Chow W M, Tzeng C M, Chen C S, Kuo S Y, Wang M Y, Pan R L
Institute of Radiation Biology and Institute of Nuclear Sciences, College of Nuclear Sciences, National Tsing Hua University, Hsin Chu, 30043 Taiwan, Republic of China.
Plant Physiol. 1992 Jan;98(1):44-52. doi: 10.1104/pp.98.1.44.
The 2',3'-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H(+)-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH(4). The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K(i) of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s(-1). The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [(3)H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.
ATP的2',3'-二醛衍生物（二醛-ATP）已被证明是来自黄化绿豆幼苗（绿豆）液泡膜H(+)-ATP酶ATP结合位点的亲和标记物。二醛-ATP导致膜结合型和可溶性ATP酶的酶活性及其相关的质子转运显著失活。这种失活是可逆的，但可以被NaBH(4)稳定。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱显示，二醛-ATP结合位点位于ATP酶的大亚基（A亚基）中。其抑制作用可被其生理底物ATP、焦磷酸和核苷酸按以下递减顺序显著保护：ATP＞焦磷酸＞ADP = AMP＞GTP＞CTP = UTP。Lineweaver-Burk图表明，抑制模式相对于ATP是竞争性的。ATP酶活性的丧失遵循假一级动力学，K(i)为4.1毫摩尔，最小失活半衰期为20秒，假一级速率常数为0.035 s(-1)。表观速率常数与二醛-ATP浓度的双对数图给出的斜率为0.927，表明失活是由于大亚基催化位点至少一个赖氨酸残基的反应所致。用[(3)H]二醛-ATP进行的标记研究表明，每摩尔ATP酶掺入约1摩尔二醛-ATP足以完全抑制ATP酶。提出了液泡ATP酶酶促机制的不等价亚基工作模型。
