From uni-site to multi-site ATP synthesis in thylakoid membranes.
The membrane-bound H(+)-ATPase from chloroplasts, CF0F1, was brought into the active, reduced state by illumination in the presence of thioredoxin and dithiothreitol. The endogenous nucleotides were removed by a washing procedure so that the active, reduced enzyme contained one tightly bound ATP per CF0F1. When [14C]ADP was added in substoichiometric amounts during continuous illumination, ADP was bound to the enzyme, phosphorylated and released as [14C]ATP, i.e., the tightly bound ATP was not involved in the catalytic turnover ('uni-site ATP-synthesis'). The rate constant for ADP binding was k = (2.0 +/- 0.5) x 10(6) M-1 s-1. The rate of ATP synthesis was measured as a function of the ADP concentration from 8 nM up to 1 mM in the presence of 2 mM phosphate during continuous illumination. A linear increase of the rate was observed up to 100 nM. Above this concentration a supralinear increase was found, indicating the occupation of a second ADP-binding site. A plateau was reached between 1.5 microM and 2.3 microM ADP with a rate of vpl = 3.7 s-1. The half-maximal rate from this plateau was observed at 780 nM. Above 2.3 microM ADP up to 1 mM ADP the data were described by Michaelis-Menten kinetics (vmax = 80 s-1; apparent KM = 32 microM). These results indicated the participation of at least two different ADP binding sites in ATP synthesis catalyzed by the membrane-bound CF0F1.
Labahn
A
A
Biologisches Institut, Universität Stuttgart, Germany.
Gräber
P
P
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D002736:Chloroplasts / Q000378:metabolism*; D007700:Kinetics; D006180:Proton-Translocating ATPases / Q000378:metabolism
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases