Inhibition by nigericin of the light-induced pH change in Rhodospirillum rubrum chromatophores.

An important aspect of energy conservation in mitochondria, chloroplasts, and chromatophores is the temporal and sequential interrelationships between electron transport, proton movements (and the resultant proton gradient), highenergy intermediates or states, and the ultimate formation of adenosine 5'triphosphate (ATP).1-4 At present, two quite dissimilar alternative postulations are under consideration. The first assumes that proton movements are a prerequisite for the establishment of a high-energy state;" 4 the other views proton movements as a consequence of the formation of a high-energy state.2' I Within recent years, a number of antibiotics have been shown to induce ion transport in mitochondria" 6 and in chloroplasts.7-9 These antibiotics are useful to investigate the interrelationship between the proton and alkali metal-cation movements and the mechanism for energy conservation. Data presented in this communication demonstrate that low concentrations of nigericin, an antibiotic which inhibits proton uptake and ATP formation in chloroplasts,7' 8 strongly inhibits the light-dependent proton movements in chromatophores without affecting the rate of ATP formation. These results suggest that proton uptake in chromatophores represents a side reaction not on the pathway of ATP formation. Methods.-Rhodospirillum rubrum, S1, was grown and chromatophores prepared as described previously.'O For measurement of light-induced pH changes, the crude chromatophore preparation was washed twice with 0.35 1M NaCl instead of 0.1 M Tris-Cl, pH 8.0, and 0.311 sucrose. ATP32 formation was assayed as described earlier."1 Bacteriochlorophyll was determined from the in vivo absorption at 880 nm.'2 pH changes in an 8-ml reaction mixture were measured with a Leeds and Northrup model 7405 pH meter with a glass electrode and a Ag/AgCl reference electrode. Red-light illumination was obtained using a Unitron model LKR illuminator with a Corning 2304 red filter, and a CS-69 infrared-absorbing filter. Light intensity was 8 X 105 ergs per square cm per sec. The gas phase was air and the temperature 220. Results.-Typical traces of the pH change observed upon illumination of suspensions of isolated chromatophores are shown in Figure 1. The steadystate level (extent) of the light-induced pH rise or proton uptake attained in a medium containing 0.1 M KCl (Fig. 1A) was rapidly reduced by addition of 4.7 X 10-8 M1 nigericin in the light. The attenuated extent corresponds to a decrease of about 70 per cent from the original extent. Complete reversal of the pH rise occurred in the dark. A second period of illumination again produced a pH rise but only to the inhibited steady-state level. Nigericin inhibited both the apparent rate of formation and the extent of the pH rise. t Von Stedingk and Baltscheffsky'3 have shown that valinomycin stimulates the rate of the light-induced pH rise in chromatophores in a medium containing KCl. This stimulation by valinomycin of the light-induced proton uptake was