Tzeng C M, Hsu L H, Pan R L
Institute of Radiation Biology, College of Nuclear Sciences, National Tsing Hua University, Hsin Chu, Taiwan, Republic of China.
Biochem J. 1992 Aug 1;285 ( Pt 3)(Pt 3):737-43. doi: 10.1042/bj2850737.
Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.
异硫氰酸荧光素（FITC）用于修饰黄化绿豆（Vigna radiata L.）幼苗液泡膜H(+)-ATPase活性位点的赖氨酸残基。FITC导致膜结合型和可溶性ATPase的酶活性及其相关的H+转运显著失活。SDS/PAGE图谱显示，FITC结合位点位于ATPase的大亚基（A亚基）上。其生理底物ATP、焦磷酸和核苷酸能以ATP＞焦磷酸＞ADP＞AMP＞GTP＞CTP＞UTP的递减顺序大幅阻止抑制作用。FITC的抑制模式相对于ATP而言是竞争性的。ATPase活性的丧失遵循假一级动力学，其抑制常数Ki为0.33 mM，最小失活半衰期为110 s，一级速率常数为0.244 s-1。表观速率常数与FITC浓度的双对数图斜率为0.913，表明失活是由于大亚基催化位点至少一个赖氨酸残基发生反应所致。标记研究表明，每摩尔ATPase掺入约1摩尔FITC就足以完全抑制ATPase。修饰ATPase后FITC发射最大值的增强和蓝移表明，标记的赖氨酸残基位于相对疏水的结构域中。
