Effect of digitonin on photophosphorylation and light-induced H+ uptake in isolated spinach chloroplasts

The effects of the presence of digitonin in the reaction mixture on photophosphorylation, light-induced H+ uptake, and the size of isolated spinach chloroplasts were studied. Digitonin inactivated photosystem II and dissipated light-induced pH increase without affecting the photophosphorylation driven by photosystem I. Digitonin increased the concentration of NH4Cl required for uncoupling. This effect of digitonin was diminished by valinomycin which barely affects phosphorylation by itself. Although such properties have all been observed with digitonin subchloroplast particles, digitonin at the concentration required for acquiring such properties had little effect on the size of chloroplasts. These results suggest that the characteristic effects of digitonin subchloroplast particles are caused by thylakoid membranes penetrated by digitonin but not by the size of membrane vesicles. Both in chloroplasts with digitonin in the reaction mixture and in digitonin subchloroplast particles, N,N'-dicyclohexylcarbodiimide, purine nucleoside di- and tri-phosphates, and organic bases with low pKa values (pyridine and aniline) made the light-induced pH increase detectable, and this was lost upon the addition of proton ionophores. These results suggest that the lack of the light-induced pH increase by chloroplasts with digitonin in the reaction mixture and by digitonin subchloroplast particles is caused both by a membrane leaky to protons and by the loss of internal buffering capacity of thylakoids. Furthermore, the phosphorylation of chloroplasts with digitonin in the reaction mixture and of digitonin subchloroplast particles is considered to be driven by the proton motive force as is that of chloroplasts without digitonin.