Slow Passive Diffusion of Orthophosphate between Intact Isolated Chloroplasts and Suspending Medium.

Isolated spinach chloroplasts purified by isopycnic centrifugation in density gradients of Percoll were found to be highly intact, to be devoid of extrachloroplastic contaminations, and to retain a high rate of CO(2)-dependent O(2) evolution.When suspended in a medium which avoided rupture of the envelope, intact purified chloroplasts progressively lost their phosphate content by passive diffusion. This led to a slow decrease in the uptake of labeled 3-phosphoglyceric acid or orthophosphate (Pi) and in the rate of CO(2)-dependent O(2) evolution by isolated chloroplasts. Under these conditions, there was a good correlation between the rate of CO(2)-dependent O(2) evolution and the concentration of Pi in the stroma space. Addition of Pi to the suspending medium at a final concentration of 10 millimolar, which counterpoised the slow efflux of Pi from the chloroplasts, slowed considerably the decrease in the rate of CO(2)-dependent O(2) evolution.