Characterization of Nucleotide Binding Sites of the Isolated H+-ATPase from Spinach Chloroplasts, CF0F1

Soluble purified CF0F1 from chloroplasts was either oxidized or reduced and then incubated with [α-32P]ATP in the presence or in the absence of Mg2+. Depending on the conditions of incubation, the enzyme showed different tight-nucleotide binding sites. In the presence of EDTA, two sites bind [α-32P]ATP from the reaction medium at different rates. Both sites promote ATP hydrolysis, since equimolar amounts of [α-32P]ATP and [α-32P]ADP are bound to the enzyme. In the presence of Mg2+, only one site appears during the first hour of incubation, with characteristics similar to those described in the absence of Mg2+. However, after this time a third site appears also permitting binding of ATP from the reaction medium, but in this case the bound ATP is not hydrolyzed. Covalent derivatization by 2-azido-[α-32P]ATP was used to distinguish between catalytic and noncatalytic sites. In the presence of Mg2+, there are at least three distinct nucleotide binding sites that bind nucleotide tightly from the reaction medium: two of them are catalytic and one is noncatalytic.