Yeast Cell-free Extracts Research Articles

I. Preparation and properties of actively respiring cell-free yeast extracts

1. 1. Respiring cell-free baker's yeast extracts have been prepared by 10-sec high-speed mechanical disintegration. 2. 2. Increasing the disintegration period reduces the oxidate ability of the extracts. 3. 3. Whole extracts have a low blank respiration at or above pH 7·4. At pH 6·5–7·1 a high blank respiration is found. Addition of small amounts of ATP or A–5–P to the blank at pH 7·4 also induces this high respiration. 4. 4. The blank respiration is due to catabolism of polysaccharide. The R.Q. of the reaction is about 1 or sometimes higher. Considerable amounts of pyruvate and acetate are formed, but these are insufficient to account for either carbohydrate disappearance, O 2 uptake, or CO 2 formation. Some pentose formation occurs. 5. 5. Various acids of the Krebs tricarboxylic acid cycle are oxidized by the extracts, succinate most and malate least rapidly. 6. 6. Quantitative balances of the metabolism of each of the Krebs cycle acids show that they can largely be accounted for by the reactions of the cycle. Only small amounts of citrate are formed from oxaloacetate, acetate and CoA. Considerable amounts of glutamate are formed from α-ketoglutarate but not from citrate. 7. 7. The results are discussed in the light of the Krebs cycle being a major pathway of yeast respiration. Other pathways exist and may account for the high blank respiration observed under certain conditions.

The Metabolism of Squalene by Cell-free Yeast Extracts1,2

The Metabolism of Squalene by Cell-free Yeast Extracts^1,2

METABOLISM OF RIBOSE-1-C14 BY CELL-FREE EXTRACTS OF YEAST

METABOLISM OF RIBOSE-1-C14 BY CELL-FREE EXTRACTS OF YEAST

Synthesis of Lipids in Cell-Free Extracts of Yeast.

SummaryCell-free preparations, capable of incorporating acetate into fatty acids and non-saponifiable lipids, have been obtained from the yeast, Saccharomyces cerevisiae.

Effects of acetate and other short-chain fatty acids on yeast metabolism

The inhibition by short-chain fatty acids of several metabolic reactions in yeast and cell-free yeast extracts is demonstrated. Some properties of these inhibitions are shown and their similarity to the binding of short-chain fatty acids to proteins is discussed.

The effect of 6-deoxy-6-fluoro- d-glucose on yeast fermentation and on hexokinase

6-Deoxy-6-fluoro- d-glucose (6FG), at molar concentrations comparable to that of glucose or fructose present, produced a marked inhibition of the rate of fermentation of intact yeast. In contrast, 6 FG has a much smaller inhibitory effect on the rate of fermentation by a cell-free yeast extract or on the activity of yeast hexokinase. The inhibition by 6FG of intact yeast fermentation was competitive with glucose or fructose, and was overcome by increasing the concentration of these substrates. The rate of fermentation of glucose or of fructose by intact yeast was decreased by reduction in substrate concentration in a concentration range where the rate of fermentation by a yeast extract or the activity of hexokinase was unaffected. These results support the postulate that 6FG inhibits a specific process, not limited by hexokinase activity, which controls the rate of entry of glucose and of fructose into the yeast cell. The uptake of glucose by rat diaphragm was much less sensitive to the presence of 6FG than was fermentation of glucose by intact yeast. Notatin oxidized 6FG at a rate about 3% of that observed with glucose. 6FG was only moderately toxic to rats and did not affect the behavior of or virus synthesis in a tissue culture preparation.

Incorporation of carboxy- 14C-acetate into krebs cycle acids in cell-free yeast extracts

Incorporation of carboxy- 14C-acetate into krebs cycle acids in cell-free yeast extracts

Distribution of enzymes in cell-free yeast extracts.

The evidence for the existence of 'mitochondria' in micro-organisms has been growing, but the criteria for thus naming cytoplasmic inclusions are by no means clear. From the biochemical point of view, mitochondria are generally considered to be sites of oxido-reduction and of organized oxidations. The 'cyclophorase' complex in the mitochondria of animal cells studied by Green and his School (Green, Loomis & Auerbach, 1948) has never been demonstrated in micro-organisms, although it has recently been found in plants (Millerd, Bonner, Axelrod & Bandurski, 1951). The failure to prepare cell-free, actively respiring yeast extracts contributes to the continuing controversy about the main respiratory mechanism of this micro-organism (cf. Krebs, Gurin & Eggleston, 1952; Martius & Lynen, 1950; and Foulkes, 1951). Methods used so far for preparing yeast extracts are probably such that the intracellular organization of enzymes, and therefore organized oxidation systems, are destroyed during cell disintegration. We have developed an ultra-rapid mechanical shaker which disintegrates 2 g. yeast almost entirely within 90 sec. (Nossal, 1953a). We failed to prepared actively respiring yeast 'mitochondria' with this machine. However, the results reported here show that the distribution of enzymes in the cell-free extracts is not unlike that in extracts of animal cells: dehydrogenases are found largely associated with the granular fraction when the extracts are prepared by very brief disintegration. While the work was in progress, Hirsch (1952)

L-Threonine Synthesis by Cell-Free Extract of Yeast

L-Threonine Synthesis by Cell-Free Extract of Yeast

Oxidate reactions in cell-free yeast extracts

Oxidate reactions in cell-free yeast extracts

The pathways of acetate oxidation

In micro-organisms considered to oxidize acetate via the dicarboxylic acid cycle or by oxidation to glycolic acid, there were found the enzymes for the synthesis of citric and for the oxidation of isocitric acid. The culture media of moulds believed to oxidize acetate via glycolic acid contained citric, α-ketoglutaric, succinic, malic, and glycolic acids. In most of these organisms malonate inhibited acetate oxidation. In the absence of malonate, citric acid was detected chromatographically from solutions where acetate was oxidized whereas in the presence of malonate only succinate was detected. Formation of succinate from acetate seems to occur by the oxidative condensation of acetylcoenzyme A to succinate. Cell-free extracts of bacteria, moulds, yeast, and animal tissues contain the enzyme which oxidizes acetate in the presence of coenzyme A, flavine adenine dinucleotide, DPN, and Mg +2. It is postulated that all cells oxidize acetate via citric acid cycle but also possess the dicarboxylic acid cycle which provides the oxalacetate required for citric acid formation.

2-Desoxy-D-glucose as an antagonist of glucose in yeast fermentation

2-Desoxy-D-glucose (2DG) has been found to inhibit strongly the anaerobic fermentation of glucose by living yeast. The degree of inhibition was found to be dependent on the molar ratio of substrate to inhibitor, but independent of the absolute quantities of substrate or yeast and of pH of the medium between 4.5 and 7.5.The effect followed the laws of competitive inhibition of an enzyme. The initial rate of fermentation of glucose by fortified cell-free yeast extract was not inhibited by 2DG. The inhibitory effect of 2DG on yeast fermentation was lost with the removal of the yeast cell wall. The evidence indicates that 2DG, acting as a structural analogue of glucose, competitively inhibits the action of a cell-wall enzyme which is essential for the transport of glucose into the cell, thus inhibiting the overall rate of fermentation by living yeast. A new substrate for yeast hexokinase has been discovered through the observation that the adenosine triphosphate-hexokinase system phosphorylated 2-desoxy-D-glucose.

The effect of nicotinamide on fermentations by fresh and by acetone-dried powders of cell-free yeast extracts

1. 1. The preparations of highly active cell-free yeast extracts and their acetone-dried powders, using both the broken Pyrex glass and the glass bead-shaker techniques, have been described. 2. 2. The presence of nicotinamide during the process of crushing yeast is essential for producing a cell-free preparation having high fermentative powers. 3. 3. Acetone powders of yeast prepared in this way have retained their fermentative action unimpaired for more than 8 months.