstrated that the changes produced by ATP are reversible, and require both substrate binding and Ca2+ binding. However, hydrolysis of the substrate is not required. It is further demonstrated that the labeled residue(s) responsible for the spectral change is not in the immediate vicinity of the ATP binding site. It is apparent that the observed spectral change is related to a conformational effect of ATP and Ca2+ on the ATPase protein, which is associated with a large free energy change occurring on binding. It is also suggested that the conformational effect extends to a significant distance from the nucleotide binding site and may be a precursory step to Ca2+ translocation.
用针对巯基的自旋标记物2,2,6,6-四甲基-4-氨基（N-碘乙酰胺）标记肌浆网（SR）的囊泡片段。对剩余巯基残基进行比色滴定并测定未结合的自旋标记物，结果表明在饱和条件下每个酶分子主要有3个残基被标记。只要在标记过程中采取预防措施防止巯基氧化，这种标记只会伴随极小的活性损失。添加ATP会在标记的SR的电子顺磁共振光谱中产生一个新的“高度受限”成分，这是之前研究中未注意到的效应。结果表明，ATP产生的变化是可逆的，并且需要底物结合和Ca2+结合。然而，不需要底物水解。进一步证明，导致光谱变化的标记残基不在ATP结合位点的紧邻区域。很明显，观察到的光谱变化与ATP和Ca2+对ATP酶蛋白的构象效应有关，这与结合时发生的大量自由能变化相关。还表明构象效应从核苷酸结合位点延伸到相当远的距离，并且可能是Ca2+转运的一个前期步骤。
