Factors affecting the ADP/O ratio in isolated chloroplasts.

(1) The effect of gradual disruption of the outer membrane of intact chloroplasts on CO2 fixation, electron transport and phosphorylation was investigated. The results suggested that whilst ferricyanide and substrate amounts of ADP enter intact chloroplasts only very slowly, methyl viologen rapidly penetrates the outer membrane. (2) Preparatwons of intact pea chloroplasts had an ATP-consuming reaction which resulted in decreased ADP/O ratios when noncyclic electron transport was measured after disruption of the outer membrane. The ATP-consuming reaction was removed into the supernatant after washing the disrupted chloroplasts. The resulting washed chloroplasts gave ADP/O ratios of 1.5-1.6 for ferricyanide and 1.9-2.0 for methyl viologen. (3) Preparations of intact spinach chloroplasts had lower activity of the ATP-consuming reaction and gave similar ADP/O ratios to washed pea chloroplasts. The ADP/O ratios of spinach chloroplasts did not alter significantly after washing. (4) An investigation of the effect of various assay conditions on the ADP/O ratio showed that the phosphate concentration was critical in obtaining optimal values for ADP/O ratio. Decreasing the phosphate concentration below 10 mM decreased the ADP/O ratio significantly. (5) It is suggested that the maximum ADP/O ratio of chloroplasts is 2.0 but that lower values can be obtained in the presence of an ATP-consuming reaction, under suboptimal assay conditions or where the chloroplasts are structurally damaged.