Abstract
Methods have been developed for identifying the pathway of assimilation of N2-derived nitrogen. The products of fixation of 13N-labeled nitrogen gas ([13N]N2), and the distribution of 13N within glutamine, were determined after short periods of labeling (approximately 1 to 120 s) and also in pulse-chase experiments. Ammonia, the amide nitrogen of glutamine, and the alpha-amino nitrogen of glutamate, in that order, were the first observed products of fixation of N2 by the cyanobacterium (blue-green alga), Anabaena cylindrica. This sequence of the formation of nitrogenous products was confirmed by the use of inhibitors. The presence of 1 mM methionine sulfoximine permitted continued formation of 13NH3, while virtually preventing 13N-labeling of amino acids. In the presence of 1 mM azaserine, glutamine was labeled, but not other amino acids. Our observations demonstrate unequivocally that N2-derived nitrogen fixed by this organism is metabolized initially by the glutamine synthetase/glutamate synthase pathway.
Highlights
From the Cyclotron Laboratory and Department Michigan 48824 of Physics, Michigan State University, East Lansing, Methods have been developed for identifying the pathway of assimilation of N,derived nitrogen
Our observations demonstrate unequivocally that N*-derived nitrogen fixed by this organism is metabolized initially by the glutamine synthetase/glutamate synthase pathway
Most studies of the pathways considered potentially capable of initial metabolism of nitrogen in cyanobacteria have been enzymological
Summary
Our observations demonstrate unequivocally that N*-derived nitrogen fixed by this organism is metabolized initially by the glutamine synthetase/glutamate synthase pathway. These changes, being slow, could have been indirect, but they did support the idea that N,-derived NH, is metabolized initially by the glutamine synthetase/glutamate synthase pathway. Studies of products of fixation of “N-labeled nitrogen gas, by Magee and Burris [17], showed that in hydrolysates of protein from the cyanobacterium Nostoc muscorum, the highest specific activity (atom per cent excess) of 15N was in glutamic acid. The period of labeling was such that several organic pr0duct.s had become labeled, the results were consistent with operation of the glutamine synthetase/glutamate synthase pathway In such studies, as increasingly shorter intervals of labeling are employed, increasingly smaller amounts of fixed “N must be detected and quantified.
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