Nitrite rotenone

Fig. 11 Effect of specific inhibitors of the respiratory chain on the nitrite reductase activity of rat liver mitochondria. Mitochondria were incubated with nitrite for 2 h under argon in the presence of succinate or glutamate/malate (Glu/Mal). Nitric monoxide derived from nitrite was trapped with hemoglobin. The concentration of NO-hemoglobin complexes was measured using low temperature ESR spectroscopy. Other details are described in Kozlov et al. [46]. CON control, ROT rotenone, TTFA thenoyltrifluoroacetone, MYX myxothiazol, AA antimycin A...

Canvin and Woo (1979) reported that under certain conditions Antimycin A (mitochondrial ATP site II inhibitor) was more effective in enhancing nitrite accumulation (75% of anaerobic control) by leaf discs than either amytal or rotenone (ATP site I inhibitors). In plant mitochondria, the malate dehydrogenase located on the outside of the inner membrane is capable of utilizing external NADH. They infer that in leaves under dark aerobic conditions, the mitochondria effectively compete with nitrate reductase for cytoplasmic NADH. Confirmation of this competition was afforded by a reconstituted system consisting of mitochondria, nitrate reductase, nitrate and NADH or NAD, malate, and malate dehydrogenase (Reed and Hageman, 1977). Nitrite production under aerobic conditions was 10% that observed under anaerobiosis. 

Other experimental data indicate that the oxygen is affecting nitrite accumulation by increasing nitrite reduction. When conditions are optimized for entry of rotenone (mitochondrial site I inhibitor) into leaf secticHis the leaves under aerobic conditicHis accumulated one-half as much nitrite as those without rotenone under anaerobic conditions (Hageman et al., 1980b Reed and Hageman, 1977). The rotenone treatment reduced respiration by... 

Because rotenone does not block mitochondrial ATP site II (site of entry of cytoplasmic NADH) these data infer that the nitrite rather than nitrate reduction is affected. Nitrite accumulated in excised leaves (gaseous milieu) under nitrogen is rapidly lost upon transfer to air (Jones and Sheard, 1978). When only the base of excised leaves are placed in a small amount of water or water containing DNF or FCCP (carbonyl-chlorideptri-fluoromethoxyphenylhydrazone) nitrite accumulation is unaffected by the treatments, under anaerobic conditions. Upon transfer to air, leaves treated with DNP or FCCP show relatively small amounts of loss of nitrite in comparison with controls (Hageman et al., 1980b). 

Estimation of Extract Content. Dilute the sample with acetone so that the final solution contains about 0-01 per cent of extract. Run 1 ml of this solution into a 25-ml conical flask containing 10 ml of concentrated sulphuric acid containing 0-01 per cent of sodium nitrite at approximately 20°. Immediately place the flask in a water-bath maintained at 40° for fifteen minutes whereupon a reddish-purple colour develops. Transfer the flask to a water-bath at 20 ° for a period of five minutes and match the colour thirty minutes after the initial mixing of the acetone solution and the acid, against the colour developed by 1 ml of standard rotenone solution, which has been similarly treated. Prepare a blank by heating and cooling 1 ml of acetone with 10 ml of the sulphuric/nitrous acid solution. 

Estimation of Rotenone Content. Dilute the sample with acetone so that the final solution contains about 0 02 per cent of extract. Pipette 1 ml of this solution into a dry 25-ml Erlenmeyer flask and add 1 ml of acetone. Add 2 ml of freshly prepared alkaline sodium nitrite solution, (one volume of 40 per cent w/v potassium hydroxide solution and seven volumes of 0 1 per cent sodium nitrite in 95 per cent ethanol), and place the flask in a water-bath at 20°. After seven minutes, add 5 ml of 25 per cent v/v sulphuric acid solution, stopper, shake sufficiently to mix, and return the flask at once to the water-bath. The bluish-red colour which develops reaches a maximum after fifteen minutes and is then stable for about one hour. 

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