Ammonia assimilation evidence

As implied in the introduction, various types of evidence can be used to resolve the question as to which pathway of ammonia assimilation is operating. In this section we wish to critically appraise the nature of such evidence. In doing this it must be borne in mind that, as in all scientific proofs, we are trying to reject invalid hypotheses rather thanprove the validity of any given one the best that we can hope to do is to show that all the available evidence is compatible with only one hypothesis. 

Certain organisms, for example fungi of the higher orders, possess two GDH enzymes, one specific for NAD, the other for NADP. A. variety of evidence (Smith et al., 1975) has indicated that in these cases, the NADP-linked enzyme is predominantly concerned with ammonia assimilation, while the NAD-linked enzyme is predominantly concerned with deamination of glutamate to 2-oxcglutarate. Control of the relative rates of amination and deamination to reactions in the cell has been observed to be exerted through control of the relative levels of the biosynthetic NADP enzyme and the catabolic NAD enzyme. This control mechanism therefore bypasses the necessity for the individual enzymes to possess elaborate allosteric control mechanisms. 

An alternative approach to estimating the metabolic capabilities of chloroplasts entails measurement of the light-dependent metabolism of radioactive tracers. Using isolated pea chloroplasts. Mills and Wilson (1978a) found that lysine, methionine, threonine, and isoleucine were synthesized from [ C]aspartate. Further evidence that aspartate was being metabolized via the anticipated pathways was provided by the demonstration that the synthesis of homoserine was inhibited by lysine and threonine (Lea et al., 1979). These results, combined with those relating to enzyme localization, lead to the concept that chloroplasts contain a complete functional sequence of enzymes which can facilitate the synthesis of the aspartate family and at least some of the branched-chain amino acids. This is consistent with the importance of chloroplasts in ammonia assimilation (Miflin and Lea, this volume. Chapter 4) and with the evidence that protein can be synthesized from CO2 in isolated plastids (Shepard and Leven, 1972 Huberer al., 1977). The actual fraction of [ ]02 which is utilized for amino acid biosynthesis in isolated plastids is usually quite small. Thus, reactions which normally occur outside of chloroplasts are considered to be of major importance in the synthesis of carbon skeletons such as oxaloacetate or pyruvate (Kirk and Leech, 1972 Leech and Murphy, 1976). 

There is thus good evidence from the inhibitor studies that glutamine synthetase plays a key role in the process of photorespiration. Possible explanations for the rapid fall in photosynthetic rates will be discussed in the following section, which describes the properties of mutants lacking enzymes of the ammonia assimilation pathway. 

In summary, there appears to be evidence for both mechanisms of NH3/NH4 transport in bacterial cells. Passive difiFusion of NH3 would not generally lead to net accumulation by cells with an alkaline interior pH relative to the medium pH. Passive diffusion would be rate-limited by the low level of NH3 available at physiological pH and by the solubility properties of the cell membrane. NH4 active transport would be required for net ammonium uptake. However, net nitrogen accumulation could be accomplished by rapid assimilation of internal ammonia into amino acids. Net accumulation might be limited by the balance of the two processes uptake by active transport vs. loss by passive diffusion. 

The location of the enzymes of anunonia assimilation in organelles known to carry out the pathway is a necessary link in the chain of evidence but again it is all too easy to obtain false positive evidence or to produce negative results. Unfortunately there are very many poor papers in the literature on enzyme localization in plant tissues and there is a need for careful critical appraisal of such results. Finally it must be emphasized that because an enzyme is present it does not mean that it is functional in any given pathway or that it works in the direction it is usually assayed. Despite this, one of the most frequently heard (if not written) arguments in favor of a role for GDH in assimilation is that because the enzyme is there and can be readily assayed in the amination reaction, (albeit using strong concentrations of ammonia) that must be its function. ... 

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