No abstract

Of the yeasts that ferment D-glucose anaerobically, over 4076 can use certain glycosides and D-galactose oxidatively, but cannot ferment them. This phenomenon is here called the Kluyver effect. More than half the yeast species described which exhibit this effect do so with more than one substrate. Yeasts showing the effect with maltose, cellobiose and D-galactose were compared with fermenting strains, to determine whether enzyme inactivation or cessation of sugar uptake was responsible. The different responses of the yeasts to anaerobic conditions, with respect to their enzymic activity, sugar uptake and COz production, consistently showed that the Kluyver effect resulted from the requirement of transport for oxygen, and this seems to be the common explanation throughout the yeasts.

Work demonstrating the operation of a photorespiratory N cycle in Chlamydomonas is described. NH3 release by this process is light dependent, sensitive to changes in pO2 and pCO2, and abolished by a photosystem II inhibitor. Evidence is presented which shows that this NH3 derives its N from protein rather than from freshly synthesised glutamate. Protein turnover is shown to provide amino-N at a rate sufficient to account for the highest photorespiratory N excretion observed suggesting that changes in excretion can be accounted for by increased catabolism of normally recirculating amino acids. It is equally possible however that a direct link between photorespiration and protein turnover exists, increased NH3 excretion resulting from enhanced protein turnover. The data suggest that if similar mechanisms operate in higher plants, previous estimates of the amount of N recycled in photorespiration may have been too high.

Ammonium phosphate labelled with 15 N has been used in a single quantitative experiment to trace the pathways of ammonia assimilation and amino acid synthesis in food yeast. Methods have been developed and are briefly described, whereby the free amino acids and amides, and the amino acid residues of the proteins, may be extracted from the yeast, separated by ion-exchange chromatography, quantitatively estimated, and the nitrogen of their α -amino groups specifically liberated for isotopic analysis. The yeast was cultured in shake-flasks on a minimal medium containing glucose, ammonium phosphate and mineral salts. By analysis of cells removed from the culture at various times it was shown that the percentage composition remained sensibly constant throughout the part of the exponential phase investigated, and hence the yeast was assumed to be in steady-state growth. For the isotopic experiment the yeast culture was transferred to medium containing ( 15 NH 4 ) 2 HPO 4 and samples where then removed at intervals for analysis of the free and protein amino acids and for measurement of their 15 N-abundance. After 30 min the remaining yeast was transferred back into unlabelled medium and further samples were then taken. This double-transfer procedure was used in order to permit more stringent tests of metabolic relationships to be made in the subsequent kinetic analysis. The quantitative analysis of the isotopic data was made by comparison with a model reaction system. The model consists of a series of branching reaction chains linking steady-state pools of intermediates from which material is randomly withdrawn in subsequent reactions; primary products of nitrogen assimilation can give rise to secondary and tertiary derivatives which, as amino acids, can act as precursors in protein synthesis. A series of kinetic equations have been derived, relating the isotopic abundance of a component in the model to the rates of the various reactions involved in its biosynthesis. By substituting numerical values in these equations and comparing the results with the experimental data it has been possible to assign to each amino acid a position in the model and to make an estimate of its rate of synthesis. This estimate can then, as a further test, be compared with the rate known to be necessary to maintain steady-state growth. The kinetic analysis indicates that glutamic acid and glutamine are the only amino acids to derive their α -nitrogen directly from ammonia; they are synthesized at a rate sufficient to provide all the α -amino nitrogen required for growth of the yeast but not to meet the total nitrogen requirements, so that other pathways for the assimilation of nitrogen must also operate. All the other amino acids apparently derive their α -amino-N from glutamic acid, many of them directly. For some of the amino acids, the labelling of the residues in the protein is consistent with their having come directly from the pool of free amino acid; this emphasizes the very small size of any pools of intermediates between amino acid and protein. For other amino acids a more complex relationship has been observed between the free amino acid pool and the proteins; the data are best interpreted by assuming that not all of the pool is available as an intermediate in protein synthesis, some of it being spatially separated and not further metabolized. This separate pool is here called a storage pool and is envisaged as functioning as part of the regulatory mechanisms of the cell by removing any small overproduction of amino acid. The results are further considered in relation to known pathways of amino acid biosynthesis in micro-organisms. The data for alanine, aspartic acid, glycine, leucine, isoleucine, valine, tyrosine and phenylalanine are consistent with these amino acids, having been formed directly by transamination from glutamic acid. Similar transaminations, but followed by other reactions, can account for the synthesis of histidine, lysine, serine and methionine; there is no evidence for alanine-hydroxypyruvate transamination in serine synthesis or for the operation of the cystathionine pathway to methionine. Threonine does not apparently derive its nitrogen from aspartic acid in this experiment, and the operation of the pathway from aspartic acid via homoserine to threonine is questioned for yeast grown on a minimal medium. The very low isotopic abundance in free ornithine suggests that this amino acid pool, or at least 97 % of it, is not an intermediate in arginine synthesis. Other mechanisms for the formation of citrulline and arginine are put forward. Proline is apparently formed from glutamic acid. The results are generally at variance with the concept of amino acid families proposed by the Carnegie Institution group; with the possible exception of the glutamic acid family there is no evidence for the transfer of nitrogen from the family head to member amino acids. It is suggested therefore that these are really keto acid families and that transamination reactions are of major importance in amino acid biosynthesis from inorganic nitrogen.

The food yeast, Candida utilis, is a highly adaptable organism possessing efficient and flexible pathways for the assimilation and interconversion of nitrogenous compounds. Earlier studies (Sims, Folkes & Bussey, 1968) indicated some of the ways by which appropriate rates of glutamate and glutamine synthesis could be maintained under different nutritional conditions. We now consider some aspects of the regulation of the enzymes glutamine synthetase (GS) and the deaminating NAD-specific glutamate dehydrogenase Candida utilis is similar to Saccharomyces cerevisiae (Kohlhaw, Dragert & Holzer, I 965) in that when it is grown on glutamate or some other amino acids there is extensive derepression of GS. There is similar derepression of GDHNAD, the enzyme that under such conditions probably supplies the ammonia required for amide synthesis (Hierholzer & Holzer, I 963). The cellular concentration of free ammonia is very low, and it is believed that the increased synthesis of the two enzymes occurs directly or indirectly in response to this reduced availability of ammonia. We have found that the addition of either ammonia or glutamine to yeast so adapted can result in the rapid and extensive inactivation of both GS and GDHNA,. Our reasons for believing that inactivation of both enzymes is necessary for the readaptation of these yeasts to increased supplies of ammonia are discussed. (GDHNAD). METHODS Organisms. Candida utilis (originally designated C. utilis BP 60) was obtained from Professor Lillian Hawker, Department of Botany, University of Bristol ; Saccharomyces cerevisiae x 2 I 80 from Dr W. Duntze, Biochemisches Institut, Universitat Freiburg im Breisgau; and Torulopsis candida N C Y C~~~ from Dr J. A. Barnett, ARC Food Research Institute, Norwich. Growth conditions. Batch cultures were grown at 27' in a Gallenkamp orbital shaker. The medium used was of the following composition: glucose, 55 mM;