Effect of Ammonium Chloride and Methionine Sulfoximine on the Acetylene Reduction of Detached Root Nodules of Peas ( Pisum sativum )

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Abstract
Acetylene-reducing activity of detached pea nodules was determined by submerging the nodules in buffer solution [tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4] containing 100 mM sodium succinate and incubating under a gas phase of 90% O2 and 10% C2H2. The nitrogenase activity was 4 to 8 μmol of C2H4 formed per g of nodule fresh weight per h and remained constant for at least 4 h. Addition of NH4Cl to the buffer solution (at a concentration of 10 mM or more) resulted in a significant decrease of nitrogenase activity, which was more pronounced at higher concentrations of ammonium chloride. The inhibition of nitrogenase activity by NH4Cl was reversible; when the NH4Cl-containing buffer solution was replaced by buffer without NH4Cl, the original activity was partly restored. Treatment of the nodules with NH4Cl had almost no effect on the amount of nitrogenase, as measured by the acetylene-reducing activity of ethyl-enediaminetetraacetate-toluene-treated bacteroid suspensions. The effect of NH4Cl was largely eliminated by simultaneous addition of 10 mM methionine sulfoximine to the assay solution. This suggests that the assimilation of ammonium ions by glutamine synthetase controls the functioning of nitrogenase activity in the nodules. However, no effect of glutamine, glutamate, or aspartate on the acetylene reduction by detached nodules could be detected.