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Oxidative phosphorylation by subcellular particles from yeast
... Comme le niveau de ploïdie, l'ADN-mt a de nombreux effets sur la physiologie de la levure. La mitochondrie a un rôle dans la phosphorylation oxydative (Utter et al., 1958), l'homéostasie , la maturation et l'assemblage des protéines (Lill and Kispal, 2000). Les protéines codées par le génome mitochondrial interagissent directement avec des protéines codées par le génome nucléaire pour la formation de complexes mitochondriaux qui assurent le bon fonctionnement de la mitochondrie (cytochrome c, cytochrome bc, ATPase) (Borst and Grivell, 1978). ...
La levure S. cerevisiae est la seule espèce capable de terminer la fermentation alcoolique du jus de raisin qui est l’étape principale de la vinification. A cause de la forte variabilité technologique retrouvée chez cette espèce, des travaux de sélection sont réalisés dans le but d’utiliser des levains performants pour l’industrie. Ces souches montrent des différences importantes à la fois dans leurs cinétiques fermentaires et leur bilans en métabolites, ce qui impacte la qualité des vins. La réponse phénotypique des levures varie également de manière considérable et non homogène face aux variations environnementales. La compréhension des mécanismes génétiques expliquant cette réponse différenciée est une question scientifique non triviale. Elle revêt une importance particulière en oenologie, où les conditions de vinifications sont très changeantes (millésimes, cépages, terroirs, conduites de vinifications…). Afin de pouvoir proposer des levains garantissant le succès des fermentations dans un large éventail de conditions, nous proposons ici de mieux comprendre ces mécanismes d’interaction Gène x Environnement dans un contexte oenologique. L’identification de locus génétiques (Quantitative Trait Loci (QTL)) contrôlant des caractères quantitatifs est rendue possible par des approches de cartographie de QTLs. Celles-ci nécessitent l’étude d’une vaste descendance en ségrégation qui doit être caractérisée sur le plan génétique et phénotypique. L’établissement d’un lien statistique entre des marqueurs génétiques et un phénotype permet la localisation de QTLs influant les caractères étudiés. Au cours de cette thèse, une méthode de phénotypage pour suivre les fermentations de plusieurs centaines d’individus a été mise au point. Grâce à elle, les performances fermentaires de deux descendances génotypées par séquençage à haut débit ont été mesurées en faisant varier les conditions de fermentations. Cela a permis l’identification de nombreux QTLs et d’estimer leur impact sur la robustesse des souches. L’implication des allèles de trois gènes qui montrent une forte interaction avec l’environnement et qui possèdent des effets pléiotropiques liés au métabolisme du SO2 a été prouvée moléculairement. Les résultats obtenus font l’objet d’une discussion générale sur l’utilisation de QTLs pour la sélection de levures plus performantes.
... In other words, we must wait up on the slow development of direct, quantitative cellular chemistry. (Cowdry, 1924) In parallel, mitochondrial functions, such as their respiratory or biosynthetic features, were analysed across large numbers of mitochondria in organisms and tissues, or isolated from those sources (Utter et al., 1958;Ohnishi and Hagihara, 1964;Weiss et al., 1970;Werner and Neupert, 1972). The fundamental divide between cell biological and biochemical approaches to mitochondria has left a void at the centre of organelle biology, leaving the question open of how exactly the individual mitochondrion carries out its functions as a physically discrete unit. ...
Mitochondria host vital cellular functions, including oxidative phosphorylation and co‐factor biosynthesis, which are reflected in their proteome. At the cellular level plant mitochondria are organized into hundreds of discrete functional entities, which undergo dynamic fission and fusion. It is the individual organelle that operates in the living cell, yet biochemical and physiological assessments have exclusively focused on the characteristics of large populations of mitochondria. Here, we explore the protein composition of an individual average plant mitochondrion to deduce principles of functional and structural organisation. We perform proteomics on purified mitochondria from cultured heterotrophic Arabidopsis cells with intensity‐based absolute quantification and scale the dataset to the single organelle based on criteria that are justified by experimental evidence and theoretical considerations. We estimate that a total of 1.4 million protein molecules make up a single Arabidopsis mitochondrion on average. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40,000 for Voltage‐Dependent Anion Channel 1 (VDAC1) to sub‐stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat (PPR) proteins that modify mitochondrial transcripts. For our analysis, we consider the physical and chemical constraints of the single organelle and discuss prominent features of mitochondrial architecture, protein biogenesis, oxidative phosphorylation, metabolism, antioxidant defence, genome maintenance, gene expression and dynamics. While assessing the limitations of our considerations, we exemplify how our understanding of biochemical function and structural organization of plant mitochondria can be connected in order to obtain global and specific insights into how organelles work.
... Those studies showed that the conversion of ADP to ATP was accomplished through electron transfer from oxidized substrates with oxygen [7]. Microbiologists evaluated the respiratory chain of bacteria, particularly Escherichia coli and found main structural similarities in the respiratory chain of mitochondria and their mechanisms [8,9]. For instance, since the bacterial respiratory chain is located in the cytoplasmic membrane and elec-tron transfer occurs from cytoplasm to periplasmic space, the chemiosmotic theory, similar to mitochondria, could be applicable for bacteria as well. ...
Adenosine triphosphate (ATP) production in living cells is very important. Different researches have shown that in terms of mathematical modeling, the domain of these investigations is essentially restricted. Recently the thermodynamic models have been suggested for calculation of the efficiency of oxidative phosphorylation process and rate of energy loss in animal cells using chemiosmotic theory and non-equilibrium thermodynamics equations. In our previous work, we developed a mathematical model for mitochondria of animal cells. In this research, according to similarities between oxidative phosphorylation process in microorganisms and animal cells, Golfar's model was developed to predict the non-equilibrium thermodynamic behavior of the oxidative phosphorylation process for bacteria in aerobic condition. With this model the rate of energy loss, P/O ratio, and efficiency of oxidative phosphorylation were calculated for Escherichia coli in aerobic condition. The results then were compared with experimental data given by other authors. The thermodynamic model had an acceptable agreement with the experimental data.
... lysodeikticus as described here shares a number of properties with similar systems of other microorganisms. The maximal P :0 ratios observed did not exceed 1.0, as is also the case for preparations from A. faecalis (l-4), A. vinelandii (&lo), Proteus vulgaris and Aerobacter aerogenes (32), Xaccharornyces ceretisiae (32), and Escherichia coli (33,34), suggesting the possibility that the respiratory chains of these microorganisms are simpler and may have fewer phosphorylation sites than those of animal tissues, or alternatively, that microbial systems are much more readily damaged or uncoupled. ...
... Zur Gewinnung der enzymatisch aktiven Partikelfraktion aus Hefe wurden die Zellen im Homogenisator nach Schlossmann [8] aufgeschlossen, zun/ichst 20 min bei 15 000 g zentrifugiert und aus dem Uberstand die aktive Fraktion durch 60 min Zentrifugieren bei 105 000 g sedimentiert [9]. Nach Vorversuchen, in denen wir die Zeit-und Enzymabh/ingigkeit der Bildung von PSPP bestimmten, erwies sich folgende Zusammensetzung fiir die Pr/iparation des PSPP als geeignet. ...
Presqualenyl-pyrophosphate (PSPP) was synthesized from farnesyl-pyrophosphate (FPP). The purified product could be transformed into a squalene in 90% yield. The structure of PSPP was established by the following experiments: 1.1) PSPP displays a remarkably acid-labile phosphate—ester bond. Analogous to FPP it was possible to stabilize this bond by the addition of bromine. This indicates a structure of the allylester type.2.2) On acid hydrolysis PSPP splits off not only the pyrophosphate but also water. This may be best explained by the presence of a tertiary hydroxyl group in PSPP.3.3) The pyrophosphate group of PSPP could be split by prostatic phosphatase. In gas-liquid-chromatography the trimethyl-silylated product gave a single peak. From the mass spectra of this compound and of the corresponding alcohol the expected molecular weight of 426 could be verified for the latter.4.4) Determining the 3H/14C ratio in 1-3H2-414C-FPP and in PSPP and squalene, synthesized from it, only three 3H-atoms could be detected in PSPP and in squalene.5.5) Ozonisation of PSPP, synthesized from 1-14C-FPP, gave radioactive malondialdehyde, which was characterised as azobenzene-malondialdehyde and 4-azobenzene-1-phenyl-pyrazole. The experimental results are in agreement with the following structure of 2, 6, 10, 15, 19, 23-hexamethyl-n-tetracosa-2, 6, 11, 14, 18, 22-hexaen-10-yl-pyrophosphate for PSPP.
Mitochondria from the yeast S. cerevisiae have been shown to be particularly suited for cryobiological studies, since they may be frozen and thawed without detectable injury to either structure or function. Mitochondria are frozen in the usual isolation medium containing mannitol and serum albumin, and no penetrating cryoprotective agent such as dimethyl sulfoxide or glycerol is required.Yeast cells have been stored at 4°C for at least 9 days, and isolated mitochondria for 30 days or more, in liquid N2 without appreciable change in mitochondrial Qo2, ADP:O, or adenosine diphosphate-linked respiratory control. Stored mitochondria retain normal morphology and capacity for adenine nucleotide translocation. Respiratory control has been preserved for as long as 53 days. Improved isolation methods together with these storage techniques have permitted study of intact yeast mitochondria with a facility virtually unattainable with mitochondria from other cell types.Mitochondria isolated from a yeast mutant with deficient aerobic energy metabolism exhibit increased lability during freezing and thawing. Both Qo2 and respiratory control are greatly reduced.
A method is described for preparing yeast mitochondria rapidly (within one hour) by using the MSK Bronwill Cell homogenizer. Yeast mitochondria obtained by the method exhibit relatively good respiratory controls and ADP/O ratios. The method is convenient for small or large amounts of yeast cells (from 5 to a hundred grams, wet weight) and gave a yield of 3 to 5 mg protein/g wet weight of yeast mitochondria.
Yeast mitochondria isolated by treatment of yeast cells, S. carlsbergensis, with snail gut juice were studied with the negative staining and thin sectioning methods. The mitochondrial fraction was little contaminated by non-mitochondrial fractions, and the mitochondrial structure seemed to be fairly undamaged.With the negative staining method, knob-like structures on the surface of cristae were clearly observed; the particles on the stalks were rather non-spherical and their mean size was about 80 × 60 Å, i.e., somewhat smaller than those reported in other organisms.
Well preserved mitochondria were isolated from Saccharomyces carlsbergensis by a procedure which involves digestion of the cell wall with snail gut juice. The yeast mitochondria thus prepared oxidized members of the tricarboxylic acid cycle, reduced nicotinamide adenine dinucleotide, d- and l-lactate, and tetramethyl-p-phenylenediamine reduced by ascorbate. Respiratory control in response to the addition of adenosine diphosphate was observed with all of these substrates. The highest respiratory control ratio (5 to 6) was obtained with α-ketoglutarate as substrate. The yeast mitochondria actively oxidized externally added NADH, and this oxidation likewise showed respiratory control. The ADP to oxygen or phosphorus to oxygen ratios observed in this system were about 0.9 with tetramethyl-p-phenylenediamine and lactate; 1.7 with succinate, NADH, citrate, and pyruvate plus catalytic amount of malate; and 2.5 with α-ketoglutarate. These respirations were completely insensitive to Amytal and rotenone. It is suggested that phosphorylation Site I is absent from these yeast mitochondria. Effects of uncouplers and inhibitors of oxidative phosphorylation on yeast mitochondria were very similar to those observed with mammalian mitochondria. The P:O ratios in the respiration with NADH as a substrate showed constant values when measured between pH 5.4 and 7.5, and at a range of tonicity between 0.3 and 1.0 m sorbitol. The yeast mitochondria could be stored for a long period of time without appreciable loss of phosphorylation activity. The components of electron transport of isolated yeast mitochondria were studied by means of difference spectrophotometry. Electron micrographs of the isolated yeast mitochondria and mitochondria in situ were also presented.
Methods for the preparation of mitochondria from Prototheca have been investigated and the state of these organelles prepared by various means compared by electron microscopy and measurement of succinoxidase activity. Ubiquinone and cytochrome (a+a3), b and c were present. The mitochondria oxidized NADH and succinate, and mammalian cytochrome c would act as an electron acceptor in the system. The P:O ratios for various tricarboxylic acid cycle intermediates and related metabolites were determined.
1. Tightly coupled mitochondria were isolated from Aspergillus niger by using an all-glass homogenizer followed by differential centrifugation. 2. The mitochondria oxidized the common intermediates of the tricarboxylic acid cycle, NADH(2) and the ascorbate-tetramethyl-p-phenylenediamine system. 3. High P/O ratios and control of respiration by ADP were obtained with all substrates tested. The average P/O ratios observed were: 1.5-1.8 with succinate as substrate [respiratory control ratio (RC) 2-4]; 0.8-1.0 with ascorbate-tetramethyl-p-phenylenediamine (RC 1.2-1.5); 1.4-1.8 with NADH(2) (RC 2-3); 2.4-2.8 with alpha-oxoglutarate (RC 3-5). 4. Bovine serum albumin (0.05-0.2%) was essential for tightly coupled respiration to be observed. 5. Coupled oxidation of exogenous NADH(2) was relatively insensitive to rotenone and Amytal. 6. The mitochondria responded to specific inhibitors and uncoupling agents in a manner similar to that of mammalian mitochondria. 7. It was concluded that the isolated mitochondria from A. niger show respiratory properties similar to those reported for intact yeast and mammalian mitochondria.
Pyrrolnitrin at 10 mug/ml inhibited the growth of Saccharomyces cerevisiae, Penicillium atrovenetum, and P. oxalicum. The primary site of action of pyrrolnitrin on S. cerevisiae was the terminal electron transport system between succinate or reduced nicotinamide adenine dinucleotide (NADH) and coenzyme Q. At growth inhibitory concentrations, pyrrolnitrin inhibited endogenous and exogenous respiration immediately after its addition to the system. In mitochondrial preparations, the antibiotic inhibited succinate oxidase, NADH oxidase, succinate-cytochrome c reductase, NADH-cytochrome c reductase, and succinate-coenzyme Q(6) reductase. In addition, pyrrolnitrin inhibited the antimycin-insensitive reduction of dichlorophenolindophenol and of the tetrazolium dye 2,2'-di-p-nitrophenyl-(3,3'-dimethoxy-4,4'-bi-phenylene)5,5'-diphenylditetrazolium. The reduction of another tetrazolium dye, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride, that was antimycin-sensitive, was also inhibited by pyrrolnitrin. The antibiotic had no effect on the activity of cytochrome oxidase, and it did not appear to bind with flavine adenine dinucleotide, the coenzyme of succinic dehydrogenase. In whole cells of S. cerevisiae, pyrrolnitrin inhibited the incorporation of (14)C-glucose into nucleic acids and proteins. It also inhibited the incorporation of (14)C-uracil, (3)H-thymidine, and (14)C-amino acids into ribonucleic acid, deoxyribonucleic acid, and protein, respectively. The in vitro protein synthesis in Rhizoctonia solani and Escherichia coli was not affected by pyrrolnitrin. Pyrrolnitrin also inhibited the uptake of radioactive tracers, but there was no general damage to the cell membranes that would result in an increased leakage of cell metabolites. Apparently, pyrrolnitrin inhibits fungal growth by inhibiting the respiratory electron transport system.
The effect of different respiratory inhibitors on the ergosterol content of microaerobically grown non-proliferating yeast cultures was monitored during adaptation to oxygen. It was found that dinitrophenol, azide, and cyanide, which act on the mechanism of the respiratory chain, cause a marked stimulation of sterol production. Acriflavine and chloramphenicol, which affect the synthesis of the respiratory apparatus, caused a delay in the onset of ergosterol synthesis or a marked decrease in sterol content. The data obtained provide presumptive evidence that a component of sterol formation is synthesized on the 70S ribosomal system of the mitochondrion and induced in the presence of oxygen.
The isolation, purification, physical and chemical properties of a mitochondrial DNA of Saccharomyces cerevisiae are described and compared with the nuclear DNA of the same organism. The two entities are clearly distinct in absorption spectra at various pH values, thermal transition temperature in two different solvent systems, buoyant density in CsCl and base composition, all of which are consistent with a GC content of 35 % for nuclear and 21 % for mitochondrial DNA. The latter, a normal double helical molecule, occurs integrated into respiratory particles which renders it DNase resistant. It is present to the extent of 0.71 μg per mg of particle protein in highly aerobic cells of the wild type, decreases during glucose repression and is greatly reduced (⩽ 0.06 μg/mg protein) in a cytoplasmic respiratory-deficient mutant (vegetative petite, ϱ−). The So20,w of the isolated DNA equals 33 s, hence its molecular weight is approximately 2 × 107. Evidence is also presented for its preferential interaction with acridines. The implications of all these findings in the problems of mitochondrial autonomy and self-duplication are discussed.
Electron transport and oxidative phosphorylation in mitochondria isolated from a cytochrome c-deficient mutant of the yeast, Saccharomyces cerevisiae, may be efficiently reconstituted by addition of purified yeast or horse heart cytochrome c. Added cytochrome c is tightly bound and is retained during reisolation of mitochondria.
Reconstituted mutant mitochondria carry out oxidative phosphorylation with substrates linked to nicotinamide adenine dinucleotide, flavoprotein, and cytochrome c as efficiently as mitochondria isolated from wild type yeast under the same conditions.
Titration of mutant mitochondria with yeast cytochrome c leads to progressive increases in P:O ratios as well as to increases in rates of phosphate and oxygen uptake. The end points of such titrations indicate that reconstitution is stoichiometric at a ratio of 0.2 mµmole of cytochrome c per mg of mitochondrial protein, approximately the amount of cytochrome c in normal wild type mitochondria.
Respiration in mutant mitochondria can be restored equally well by two types of yeast cytochrome c (iso-1- and iso-2-cytochrome c) and by horse heart cytochrome c. The two types of yeast cytochrome c are virtually equivalent in restoring phosphorylation coupled to succinate oxidation, whereas the horse heart enzyme is less effective.
Mutant mitochondria reconstituted with iso-1-cytochrome c catalyze phosphorylating oxidation of succinate, α-ketoglutarate, glutathione plus tetramethyl-p-phenylenediamine, pyruvate plus malate, and d-lactate plus malate with efficiency comparable to that of normal yeast mitochondria. Ethanol oxidation was partially restored. Oxidative phosphorylation with l-lactate, even in the presence of added malate to provide citric acid cycle intermediates, was poorly restored by either of the isocytochromes c.
1.1. The apparent absence of succinic dehydrogenase activity in anaerobically grown yeast, reported in the literature, was due to the employment of assay methods unsuitable for the measurement of the activity of this enzyme. With the aid of phenazine methosulfate, a reagent capable of reacting directly with the primary dehydrogenase, it has been shown that anaerobic yeast contains a high level of succinic dehydrogenase.2.2. Slonimski's observation of the virtual absence of succinic-cytochrome c reductase in anaerobically grown cells has been confirmed.3.3. During oxygenation of anaerobically grown cells a rapid, induced synthesis of the succinic-cytochrome activity occurs, which may be accompanied by a moderate increase in dehydrogenase activity. At the end of O2 adaptation the ratio of succinic dehydrogenase to succinic-cytochrome c reductase activities is the same as in aerobically grown cells.4.4. The virtual absence of succinic oxidase in anaerobically grown cells is thus entirely due to the absence of the electron-transport system and not that of the dehydrogenase. O2 adaptation thus entails synthesis of the hemoproteins and the incorporation of succinic dehydrogenase in the respiratory chain.5.5. The possible relevance of these observations to the apparent absence of other cytochrome-reducing dehydrogenases in anaerobic yeast has been pointed out.
When Saccharomyces cerevisiae was grown under anaerobic conditions with energy and carbon supplied by glucose, it was established that two classic uncouplers of oxidative phosphorylation, 2,4-dinitrophenol and azide, are potent inhibitors of protein and ribonucleic acid (RNA) synthesis. During the inhibition it was found that the cells maintained high levels of adenosine triphosphate (ATP), amino acids, and nucleotides. All of the reactions of in vitro amino acid incorporation and stimulation of incorporation by addition of polyuridylic acid (poly U) were insensiliveto the uncouplers. The incorporation of added uracil into uridine triphosphate (UTP) and the activity of an isolated deoxyribonucleic acid dependent RNA polymerase were unaffected by the poisons. A uniform depression of [ 14C]uracil and [ 14C]leucine incorporation throughout the polysomes and monosomes was revealed by sucrose density gradient studies. Several possible explanations are considered including the idea that high-energy intermediates of oxidative phosphorylation common to ATP production and macromolecular synthesis, may exist.
Relatively fragile parts of the yeast cell have been isolated by releasing them from protoplasts by means of brief ultrasonic treatment. Particular attention has been paid to the possibility of separating DNA and RNA in association with particles of visible dimensions. The primary vacuole and various accessory vacuoles, each in a state of relative preservation, figures prominently among the products released at pH 6–7, whereas at pH 5 the most stable product exhibited a characteristic refractile cap borne by a membrane which, in various instances, could be clearly discerned as a spheroidal vesicle of about 2–3 μ diameter distinct from the main vacuole.Two further methods of isolating the refractile bodies were developed, one involving the use of formamide as a suspending medium and the other a preliminary lysis of the protoplasts. In each case the refractile bodies, stabilized in the presence of magnesium and calcium ions, were found to be associated with an approximately characteristic amount of DNA probably representing the whole of that found in the protoplast. It is suggested that these bodies are derived fairly directly from the yeast nucleus, which appears to contain only about 1 per cent of DNA by weight, but several times this amount of RNA.
A specially designed high-speed blendor and glass beads have been used to disintegrate yeast cells. The method enables large quantities of cells to be fragmented quickly at low temperature, and cell-free mitochondrial particles to be prepared in high yield. The particles are isolated in a sucrose-Tris-EDTA medium and extensively refractionated in the same medium. The success of the fractionation is dependent upon the presence of the Tris buffer, as the latter prevents the aggregation of the particulate material. Two morphologically and enzymatically different particle types have been obtained: a heavy fraction corresponding to mitochondria in size and internal organization, and a light fraction consisting of vesicular, single-membrane particles of a smaller size. The light particles oxidize DPNH and succinate, but do not oxidize pyruvate-malate, and lack the capacity for phosphorylation. The heavy particles oxidize pyruvate-malate as well as the citric acid cycle intermediates, although their alpha-ketoglutaric dehydrogenase activity is low. Oxidation by the heavy particles is coupled to phosphorylation, and P/O ratios of about 1.5 have been obtained. Lactic acid dehydrogenase is also present in the heavy fraction, and lactate is oxidized with a P/O ratio of about 0.7.
A subcellular particulate fraction isolated from baking yeast and having the properties of mitochondria, was found to oxidize succinate simultaneously with the esterification of inorganic phosphate. l-Thyroxine was found to depress the ratio significantly.Attempts were made to establish optimum conditions for a demonstration of this action of the hormone. The osmolarity of the pretreatment medium, the concentrations of sucrose, Mg, and albumin, and the pH of the incubation mixture were varied; the effects of these modifications on the uptake of oxygen and inorganic phosphate were determined, as influenced by l-thyroxine. It was observed that conditions producing a maximal rate of phosphorylation were not the most favorable ones for a demonstration of the uncoupling action of the hormone.Under conditions which produced a low rate of phosphorylation, l-thyroxine stimulated the oxidation of succinate markedly.
Yeast cells grown ailaerobically on glucose suppleillented with yeast extract, hydrolyzed casein, and oleic acid developed a respiratory capacity on exposure to air. 'The time course of development of respiration was characteristic with an abrupt onset of oxygen consumption. The magnitude of the uptake of oqrgen and the kinetics of its onset were determined by the concentration of glucose to which the yeast was exposed during growth and by the relative amounts of glucose and yeast present during the time of adaptation. A source of amino acids was necessary during the time of adaptatioil for the consistent development of respiration. Under the growth conditions used, adaptive developlllent of respiration occurred 111ost efficieiltly when the cells were harvested immediately prior to the onset of the stationary phase. C>'tochrome oxidase was virtually absent fro111 the anaerobically grown cells. The liinetics of its adaptive formation were not the same as those for the development of respiration. Cytochrome oxidase appearecl before respiratioil became appreciable. ii'hen these anaerobically grown >,east cells were added to the adaptation n~ediuill there was a burst of gas output. 'The identity of the gas is un1;nown but it is likely to be COs, HZS, or Hs. 'I'he role of glucose and other factors in the adaptive development of respiration in yeast is discussecl.
A particle-bound, Mg2+, DNP-activated ATPase has been obtained from baker's yeast (fresh or dried) by mechanical disintegration and differential centrifugation of the cell extract. The isolated fraction hydrolyses ATP, GTP and ITP but is not effective with CTP. ADP inhibits the hydrolysis of ATP and ITP; IDP does not. The addition of an ADP-trapping system prevents the inhibitory action of ADP and keeps the rate of hydrolysis of ATP linear. ATPase activity is released by Mg2+ and Mn2+ but not by Ca2+. In the presence of 2.5 mM Mg2+, maximum activity is at pH 9.0–9.5. DNP produces significant activation of the enzyme with ATP as substrate but not with GTP, CTP or ITP; the stimulation of ATPase by DNP is most effective at pH 5.5–6.5. In the presence of Mg2+, 50 mM fluoride inhibits, by 53%, the enzymic activity. The properties of the particle-bound ATPase are considered in relation to the surface-located, and soluble, yeast ATPases and animal mitochondrial ATPase.
Some properties of particle-bound intracellular ATPase from baker’s yeast. Biochimica et Biophysica Acta (BBA) - Specialized Section on Enzymological Subjects, 89(3), 532–539. doi:10.1016/0926-6569(64)90079-3
Treatment of yeast with filipin causes an inhibition of the terminal electron transport system. Mitochondria prepared from filipin treated yeast are unable to oxidize NADH or succinate; however, they retain about 50% of their NADH and succinate-cytochrome c reductase (EC 1.6.2.1 and EC 1.3.99.1 respectively) activities and about 60% of their cytochrome oxidase (EC 1.9.3.1) activity. Partial restoration of NADH oxidase system but not of succinate oxidase system activity occurs with exogenous cytochrome c. Mitochondrial preparations from yeast inactivate filipin.
Mitochondrial particles, to which the bulk of the succinoxidase and cytochrome oxidase activities were exclusively bound,
were isolated from Candida albicans after disrupting the growing cells in a mortar with quartz sand. Electron-microscopic examination of the mitochondrial preparation
showed that it mainly contained native and submitochondrial particles. Mitochondria thus prepared could couple phosphorylation
to oxidation of various substrates including TCA cycle members, exogenous NADH, glutamate, ethanol, and D- and L-lactate.
P/O ratios obtained with these substrates corresponded closely to those reported for mitochondria from other fungi, approximately
1 unit lower than P/O ratios with mammalian intact mitochondria, whereas much lower respiratory control ratios were observed.
Oligomycin strongly inhibited oxidative phosphorylation and Mg++-induced ATPase activity. The effects of inhibitors of electron transfer such as amytal, rotenone, antimycin A or sodium azide
on Candida mitochondria were similar to those on mammalian mitochondria and it is of note that the sensitivity of oxidation of both
a-ketoglutarate and NADH by C. albicans mitochondria to rotenone exceeded those of any mitochondrial preparations of other fungi.
A comparison of the structural and functional integrity of yeast mitochondria prepared by a mechanical disintegration method with that reported for mitochondria prepared by enzyme methods has been made.The mitochondria prepared by mechanical disruption show at least as great a degree of functional integrity as that reported for enzymically prepared mitochondria. This was judged by the substrates oxidised, the rates of these oxidations, the respiratory control and ADP:0 ratios and difference spectra.The respiration of citrate and of ethanol by the mechanically prepared mitochondria has been shown to be stimulated by exogenous NAD; in the case of the latter substrate a degree of respiratory coupling became apparent as a consequence of this stimulation.The penetration of NADH2, and of citrate or of ethanol in the presence of exogenous NAD, into the mitochondria has been examined.The results are consistent with the reported existence of a NADH2 dehydrogenase system located in the outer area of the inner mitochondrial membrane. The NAD stimulation of ethanol respiration also suggests the occurrence of an external alcohol dehydrogenase in the mitochondrial preparation; similar results with citrate suggest external forms of aconitic hydratase and isocitric dehydrogenase to be present.Unlike animal mitochondria the rate of penetration of oxidisable substrates in uninhibited suspensions appeared to exceed respiration and/or outward diffusion rates in this preparation.Evidence of a malate stimulated citrate and isocitrate transport system similar to that reported for animal mitochondria is presented, but the specificity of the system for tricarboxylate anion appears to differ, at least in respect of propane 1.2.3 tricarboxylate (tricarbalyllate).
Measurements of difference spectra and enzymatic activities of aerobically and anaerobically grown Saccharomyces cerevisiae show that: 1.1. The yeast grown under both conditions synthesizes cytochrome pigments and flavoproteins which are rapidly oxidized on addition of oxygen and rapidly reduced when oxygen in solution is exhausted. The absorption spectra of the cythochromes of the two kinds of yeast are different. Although cytochromes α + α3 are missing from anaerobically grown yeast, there is evidence that another oxidase may be present.2.2. Both kinds of yeast contain active primary dehydrogenases, but they show different reactivities with added redox systems in anaerobically grown yeast, as compared with yeasts grown aerobically.3.3. The cytochrome system and flavoproteins which react rapidly with oxygen are associated with different kinds of cellular structures in the two kinds of yeast, and this may indicate differences in the structural arrangements of membrane-bound systems.4.4. Additional pigments are found in both aerobically and anaerobically grown yeast which show changes in absorption spectrum on addition or deletion of oxygen. However, these changes are very slow compared with those of the respiratory system.5.5. Both aerobically and anaerobically grown yeast synthesize a pigment with properties like one found in the microsome fraction of rat liver.6.6. Aerobically and anaerobically grown yeast synthesize comparable quantities of cytochrome c peroxidase (EC 1.11.1.5).
1. A procedure for the preparation of protoplasts from yeast Saccharomyces cerevisiae harvested in their stationary phase of growth on glucose was modified and mitochondria were isolated from the protoplasts.2. Oxidative activity, phosphorylation efficiency and sensitivity to 2,4-dinitrophenol and oligomycin of these mitochondria were similar as previously reported for mitochondria from lactate-grown Saccharomyces carlsbergensis.3. The mitochondria exhibited a Mg2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity with two pH optima, one at pH 6.2 and another at 9.5. Properties of the ATPase at the two pH optima with respect to activation by dinitrophenol, requirement for cations, stimulation by detergents, as well as dependence on ATP and Mg2+ concentrations, were studied. The activity at pH 9.5 was strongly inhibited by oligomycin and azide and only moderately by fluoride. Inverse sensitivity towards the three inhibitors was observed at pH 6.2.4. The mitochondria catalyzed ATP-inorganic phosphate exchange reaction which was inhibited by dinitrophenol, azide and oligomycin.
This chapter discusses two ways of releasing mitochondria from within the yeast cell walls (1) break the cell wall open by force; (2) remove the cell wall by enzymatic digestion. The various methods available and their relative merits are described in the chapter. The most frequently used methods for breaking cell walls by force are those employing small glass beads with either rapid stirring or shaking. Mitochondria are also prepared from yeast cells by either ultrasonic treatment, grinding with Carborundum, or by the use of a French press. An improved method of high-pressure extrusion, which employs the Sorvall Ribi Cell Fractionator, is also used successfully. This instrument allows large quantities of yeast cells to be processed, and the temperature at the point of breaking is carefully controlled by a stream of refrigerated nitrogen. Enzymes from the gut juice of Helix pomatia are used for the digestion of yeast cell walls. The mitochondrial fraction prepared is further purified by density gradient centrifugation. The different methods of preparation described produce mitochondria with varying degrees of structural integrity and differing properties. Yeast mitochondria purified by density gradient flotation contain DNA. A DNA-dependent synthesis of RNA has been described in yeast mitochondria.
This chapter focuses on the preparation and solubilization of bacterial particles. In contrast to the activity found with crude bacterial homogenates or mammalian mitochondria, the isolated particles from bacteria usually exhibit an additional requirement for factors which were apparently solubilized during the disruptive procedure. Bacterial systems that can be fractionated and reconstituted by the addition of essential components liberated from complex structures offer new tools for elucidating the mechanisms involved in oxidative phosphorylation and in protein biosynthesis. Although a large number of enzymes are associated with the large particulate fraction, the preparations are assayed only for their ability to couple phosphorylation to oxidation, since this activity is labile and can be used as an index of structural integrity. The particulate and supernatant fractions are separated and then tested for their ability to couple phosphorylation to oxidation. The assay system consists of the bacterial fractions, either separately or reeombined, a suitable electron donor, orthophosphate, and a phosphate aceeptor system. The consumption of oxygen and the esterification of orthophosphate are measured.
We isolated 2,4-dinitrophenol (DNP)-resistant sake yeast strains by UV mutagenesis. Among the DNP-resistant mutants, we focused on strains exhibiting high malic acid and low acetic acid production. The improved organic acid composition is unlikely to be under the control of enzyme activities related to malic and acetic acid synthesis pathways. Instead, low mitochondrial activity was observed in DNP-resistant mutants, indicating that the excess pyruvic acid generated during glycolysis is not metabolized in the mitochondria but converted to malic acid in the cytosol. In addition, the NADH/NAD(+) ratio of the DNP-resistant strains was higher than that of the parental strain K901. These results suggest that the increased NADH/NAD(+) ratio together with the low mitochondrial activity alter the organic acid composition because malic acid synthesis requires NADH, while acetic acid uses NAD(+).
IT is recognized1 that 2 : 4-dinitrophenol (DNP) stimulates the total oxidation of glucose by baker's yeast, which takes place mostly through the combined action of the Embden-Meyerhof and the citric acid cycles2. Nevertheless, the oxidation of pyruvate, a compulsory intermediate of glucose degradation in yeast, is strongly inhibited by DNP in the same experimental conditions that stimulate glucose oxidation3. In order to elucidate this apparent contradiction our previous experiments have been extended using substrates labelled with carbon-14.
1. Mitochondria exhibiting respiratory control were prepared from bakers' yeast and from Saccharomyces cerevisiae strain D-261 after disrupting the cells in a colloid mill at low speeds with glass beads. Optimal values of the variables involved were determined. Yields of 13–25 mg mitochondrial protein per 100 g packed cells were routinely obtained. Mitochondrial quality was similar to that of particles prepared by a method employing snail gut enzymes to digest cell walls.2. The functions of mitochondria prepared under the mildest homogenization conditions were only slightly affected by large alterations in pH or Mg2+ concentration of the reaction medium.3. Oligomycin and atractyloside were potent inhibitors of phosphorylating respiration in yeast mitochondria.4. Respiration was specifically stimulated by ADP, with an apparent Km = 26.5 μM. Phosphate also stimulated malate-supported respiration, with an apparent Km of 1.5 mM. Varying the phosphate concentration with succinate as substrate led to a transition from ADP-controlled respiration at high phosphate to uncontrolled respiration at low phosphate.5. P:O or ADP:O values for this preparation were about 1.8 during succinate oxidation, 1.8 during ethanol oxidation, 2.4 during α-ketoglutarate oxidation and 1.7 during oxidation of malate plus pyruvate.
Ungerminated uredospores of Puccinia graminis f. sp. tritici Erikss. & Henn. race 15B were disrupted in a buffered sucrose serum albumin solution, using a "Teflon" pestle homogenizer and glass beads 80 μ in diameter. A particulate fraction was sedimented between 2000 and 30,000 × g and its oxidation of Krebs' cycle acids measured manometrically. Endogenous respiration of washed preparations was low or negligible. Oxygen uptake was observed with succinate (40 to 60 μl/hour mg protein), α-ketoglutarate, malate, citrate, and isocitrate (10 to 30 μl/hour mg protein) but not with fumarate or pyruvate. Succinate oxidation was sensitive to heat, cyanide, and malonate.Aspects of the extraction procedure were examined for effects on yield and activity. Serum albumin in the grinding medium, very low grinding speed, and short grinding time favored high activity per mg protein. Glass beads increased the yield with the "Teflon" pestle while an all-glass homogenizer gave preparations of low activity.Oxidatively active particle suspensions reduced Janus green B and were shown by electron microscopy to consist of vesicles and some mitochondria.
A specially designed high-speed blendor and glass beads have been used to disintegrate yeast cells. The method enables large
quantities of cells to be fragmented quickly at low temperature, and cell-free mitochondrial particles to be prepared in high
yield. The particles are isolated in a sucrose-Tris-EDTA medium and extensively refractionated in the same medium. The success
of the fractionation is dependent upon the presence of the Tris buffer, as the latter prevents the aggregation of the particulate
material. Two morphologically and enzymatically different particle types have been obtained: a heavy fraction corresponding
to mitochondria in size and internal organization, and a light fraction consisting of vesicular, single-membrane particles
of a smaller size. The light particles oxidize DPNH and succinate, but do not oxidize pyruvate-malate, and lack the capacity
for phosphorylation. The heavy particles oxidize pyruvate-malate as well as the citric acid cycle intermediates, although
their α-ketoglutaric dehydrogenase activity is low. Oxidation by the heavy particles is coupled to phosphorylation, and P/O
ratios of about 1.5 have been obtained. Lactic acid dehydrogenase is also present in the heavy fraction, and lactate is oxidized
with a P/O ratio of about 0.7.
This chapter discusses the different aspects of oxidative phosphorylation systems. Microorganisms, like other living matter, are dependent on electron-transport systems for the conversion of energy into a chemically utilizable form. In contrast to animal tissues which require organic compounds as sources of energy, various microorganisms obtain energy from the oxidation of a wide variety of compounds, either organic or inorganic. Esterification of phosphate can also be determined by measuring the formation of ATP or glucose-6-phosphate or the incorporation of P32 into these compounds. The ratio between inorganic phosphate disappearance and the atoms of oxygen consumed is the P/O ratio. Coupled phosphorylation with bacterial systems requires the presence of the bacterial extract, a phosphate acceptor system, Mg++, and fluoride to inhibit ATPase. In contrast to mammalian mitochondria, the oxidation observed with bacterial systems capable of coupled activity is usually independent of the presence of a phosphate acceptor system. The effects of both chemical and biological uncoupling agents on oxidative phosphorylation with extracts from M. phlei are also shown in the chapter.
Zellfreie Hefeextrakte führen markierte Mevalonsäure in Squalen über. Es wird über Versuche berichtet, in denen zwei neue Zwischenprodukte isoliert und als Isopentenyl-pyrophosphat und Farnesyl-pyrophosphat identifiziert werden konnten. Die präparative Synthese von Isopentenyl-pyrophosphat wird beschrieben und seine biologische Umwandlung in Farnesyl-pyrophosphat und Squalen diskutiert. Die markierte Verbindung ließ sich mit Enzymen anderer Herkunft in Cholesterin und Kautschuk inkorporieren und erwies sich damit als der universelle Baustein der unter die „Isopren-Regel” fallenden Naturstoffe.
Subcellular fractionsof aerobic yeast contain two forms of l-lactic dehydrogenase with different properties: the particulate enzyme reduces cytochrome c with a smaller velocity relative to ferricyanide reduction than the supernatant l-lactic dehydrogenase; also, the particle-bound l-lactic dehydrogenase is not inhibited by anticristalline-l-lactic dehydrogenase serum which inactivates soluble l-lactic dehydrogenase. By liberating the bound l-lactic dehydrogenase with the help of a detergent, it is shown that the differences between the two forms are due to the linkage of the enzyme to the particles. Bound l-lactic dehydrogenase seems to be absent from the particles in the course of respiratory adaptation.RésuméLes fractions subcellulaires de la levure aérobie contiennent deux formes de la l-lacticodehydrogenase qui se distinguent par deux propriétés: 1. La l-lacticodehydrogenase liée aux particules respiratoires réduit le cytochrome c avec une vitesse relative par rapport au K3Fe(CN)6 plus faible que la l-lacticodéshydrogénase du surnageant. 2. La l-lacticodéshydrogénase liée résiste aux immunsérums anti-l-lacticodéshydrogénasecrist. qui inhibent l'enzyme soluble. En détachant, par un détergent, la l-lacticodéshydrogénase liée, on montre que ces différences sont dûe á la liasion de l'enzyme avec les particules. La l-lacticodéshydrogénase liée semble être absente des particules au cours de l'adaptation respiratoire.
This chapter contains section titled:
IntroductionBiosynthesis of Cholesterol from AcetateEarly Stages of the BiosynthesisIntermediary Stages of Squalene Synthesis from MevalonateGeneral Mechanism of Squalene BiosynthesisCyclization of SqualeneLanosterol to CholesterolPerspective
Two commercially available ultrasonic instruments are described, in which micro-organisms and other cells may be disintegrated.Sound waves (20 kc/s) are generated in the cell suspension by means of titanium velocity transformers (probes) coupled to a magnetostriction transducer. One instrument operating at 500 W will disintegrate from 2 to 500 ml, the other operating at 50 W from 1 ml to 30 ml.Yeast was used as a test organism but results on some other organisms are also given. Cell rupture is shown to be independent of sonically generated free radicals, but enzyme inactivation (alcohol dehydrogenase) is accelerated by free radicals. Increasing the viscosity, decreasing surface tension of the suspending medium, or the presence of CO2 decreases disintegration. The addition of solid nuclei such as powdered glass, as well as small air bubbles, increases disintegration.This is consistent with disintegration being due to cavitation, but does not indicate the precise mechanism of cell rupture. Some effects of sonic disruption are compared with disruption by other methods.
Baker's yeast oxidizes pyruvic and acetic acids mostly through the Krebs cycle. The substrate carbon is partly assimilated into reserve carbohydrates and into amino acids derived from the cycle intermediates. At pH 4.8 (or less), 0.05 mM DNP (2,4-dinitrophenol) inhibits the oxidation and assimilation of acetic and pyruvic acids by starved yeast. Glucose oxidation is not affected or stimulated by DNP, notwithstanding that pyruvate and acetate are intermediates of glucose oxidation. DNP interferes with the accessibility of exogenous pyruvic acid to the enzyme systems concerned with its oxidation and decarboxylation, while with acetic acid DNP prevents its incorporation into citrate and its further oxidation in the Krebs cycle. The oxidation of endogenous substrates is promoted by simultaneous oxidation of exogenous acetic and pyruvic acids; DNP prevents this effect of pyruvic acid.
Immunology and Cell Biology focuses on the general functioning of the immune system in its broadest sense, with a particular emphasis on its cell biology. Areas that are covered include but are not limited to: Cellular immunology, Innate and adaptive immunity, Immune responses to pathogens,Tumour immunology,Immunopathology, Immunotherapy, Immunogenetics, Immunological studies in humans and model organisms (including mouse, rat, Drosophila etc)
RESPIRATORY chain phosphorylation has been demonstrated in crude extracts of a few bacterial species1-4, and in one case the extract was separated into three components which were all necessary for phosphorylation coupled with the oxidation of reduced diphosphopyridine nucleotide2. However, the elements supporting the system of enzymes responsible for oxidative phosphorylation in bacterial cells is still unknown. The present communication describes some preliminary experiments in which a crude water-extract from Azotobacter vinelandii was fractionated by high-speed centrifugation into particles and `soluble' fractions. This organism was chosen because it is strictly aerobic (cf. ref. 2) and has a very high respiratory activity.
1.1. A procedure for the isolation of actively respiring particles from yeast is described. These particles are considered to be mitochondria. They stain vitally with Janus green B and oxidize the main citric acid cycle substrates as well as pyruvate and acetate.2.2. The washed mitochondria oxidize pyruvate only in the presence of a catalytic amount of one of the Krebs cycle substrates. In the presence of catalytic amounts of malate, pyruvate is oxidized to carbon dioxide and water.3.3. The requirements for acetate oxidation are more specific than those for pyruvate. Unlike pyruvate, only α-ketoglutarate and citrate initiate acetate oxidation. The pathway by which acetate is dissimilated has not been established.
1.1. A high-speed shaker has been used for the preparation of actively respiring extracts of baker's yeast, Aerobacter aerogenes and Proteus vulgaris OX 19. Disintegration periods ranged from 10 to 40 seconds.2.2. 10-second yeast extracts contain granules which oxidise isocitrate, α-ketoglutarate, succinate, lactate and ethanol, and which account for a large proportion of the respiration of the whole extract.3.3. 15-second extracts of Proteus oxidise many substrates, and the isolated granules oxidise succinate, lactate and formate. With lactate as substrate, whole extracts and, to a lesser extent, high-speed supernatants, show a phosphorylation sensitive to 2,4-dinitrophenol; the granules alone show no phosphorylation.4.4. 15-second extracts of Aerobacter also oxidise a variety of substrates, but the isolated granules oxidise succinate and lactate only. Whole extracts and high-speed supernatant fractions, but not the granules, show a phosphorylation insensitive to 2,4-dinitrophenol with glucose as the substrate. The granules have slight phosphorylating activity with lactate as substrate.5.5. The relation of the granules of micro-organisms to animal mitochondria is discussed.
FRESHLY prepared suspensions of the granular components of heart-muscle (sarcosomes1, interstitial granules2 or mitochondria3) are able to oxidize a number of intermediary metabolites, and to couple this oxidation with the synthesis of adenosine triphosphate from adenosine diphosphate and inorganic phosphate (oxidative phosphorylation)4,5. In agreement with the experience of other workers using preparations from different tissues, it has been found that the heart-muscle sarcosomes rapidly lose their ability to carry out oxidative phosphorylation, especially at temperatures above 0° C. Such aged preparations oxidize succinate at practically unimpaired rate, but the accompanying phosphorylation is lost. The ability to oxidize α-ketoglutarate is lost to about the same extent as the phosphorylation associated with this oxidation, so that the P : O ratio (the number of atoms of inorganic phosphate esterified per atom of oxygen consumed) is not greatly affected. The lability of rat heart sarcosomes is indicated by the following percentages of inactivation of the α-keto-glutaric oxidase system (the enzyme complex required for the aerobic oxidation of α-ketoglutarate to succinate) : 10 min. standing at 15.5° C., 52 per cent inactivation ; 15 min. at 25° C., 94 per cent inactivation. Cat preparations are somewhat more stable (15 min. standing at 25° C., 71 per cent inactivation).
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