Isolated, intact spinach chloroplasts incubated in light in the presence of HC0 a-and NO~-have the greater proportion of their NADP present in the reduced form. The steady state concentration of NADPtt in light in these chloroplasts is significantly higher in tile presence of NO 2 than in its absence. These results invalidate earlier conclusions (Grant and Canvin, 1970) that N0~inhibits photosynthesis by preventing NADP reduction.
In a previous paper from this laboratory (Grant and Canvin, 1970) the inhibitory effect of nitrite on photosynthesis by isolated, intact chloroplasts was reported. NO2-inhibition could be partly relieved by the addition of ribose-5-phosphate but not by phosphoglyeerie acid. On the basis of these experiments it was concluded that the site of NO 2inhibition was likely to lie between the points at which these two compounds were metabolised in the Calvin cycle. Additional experiments carried out with broken chloroplasts (grana) prepared by the method of Whatley and Arnon (1963) and supplemented with chloroplast extract, indicated that NADP reduction and 03 production when NADP was the electron aeceptor were both inhibited by NO2-. On this basis it was suggested that NO 2-acted by inhibiting NADP reduction in the intact chloroplast.
Subsequent experiments, reported here, have shown that this latter interpretation is incorrect. Insofar as we can determine, NO 2-does not inhibit the reduction of NADP either in the granal system or in the intact chloroplast. Measurements made on the steady state level of NADPtI in intact chloroplasts, incubated in the presence of nitrite in the light, indicate that significantly higher levels of NADPH are present when compared to controls in which nitrite is absent.
The methods used for the growth of spinach and the isolation of intact chloroplasts have been described in a previous paper (Grant et al., 1970). The method