54 NITROGEN METABOLISM 



closely related to one of the natural intermediates. The high 

 concentration of N^^ in glutamic acid was indicative of its 

 importance in the pathway of Na-fixation. Such a conclusion 

 was in keeping with the results of previous work with other 

 organisms in which it had been established that glutamic 

 acid occupies a key position in amino-acid metabolism and 

 could be synthesized from NHg and a-ketoglutarate by the 

 glutamic acid dehydrogenase system (cf. Chap. II). 



Winogradsky was the first to note, during his experiments 

 with CI. pasteurianum, that ammonium salts inhibited N2- 

 fixation. Azotobacter responds in a similar fashion, both to 

 ammonium salts and to compounds (urea, asparagine) which 

 it can convert to NH3 [46]. Since nitrate and nitrite were 

 only inhibitory after a lag period, it was inferred that they 

 were not utilized by Azotobacter until the appropriate 

 enzymes had been formed by adaptation, and in fact there 

 was no lag period with cells which had been grown in media 

 containing NO7 and NO^ • Organic nitrogen compounds, 

 e.g. aspartic and glutamic acids, were only moderately 

 inhibitory, perhaps not unexpectedly since they are probably 

 more concerned with intermediary metabolism rather than 

 with the initial steps of the fixation mechanism. 



Further isotope experiments provided additional evidence 

 that the fixation of N 2 by CI. pasteurianum, Chromatium and 

 Nostoc muscorum is accomplished by essentially the same 

 route as in Az. vinelandii [43]. In each of these organisms 

 after exposure to Ng-^^ or N^^H4 , the dicarboxylic amino- 

 acids, and in particular glutamic, contained the highest 

 concentration of N^^. Furthermore, the results of the ex- 

 periments with CI. pasteurianum. [51] proved to be in some 

 respects comparable with those of Virtanen with nodulated 

 leguminous plants. Under certain conditions, the fixation 

 of N 2 by CI. pasteurianum and by the symbiotic system was 

 accompanied by the excretion of nitrogenous compounds 

 into the environment; the anaerobe excreted mainly NH3 

 whilst the plant excreted aspartic acid, ^-alanine and a small 

 amount of an oxime, identified as oximinosuccinic acid. 

 These substances are regarded as being products, not of 

 catabolism, but of the processes directly concerned with 



