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利用铬三磷酸腺苷对绵羊肾髓质(钠 + 钾) - 三磷酸腺苷酶的三磷酸腺苷结合位点进行表征。

Characterization of ATP binding sites of sheep kidney medulla (Na+ + K+)--ATPase using CrATP.

作者信息

Grisham C M

出版信息

J Inorg Biochem. 1981 Feb;14(1):45-57. doi: 10.1016/s0162-0134(00)80013-6.

DOI:10.1016/s0162-0134(00)80013-6
PMID:6260898
Abstract

The nucleotide substrate sites of sheep kidney medulla (NA+ + K+)-ATPase are characterized using CrATP, a paramagnetic, substitution-inert substrate analogue probe. The paramagnetic effect of CrATP on 1/T1 of water protons of water protons is enhanced upon complexation with the enzyme. Titrations of the enzyme with CrATP in the presence of Na+ and K+ yielded characteristic enhancements for the binary enzyme-CrATP and ternary enzyme-Mg2+-CrATP complexes of 3.3 and 3.6 and dissociation constants for CrATP of 5 and 12 microM, respectively. Substitution of Li+ for K+ in these titrations did not substantially alter the titration behavior. From the frequency dependence of 1/T1, the correlation time, tau c, for the dipolar water proton-CrATP interaction is 2.7 x 10(-10) sec, indicating that tau c is dominated by tau s, the electron spin relaxation time of Cr3+. The paramagnetic effect of enzyme-bound Mn2+ on 1/T1 of water protons decreases upon the addition of CrATP. Titration of the binary enzyme-Mn2+ complex with CrATP decreases the characteristic enhancement due to Mn2+ from 6.6-8.0 to 1.5. The failure to observe free Mn2+ epr signals in solutions of the ATPase, Mn2+, and CrATP demonstrate that this decrease in epsilon Mn is due to cross-relaxation between Mn2+ and Cr3+ bound simultaneously to the enzyme, and not to displacement of Mn2+ from the enzyme by CrATP. The relaxation rate, 1/T1, of 7Li+ is increased upon addition of CrATP to solutions of the ATPase, indicating that the sites for Li+ and CrATP are close on the enzyme. A Cr3+-Li+ distance of 4.8 +/- 0.5 angstrom is calculated from that data.

摘要

利用顺磁性、取代惰性底物类似物探针CrATP对绵羊肾髓质(Na⁺ + K⁺)-ATP酶的核苷酸底物位点进行了表征。CrATP与该酶络合后,其对水质子1/T1的顺磁效应增强。在Na⁺和K⁺存在的情况下,用CrATP对该酶进行滴定,得到二元酶-CrATP和三元酶-Mg²⁺-CrATP络合物的特征增强值分别为3.3和3.6,CrATP的解离常数分别为5和12 μM。在这些滴定中用Li⁺取代K⁺并没有显著改变滴定行为。根据1/T1的频率依赖性,偶极水质子-CrATP相互作用的相关时间τc为2.7×10⁻¹⁰秒,这表明τc由Cr³⁺的电子自旋弛豫时间τs主导。加入CrATP后,酶结合的Mn²⁺对水质子1/T1的顺磁效应降低。用CrATP滴定二元酶-Mn²⁺络合物会使Mn²⁺引起的特征增强从6.6 - 8.0降至1.5。在ATP酶、Mn²⁺和CrATP的溶液中未观察到游离Mn²⁺的电子顺磁共振信号,这表明εMn的这种降低是由于同时结合到酶上的Mn²⁺和Cr³⁺之间的交叉弛豫,而不是CrATP将Mn²⁺从酶上取代。向ATP酶溶液中加入CrATP后,⁷Li⁺的弛豫速率1/T1增加,这表明Li⁺和CrATP在酶上的位点相近。根据该数据计算出Cr³⁺-Li⁺距离为4.8±0.5埃。

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