Significant quantities of endogenous GDP and ADP are present on catalytic sites of the F1-ATPase isolated from M. lysodeikticus in the absence of added nucleotides.
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.
Mileykovskaya
E I
EI
Department of Chemistry, University of California, San Diego, La Jolla.
Kormer
S S
SS
Allison
W S
WS
D000244:Adenosine Diphosphate / Q000032:analysis* / Q000096:biosynthesis; D000255:Adenosine Triphosphate / Q000032:analysis*; D001665:Binding Sites; D002118:Calcium; D006153:Guanosine Diphosphate / Q000032:analysis* / Q000096:biosynthesis; D008274:Magnesium; D008837:Micrococcus / Q000201:enzymology*; D010710:Phosphates; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D010710:Phosphates; D006153:Guanosine Diphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium; D002118:Calcium