Evidence for two tightly bound nucleotides on thylakoid-bound chloroplast coupling factor 1 (CF 1): One ADP — Exchangeable upon illumination - And one non-exchangeable MgATP

Abstract

Isolated chloroplast coupling factor 1 contains a tightly bound ADP (which is exchangeable for labeled medium ADP or ATP) but contains two tightly bound nucleotides (one ADP and one ATP) after incubation with radioactive ATP in the presence of magnesium ions (Bruist, M.F. and Hammes, G.G. (1981) Biochemistry 20, 6298–6305). On the membrane-bound enzyme, only one nucleotide (mainly ADP) was found to be exchangeable during incubation with labeled ATP in the light (Bickel-Sandkötter, S. and Strotmann, H. (1981) FEBS Lett. 125, 188–192). This paper demonstrates that membrane-bound CF 1 contains another tight binding site, occupied with non-exchangeable ATP in addition to the tight ADP site (exchangeable in the light). During isolation, the incubation of CF 1 with EDTA causes the loss of the tightly bound ATP so that the normal isolation procedure yields an enzyme species with only one tightly bound nucleotide molecule (ADP). If EDTA is omitted during isolation, the additional ATP can be detected easily by the luciferin-luciferase method. Reconstitution of isolated CF 1 containing two tightly bound nucleotides with CF 1-free membranes results in a membrane-bound coupling factor with two radioactive nucleotides. Because the exchange of this ATP is very slow, it is assumed that the tightly bound ATP has structural rather than catalytic or regulatory functions.