Mileykovskaya E I, Kormer S S, Allison W S
Department of Chemistry, University of California, San Diego, La Jolla.
Biochim Biophys Acta. 1992 Mar 13;1099(3):219-25. doi: 10.1016/0005-2728(92)90030-6.
The F1-ATPase from Micrococcus lysodeikticus is isolated in the absence of exogenous nucleotides. After removing loosely bound nucleotides from the isolated enzyme by gel permeation chromatography, analysis for tightly bound nucleotides revealed in 14 experiments 0.4 +/- 0.1 mol ADP, 0.5 +/- 0.2 mol GDP, and 0.8 +/- 0.2 mol ATP per mol of F1. Incubation of the isolated enzyme with Mg2+ or Ca2+ did not alter the endogenous nucleotide composition of the enzyme, indicating that endogenous ATP is not bound to a catalytic site. Incubation of the enzyme with P(i) decreased the amount of tightly bound ADP and GDP but did not effect the ATP content. Hydrolysis of MgATP in the presence of sulfite raised the tightly bound ADP and lowered tightly bound GDP on the enzyme. In the reciprocal experiment, hydrolysis of MgGTP in the presence of sulfite raised tightly bound GDP and lowered tightly bound ADP. Turnover did not affect the content of tightly bound ATP on the enzyme. These results suggest that endogenous ADP and GDP are bound to exchangeable catalytic sites, whereas endogenous ATP is bound to noncatalytic sites which do not exchange. The presence of endogenous GDP on catalytic sites of isolated F1 suggests that the F0F1-ATP synthase of M. lysodeikticus might synthesize both GTP and ATP under physiological conditions. In support of this hypothesis, we have found that plasma membrane vesicles derived from M. lysodeikticus synthesize [32P]GTP from [32P]P(i) using malate as electron donor for oxidative phosphorylation.
从溶壁微球菌中分离出的F1 - ATP酶是在没有外源核苷酸的情况下进行的。通过凝胶渗透色谱法从分离出的酶中去除松散结合的核苷酸后，在14次实验中对紧密结合的核苷酸进行分析，结果显示每摩尔F1含有0.4±0.1摩尔ADP、0.5±0.2摩尔GDP和0.8±0.2摩尔ATP。将分离出的酶与Mg2 +或Ca2 +一起孵育不会改变酶的内源性核苷酸组成，这表明内源性ATP不与催化位点结合。将酶与无机磷酸（Pi）一起孵育会减少紧密结合的ADP和GDP的量，但不影响ATP含量。在亚硫酸盐存在下MgATP的水解增加了酶上紧密结合的ADP并降低了紧密结合的GDP。在反向实验中，在亚硫酸盐存在下MgGTP的水解增加了紧密结合的GDP并降低了紧密结合的ADP。周转率不影响酶上紧密结合的ATP的含量。这些结果表明，内源性ADP和GDP与可交换的催化位点结合，而内源性ATP与不可交换的非催化位点结合。分离出的F1的催化位点上存在内源性GDP表明，溶壁微球菌的F0F1 - ATP合酶在生理条件下可能同时合成GTP和ATP。为支持这一假设，我们发现源自溶壁微球菌的质膜囊泡以苹果酸作为氧化磷酸化的电子供体，从[32P]Pi合成[32P]GTP。
