G. Harris

The uptake of nutrients by yeasts III. The maltose permease of a brewing yeast

Abstract Maltose penetrates the cells of a brewers yeast at pH values at which it is not utilized and is finally concentrated in the cells with respect to the medium. The mechanism for concentration is highly specific for maltose as, of a number of sugars and sugar derivatives tested, only methyl α-glucoside at comparatively high concentrations inhibits the uptake of maltose. The mechanism is adaptive in nature and maltose is far more effective as the inducer than any of the other sugars examined, including methyl-α-glucoside. The uptake of maltose by adapted cells is inhibited by various agents, such as dinitrophenol, sodium azide and sodium fluoride, which uncouple the energy supply. Moreover, the aminoacid analogues, p -fluorophenylalanine and β-2-thienylalanine, markedly depress the formation of the concentrating mechanism in the presence of maltose. These findings indicate that the concentration of maltose involves a protein component and that it is brought about by a system resembling both the permeases found in some other microorganisms and the maltotriose permease already demonstrated in the same yeast. The uptake mechanism is therefore called maltose permease.

The relationship between peptidyl-nucleotidates and protein synthesis in Brewers' yeast.

Abstract Changes in the concentration of peptidyl-nucleotidates in yeast take place with the onset of growth. Moreover, the varieties of amino acids present in the peptidyl-nucleotidates, alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, leucine, lysine, proline, serine and valine, indicate a relationship to the protein of the yeast. Studies using [14C]arginine have in fact shown that this amino acid passes from the peptidyl-nucleotidates to the protein. The results taken with others showing that arginine is bound to a variety of other amino acids in peptidyl-nucleotidates, suggest that these other amino acids also enter protein via the peptidyl-nucleotidates. In confirmation, protein synthethis in the yeast in contact with [14C]arginine was inhibited by means of hydroxylamine, the inhibition resulting in an increased concentration of peptidyl-nucleotidates in the cells, accompanied by an accumulation of [14C]arginine in these compounds.

Formation and metabolism of activated peptides and protein in yeast and derived extracts.

Abstract The distribution of labelling among certain fractions prepared at intervals from yeast cells following an initial brief period of uptake of [14]arginine was consistent with the earlier idea that the amino acid entered protein via nucleotide-peptides. Moreover, the results indicate that under the conditions employed some of the compounds were involved in protein turnover. Thus an enzyme system isolated from extracts of yeast was found to form activated peptides, even though it failed to incorporte labelling from free amino acids into these compounds. It is interesting to note that its activity was at first independent of added nucleoside triphosphates even after prolonged dialysis and precipitation. Fractionation of the crude protein by chromatography on hydroxyapetite separated it into fractions capable of forming activated peptides and other which yielded activated amino acids. The activities of all these purified fractions were then dependent upon added ATP.

Dynamic aspects of the nucleotide pool of Brewer's yeast during growth.

Chromatography of ethanolic and aqueous trichloracetic acid extracts prepared successively from a particular strain of yeast at various stages of growth revealed, in addition to nucleotides already recognised in other yeasts, a number of new compounds of this nature including derivatives of uridine diphosphate, various nucleotide-peptide anhydrides which may be intermediates in protein synthesis, and polynucleotides. During the onset of growth of the yeast the pool of alcohol-soluble nucleotides decreased, while the reservoir of nucleotides soluble in trichloacetic acid increased; but later a steady state was attained. The consequent hypothesis that the alcohol-soluble compounds are precursors of the nucleotides soluble in trichloracetic acid, which in turn are converted into insoluble compounds, was supported by the results of experiments in which the uptake and subsequent metabolism of [14C]adenine and uracil were examined. Entry of [14C]uracil into a nucleotide-peptide anhydride was observed.

Changes in peptidyl-nucleotidates during development of yeast cultures.

Abstract Peptidyl-nucleotidates have been extracted successively by means of aqueous ethanol and trichloroacetic acid from yeast cultures in various phases of development. The aqueous ethanolic extracts showed a general decrease in 5′-phosphoanhydride content and an increase in ribose esters as the age of the culture increased. It is suggested that some of these esters are involved in the re-cyclisation of proteins.

The nature of activated compounds in yeast in an early growth phase

Abstract The periodate consumption of concentrates of the peptidyl-nucleotidates from yeast in an early phase of growth increased only slightly after cleavage of the activated bonds. It therefore appears that 2′- or 3′-ribose ester bonds account for only 11% of the total activation in contrast to the situation in an older culture. This finding is discussed in relation to earlier results and to the question of protein biosynthesis.

Utilization of 8-azapurines by yeasts

At concentrations normally inhibitory to certain bacteria, 8-azaadenine and 8-azaguanine are metabolised by the yeast strains Pichia membranaefaciens, Candida utilis, Rhodotorula glutinis, Saccharomyces carlsbergensis and Saccharomyces cerevisiae. With the first, a longer lag phase than normal is induced when the azapurine is used as the sole source of purine, whereas with the other yeast strains growth commences normally. 8-Azaguanine is principally deaminated to 8-azahypoxanthine and a mixture of compounds which are highly fluorescent under ultraviolet light. 8-Azaadenine yields similar fluorescent materials none of which were identified. In no case did it appear that the azapurines were incorporated into ribosides, nucleotides or nucleic acid in the yeasts.

New activated compounds from brewers' yeast

Abstract Aqueous-ethanolic extracts obtained from yeast in either late or early phases of growth contain in addition to the basic peptidyl-nucleotidates earlier isolated two further groups of substances capable of acylating hydroxylamine at neutrality. One of these consists of acidic peptidyl-nucleotidates and the other of very basic peptide derivatives and both these groups are present in greater amounts in the extracts derived from the cells in the later phase. Structural studies on the materials present in these extracts show that the acidic compounds, unlike the peptidyl-nucleotidates formerly obtained, are composed entirely of 2′- or 3′-linked esters of ribonucleotides. The basic peptidyl-nucleotidates in these extracts are resolvable by electrophoresis into theri ester and anhydride components and the amounts of these respective groups are in conformity with previous estimates made by analysis of the mixture. The extracts contain in addition to substances which acylate hydroxylamine at neutrality further materials which react with this base only at pH 7·7 in presence of sodium borate and, being unreactive towards ninhydrin, are perhaps phosphoramides.

The uptake of adenine by brewers' yeast and a respiratory-deficient mutant

Abstract On suspending “resting” cells of a strain of brewing yeast and of a derived respiratory-deficient mutant in an adenine-containing medium, there resulted no ultimate concentration of free adenine within the cells although the total concentration of adenine derivatives increased when glucose or certain other carbohydrates were incorporated into the medium. The increase resulted from a stimulation of the conversion of the adenine into nucleotides. After keeping in the presence of adenine and glucose and then transferring to a medium free of glucose, the yeasts released hypoxanthine and not adenine, even though adenine was present within the cells in a form soluble in cold trichloroacetic acid. There was no evidence that the uptake of adenine by these yeasts was dependent upon a specific concentrating mechanism as is the case for certain carbohydrates. It appears rather that the linkage between uptake of adenine and glucose metabolism is due to combination of the purine with a dissimilation product of glucose, possibly 5-phosphoribosyl-1-pyrophosphate, to form nucleotides.