Yeast Cell-free Extracts Research Articles

Comparative investigations on the metabolism of formaldehyde in the presence of ribose-5-phosphate in cell-free extracts of yeasts grown on methanol

Comparative investigations on the metabolism of formaldehyde in the presence of ribose-5-phosphate in cell-free extracts of yeasts grown on methanol

Über den Abbau von L-Äpfelsäure durch Hefen verschiedener Gattungen mit Malatenzym

(1) The aerobic assimilation of malic acid is not a character of certain yeast genera or species as was shown by testing more than 300 different strains. Single strains of the following-species were found to grow on malic acid as the only carbon source: Candida pulcherrima, C. utilis, C. mycoderma, Torulopsis famata, Pichia membranaefaciens, P. wickerhamii, Hansenula capsulata, Trigonopsis variabilis , and Zygosaccharomyces chevalieri . (2) During fermentation C. pulcherrima and T. famata decompose up to 40% and C. utilis up to 80% of the L-malic acid that is present in the medium. (3) L-Malic acid is decomposed to CO 2 and the corresponding amounts of ethanol or pyruvate by cell free extracts of yeasts of the genera Pichia, IIansenula, Trigonopsis, Zygosaccharomyces, Kloeckera, Candida , and Torulopsis . (4) The enzymatic activity of malate dehydrogenase (L-malate: NAD oxidoreductase, E.C.1.1.1.37) was separated from the decarboxylating enzyme by gel chromatography of extracts of some strains of the genera Pichia, Candida , and Hansenula . It is assumed that malic acid is decomposed by a malic enzyme (L-malate: NAD oxidoreductase, decarboxylating E.C. 1.1.1.38) that was partially purified and found to have a molecular weight of 120000–130000. (1) Die Untersuchung der aeroben Verwertung von L-Äpfelsäure bei über 300 Hefestämmen verschiedener Gattungen und Arten ergab, daß diese Eigenschaft nicht gattungs- oder artipezifisch ist. Einzelne Stämme folgender Arten wuchsen aerob mit Äpfelsäure als einziger Kohlenstoffquelle: Candida pulcherrima, C. utilis, C. mycoderma, Torulopsis famata, Pichia membranaefaciens, P. wickerhamii, Hansenula capsulata, Trigonopsis variabilis und Zygosaccharomyces chevalieri . (2) Unter Gärbedingungen wurden unterschiedliche Mengen von L-Äpfelsäure abgebaut. C. pulcherrima und T. famata vermögen bis zu 40% und C. utilis bis zu 80% der im Medium vorhandenen L-Äpfelsäure umzusetzen. (3) Zellfreie Extrakte aus Hefen der Gattungen Pichia, Hansenula, Trigonopsis, Zygosaccharomyces, Kloeckera, Candida und Torulopsis bilden aus L-Äpfelsäure in Abhängigkeit von Mn ++ und NAD CO 2 und in entsprechenden Mengen Äthanol oder Pyruvat. (4) Bei einigen Stämmen der Gattungen Pichia, Candida und Hansenula konnte durch Gel-Chromatographie die Äpfelsäure decarboxylierende Aktivität von der Malat-Dehydrogenase (L-Malat: NAD Oxidorekdutase E.G.1.1.1.37) abgetrennt und angereichert werden, woraus auf das Vorkommen eines Malatenzyms (L-Malat: NAD Oxidoreduktase, decarboxylierend E.C.1.1.1.38) geschlossen werden kann. Das Molekulargewicht dieses Enzyms liegt bei 120-130000.

Kinetic properties of S-adenosylmethionine:Δ 24-sterol methyltransferase enzyme(s) in mitochondrial structures of Saccharomyces cerevisiae

The inhibition of S-adenosylmethionine:Δ 24-sterol methyltransferase (EC 2.1.1.41) activity by endogenous cellular components has been studied in vitro. The principal inhibitors were Na + and K +; Cs +, NH 4 + and Li + were also shown to inhibit the reaction. The possible significance of inhibition by sodium and potassium is discussed. Evidence is presented for the presence of more than one enzyme capable of methylating sterols in cell-free extracts of yeast. Three enzymatic activities are described which differ in their respective Michaelis constants for S- adenosyl- l-methionine , pH optima, and affinity for zymosterol. Based on differences in the apparent Michaelis constants for zymosterol, it appears that only one may be responsible for in vivo methylation of this ergosterol precursor.

Structural specificity of bile acid for inhibition of sterol synthesis in cell-free extracts of yeast

Inhibitory effects of about 40 species of bile acids and related compounds or 14C incorporation from [14C]acetate into the nonsaponifiable fraction were examined with cell-free extracts of yeast. The presence of a 3α- or 7α-hydroxyl group and of a carboxyl group at the terminal of the side chain was required for the inhibitor yactivity. Introduction of an additional hydroxyl group in the 6α- or 12α-position abolished the inhibition. Bile acids with a 3β-hydroxyl group showed scarcely any inhibition. With regard to length of the side chain, the presence of a C4- or C5-side chain at the 17α-position was necessary. Conjugation with taurine scarcely affected the inhibition while conjugation with glycine lowered the inhibition to about one half. These specific structural requirements of bile acids for the inhibition indicate that the inhibition by bile acids is not a non-specific action due to their detergent action.Evidence was also presented suggesting that the site of inhibition by bile acids in the conversion of acetate into sterol was the reduction step of 3-hydroxy-3-methylglutaryl-CoA to mevalonate.

Cell-free enzymic esterification of 5-carboxymethyluridine residues in bulk yeast transfer RNA

1. There is esterification of 5-carboxymethyluridine residues, to yield methyl 5-carboxymethyluridine residues, when bulk yeast tRNA and S-adenosylmethionine are incubated with cell-free extracts of yeast or wheat embryo. 2. Synthesis of methyl 5-carboxymethyluridine is augmented several fold if preexisting methyl carboxylate ester linkages in yeast tRNA are saponified before the tRNA is subjected to methylation, in vitro. 3. The ester that is formed by methylation, in vitro, is largely confined to a Py-mcm 5U sequence, as previously shown to be the case when the ester is formed, in vivo.

Enzymic synthesis of saccharopine- 14C

A simple enzymic method for the preparation of radioactive saccharopine has been described using l-lysine-UL- 14C, α-ketoglutaric acid, NADH + H +, and a yeast cell-free extract. Radioactive saccharopine was purified by chromatography on Dowex formate and crystallized from ethanol.

Polymorphism of malate dehydrogenase in cytoplasmic and mitochondrial fractions ofBlastomyces dermatitidis

Malate dehydrogenase isozymes were studied in cell-free extract (crude extract), cytoplasmic, and mitochondrial fractions of yeast and mycelial phases of Blastomyces dermatitidis. All 3 fractions were subjected to disc electrophoresis on polyacrylamide gels. Different molecular forms of malate dehydrogenase were clearly demonstrated in both cytoplasmic and mitochondrial fractions of the yeast phase. The isozyme patterns in the yeast and the mycelial phases were different in number of bands.

Inhibition of ergosterol synthesis in cell-free extracts of yeast by bile acid

The inhibition of the conversion of 14C-acetate into the nonsaponifiable fraction by bile acid with the cell-free extracts of yeast was demonstrated not to be due to the non-specific detergent nature of it on the basis of the following observations: 1) The inhibition by various bile acids showed a greater variation than expected from their critical micellar concentrations. 2) The inhibition seemed specific to the reduction step of 3-hydroxy-3-methyl-glutaryl-CoA to mevalonate and was reversed by the addition of serum albumin, differently from the non-specific inhibition by synthetic surfactants, such as sodium lauryl sulfate and benzalkonium chloride.

The biosynthesis of thiamine conversion of 2-methyl-4-amino-5-formylpyrimidine to 2-methyl-4-amino-5-hydroyxmethylpyrimidine by cell-free extracts of baker's yeast

1. 1. Cell-free preparations of Saccharomyces cerevisiae, were shown to catalyze the reduction of 2-methyl-4-amino-5-formylpyrimidine to the corresponding 5-hydroxymethyl derivative, a known intermediate in thiamine biosynthesis. Either NADH or NADPH served as a cofactor. The pH optimum of the system was 6.5–7.0 and the apparent K m for the formylpyrimidine substrate was 3.3·10 −4 M. 2. 2. Details of a microbiological assay for the pyrimidine moiety of thiamine, using Escherichia coli Strain M70-17, are described.

Biochemical mechanisms underlying the metabolic oscillations in yeast

Metabolic oscillations associated with the glycolytic pathway in intact cells and cell-free extracts of yeasts probably represent the first observation of prolonged self-sustained biochemical oscillations in vivo and in vitro. These oscillations have many characteristics similar to those of endogenous biorhythms which, others have postulated, might be driven by oscillations at the biochemical level. Among these characteristics are a stable frequency, a self-sustained nature, a susceptibility to phase shifting, fadeout, and reinitiation. The frequency is, however, temperature sensitive and high (approximately 1.8 minutes−1 in vivo and 0.17 minutes−1 in vitro).The metabolic oscillations, which represent a pulsing of the glycolytic flux, appear to arise by a mechanism involving an initial step of constant and specific velocity together with an activation of the enzyme phosphofructokinase by its product adenosine diphosphate. Other coupled reactions are also involved which may explain the appearance of "beats" and pulse-type oscillations, as well as the normal sinusoidal type of waveform. These oscillations are an interesting metabolic control phenomenon and allow the demonstration of apparent metabolic coupling between individual cells. They may also be useful as a model for a driving oscillation for endogenous biorthythms.

The synthesis of cytidine diphosphate diglyceride by cell-free extracts of yeast

The synthesis of cytidine diphosphate diglyceride by cell-free extracts of yeast

A method for extraction of sterols from enzymically active cell-free preparations

A method has been presented for the extraction of sterolic components from cell-free extracts of yeast. This method has been shown to give two-fold greater yields than saponification, and the yields have also proved to be much more consistent than other methods. The technique involves the suspension of the TCA-precipitated fraction of the cell-free reaction in DMSO, followed by a short period of heating, adjusting to alkaline pH, and then extraction with petroleum ether.

A study of acetyl-CoA condensation with α-keto acids

The condensation of acetyl-CoA with α-ketobutyraté and with α-ketovalerate to yield α-ethylmalic acid and α-( n-propyl)malic acid, respectively, has been demonstrated to occur in the presence of dialyzed cell-free extracts of baker's yeast ( Sacchararomyces cerevisiae). These reactions have been studied by means of a fluorometric assay procedure suitable for determination of α substituted malic acid derivatives. The reactions were shown to require α-keto acid, acetate, CoA, and ATP or the presence of α-keto acid and acetyl-CoA alone. The reaction between acetyl-CoA and α-ketobutyrate was also demonstrated by means of acetyl-CoA disappearance as measured by a decrease in absorbance at 232 mμ. Comparison of specific activities of the various ammonium sulfate fractions of the yeast extract indicated that the condensations of acetyl-CoA with α-ketobutyrate and α-ketovalerate are catalyzed by the same enzyme fraction. In the light of these results and results of experiments reported previously the condensation of acetyl-CoA with α-keto acids may be viewed as a general type of enzyme reaction.

Studies on the Biosynthesis of Ergosterol in Yeast: FORMATION OF METHYLATED INTERMEDIATES

Cell-free extracts of yeast convert 14C-mevalonic acid into squalene, lanosterol, zymosterol, and sterols more polar than ergosterol. Formation of two of the more polar sterols (Es1 and Es2) has also been demonstrated with methyl-labeled methionine and with 14C-adenosylmethionine as precursors. The two polar sterols, Es1 and Es2, formed from radioactive methyl donors yield labeled formaldehyde on chemical degradation. Es2 is reduced to a mixture of ergosterol and ergostanol on partial reduction with Raney nickel and hydrogen. The tentative structure 5,7,22,24(28)-ergostatetraene-3β-ol is assigned to one of the sterols (Es2). Intact yeast cells convert zymosterol to Es2 anaerobically, and aerobically they convert Es2 to ergosterol. Evidence for the role of Es1 and Es2 as intermediates in ergosterol biosynthesis is discussed, and it is suggested that a C27 sterol is the acceptor in the transmethylation reaction.

Biosynthesis of α-ketoadipic acid by extracts of Baker's yeast

A cell-free extract of baker's yeast is capable of converting the carbon atoms of acetate and α-ketoglutarate to α-ketoadipic acid. The co-factors required are coenzyme A, diphosphopyridine nucleotide, and adenosine triphosphate. When the substrates employed were labeled in specific positions with carbon 14 the distribution of the label in the α-ketoadipate formed was consistent with its formation via the Strassman-Weinhouse mechanism. This involves the condensation of acetyl CoA with α-ketoglutarate to form a seven-carbon homologue of citric acid which is then degraded through a series of reactions similar to those of the citric acid cycle to yield α-ketoadipic acid.

AMINO ACID INCORPORATION INTO PROTEIN BY CELL-FREE EXTRACTS OF YEAST.

AMINO ACID INCORPORATION INTO PROTEIN BY CELL-FREE EXTRACTS OF YEAST.

The conversion of stearic to oleic acid by liver and yeast preparations

1. 1. Rat-liver homogenates, capable of converting stearic acid to oleic acid, were fractionated by differential centrifugation. The isolated microsomes converted stearyl-CoA to oleate in the presence of oxygen and reduced TPN. The supernatant solution contained the stearate-activating enzymes. The oleic acid formed was isolated and degraded and shown to be the natural Δ 9-isomer by gas-chromatographic identification of the radioactive azelaic acid formed. 2. 2. Rat-liver homogenates, yeast cells, and cell-free yeast extracts which converted stearate to oleate in about 13% yield, all converted 1–2% of the stearate to a hydroxystearic acid. Preliminary results indicated that the product was a mixture of hydroxystearic acids containing both 9- and 10-hydroxstearate as well as several others. 3. 3. The following labelled compounds were incubated with liver homogenates, yeast cells, and cell-free extracts of yeast; 9-hydroxystearic acid, 10-hydroxystearic acid, 9,10-epoxystearic acid, and 9,10-dihydroxystearic acid. The labelled fatty acids produced from these were isolated and measured by gas-liquid chromatography arranged for the automatic counting of each acid. Both 9- and 10-hydroxystearic acids were converted to oleic acid, although in less than 4% yield by all three systems. A second major product formed exhibited the chromatographic behaviour of a monohydroxy mono-unsaturated C 18 acid but was not further identified. In all systems tested, except intact yeast cells, 9- or 10-hydroxystearic acid was also converted to a slight extent (2% or less) to stearic acid. The CoA derivatives of 9- and 10-hydroxystearic acids were not converted to oleate to any greater extent than the free acids. 9,10-Epoxystearic acid was converted to both oleic and stearic acids by liver but not by yeast preparations; while 9,10-dihydroxystearic was inert in both systems. 4. 4. These results support the hypothesis of a hydroxystearic acid as an intermediate in the formation of oleic acid from stearic acid only if it is assumed that the intermediate is irreversibly bound to the enzyme.

Photoreactivation of transforming DNA by an enzyme from bakers' yeast.

Ultraviolet-inactivated Hemophilus influenzae transforming DNA recovers its activity when mixed with cell-free extracts of bakers' yeast and exposed to visible light. The active agent in the extract is not used up in the reaction, and purification has not separated it into more than one non-dialyzable component. It differs from the agent in Escherichia coli extract, which produces very similar photoreactivation, but which can be resolved into non-dialyzable and dialyzable components, the latter being used up during illumination. The yeast agent can be salted out of solution and recovered quantitatively; it is inactivated by crystalline trypsin and chymotrypsin and by brief heating at 60 degrees C.-all facts suggesting that it is an enzyme for which ultraviolet lesions in the DNA serve as substrate. The kinetics of recovery are also consistent with such an assumption. This enzyme is unusual both because it is involved in a light-dependent reaction and because it has a non-destructive action on DNA outside an intact cell.

Activation of amino acids and amides by cell-free preparations

Cell-free extracts of pig liver, yeast, and pea seeds catalyze a pyrophosphate-ATP exchange which is promoted to varying degrees by every amino acid and amide normally occurring in protein. Attainment of this exchange is markedly dependent upon the methods for preparation and assay of the enzymes. The preparations also catalyze the formation of amino acid hydroxamates and of apparent amino acid polynucleotide compounds. The amino acid-polynucleotide compounds are able to transfer their amino acid to the protein of isolated ribonucleoprotein particles in every case examined. The protein-bound amino acid can be removed by incubation with non-labeled amino acid-polynucleotide, but not with the free amino acid.

Studies of lipide synthesis in cell-free yeast extracts

Yeast cell fractions prepared in buffer show a random distribution of lipide-synthesizing enzymes. When the cell fractions are obtained with the aid of a saturated lactose solution to afford protective conditions, most of these enzymes are found in the particulate, or mitochondrial, fraction which is sedimentable by a centrifugal force of 11,500 × g. A requirement of coenzyme A (CoA) for sterol synthesis from acetate by cell-free yeast extracts can be demonstrated only when the extracts are partially depleted of the cofactor by treatment with Dowex 1-Cl. Further supplementation with CoA is required for the conversion of squalene to the fatty acids.