E. W. YEMM 



It may be noted that nitrates, nitrites and, to a less extent, hydroxylamine increase 

 the rate of respiration in barley roots. But here there is evidence of greater complexity 

 compared with the effects of ammonium salts. The respiratory quotient rises con- 

 siderably above unity with nitrates and nitrites, suggesting that they act as hydrogen 

 acceptors in the oxidation mechanism. The action of hydroxylamine is complicated 

 by its toxic effects even at low concentrations. 



An account of the experiments with barley roots has been given by Willis (1950, 



*950- 



THE PRODUCTS OF NITROGEN ASSIMILATION 



In an attempt to identify some of the reactions associated with the high rates of 

 cellular oxidation, the products formed in the cells during the early phases of assimila- 

 tion have been investigated. For this purpose analyses of the soluble and insoluble 

 nitrogenous constituents were made, so that it is possible to give some account of the 

 changes of amino-acids and proteins. It has been found consistently in experiments 

 with both yeast and root tissues that glutamic acid and its amide, glutamine, are 

 rapidly formed in the early stages of assimilation, corresponding fairly closely in time 

 with the highest rates of cell oxidation. The results of an experiment in which yeast 

 cultures were supplied with ammonium phosphate are given in Figure 3. 



During the first 30 min. a marked increase of glutamic acid and glutamine occurs 

 with a smaller accumulation of alanine; together these constituents account for 

 about 70 per cent, of the total nitrogen assimilated by the cells over the initial period. 

 Subsequently, they are maintained at a fairly steady or falling level. There is a gra- 

 dual formation of other, as yet unidentified, soluble-N, and a small increase in the 

 tripeptide, glutathione, was observed in some of these experiments (Yemm and 

 Folkes, 1954). The progressive rise in complex insoluble-N in the cells indicates an 

 active synthesis of protein during the course of the experiment. 



At present, the identification of amino-acids and amides in the yeast rests mainly 

 on separations by paper chromatography, or on the use of specific enzymes for ana- 

 lysis. Most of the estimates of glutamic acid and glutamine were made by means of 

 glutaminase and glutamic decarboxylase, prepared from Clostridium welchii by the 

 method described by Krebs (1948). 



Analytical data from a similar experiment with barley roots are shown in Figure 4. 

 On a much longer time-scale they have several features in common with the data 

 for yeast. Ammonia-N accumulates temporarily in the roots, but at first the main 

 product of assimilation is glutamine, which makes up about 80 per cent, of the 

 ammonia utilized in the first 12 hours. Asparagine, the other common plant amide, 

 increases at a later stage; together the two amides account for almost all of the free 

 amino-N in the tissues. 



In some of the experiments with barley roots it has been possible to obtain more 

 decisive evidence of the primary synthesis of amides from ammonia by using isotopic 

 nitrogen to trace the products of assimilation in the cells. Ammonium phosphate, 

 containing about 30 per cent, excess of 15 N, was supplied to the roots and its incorpor- 

 ation into the amide and other nitrogen fractions was estimated after varying periods 

 of assimilation. The abundance of the isotope in some of the different fractions is 

 shown in Figure 5. 



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