RESUMO
The biosynthesis of L-lactate oxidase in the Yarrowia lipolytica yeast during submerged cultivation in laboratory bioreactors ANKUM-2M has been studied. It has been shown under optimal conditions of yeast cultivation with L-lactate that 24.5 U/L enzyme accumulated in the medium and the yield was 2.0 U/(L h). An increase in the biosynthesis of L-lactate oxidase to 75 U/L and the yield to 3.2 U/(L h) was achieved in the medium with L-lactate (1%) and glucose (2%). The enzyme was purified 251 times to homogeneity by hydrophobic and ion exchange chromatography state with a yield of 45% and a specific activity of 55.3 U/mg. Techniques of gel filtration and denaturing electrophoresis showed that L-lactate oxidase from Y. lipolytica is a tetramer with a molecular mass of 200230 kDa. The enzyme showed a strict specificity to L-lactate and did not oxidize fumarate, pyruvate, succinate, ascorbate, dihydroxyacetone, glycolate, D-lactate, D, L-2-hydroxybutyrate and D, L-alanine or D-serine.
Assuntos
Proteínas Fúngicas/biossíntese , Oxigenases de Função Mista/biossíntese , Yarrowia/metabolismo , Reatores Biológicos , Cromatografia por Troca Iônica , Fermentação , Proteínas Fúngicas/isolamento & purificação , Glucose/metabolismo , Cinética , Ácido Láctico/metabolismo , Oxigenases de Função Mista/isolamento & purificação , Peso Molecular , Multimerização Proteica , Especificidade por SubstratoRESUMO
The authors' and literature data on the adaptation response of the micromycetes Yarrowia lipolytica to various stress impacts are considered in the review. The uniformity of cellular response to all stress factors is discussed.
Assuntos
Yarrowia/fisiologia , Adaptação Fisiológica , Antioxidantes/metabolismo , Catalase/metabolismo , Oxidantes/farmacologia , Oxirredutases/metabolismo , Estresse Fisiológico , Superóxido Dismutase/metabolismo , Yarrowia/efeitos dos fármacos , Yarrowia/ultraestruturaRESUMO
An effector of tissue stress of hepatocytes, prodigiozan-dependent comuton (PDC), provokes deenergiezation of liver mitochondria, preloaded by Ca2+ ions. In this case a decrease of membrane potential (MP) and Ca2+ efflux by cyclosporine A sensitive mechanism of megapore is observed. If megapore is blocked by cyclosporin A, protonofor FCCP provoked decrease of MP and Ca2+ efflux by cyclosporin A-insensitive mechanism. It is shown that PDC increases resistance of mitochondria to mentioned protonofor action by inhibition of both these effects. An inhibitory action of PDC is realized by K+ and NADH-dependent mechanism. The effector of hepatocyte tissue stress, prodigiozan-dependent comuton (PDC), evokes deenergizing liver mitochondria preloaded with Ca2+, both membrane potential (MP) decrease and Ca2+ release in according to cyclosporine A-sensitive mechanism of megapore being observed. If megapore is blocked by cyclosporin A, protonophore FCCP reduces of MP and Ca2+ release in according to cyclosporin A-insensitive mechanism. PDC is shown to increase the resistance of mitochondria against protonophore action mentioned above by means of inhibition of both these effects. Inhibitory action of PDC is realized due to both K+ and NADH-dependent mechanism. Protective effect takes place only in intact mitochondria of these cells providig (on condition that) its megapore mechanism is not activated. Moreover, the results obtained are evidence of PDC can function as protector due to intensification of energy generation in damaged.
Assuntos
Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Prodigiozan/farmacologia , Ionóforos de Próton/farmacologia , Animais , Cálcio/metabolismo , Ciclosporina/farmacologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Substâncias Protetoras/farmacologia , RatosRESUMO
Endotoxine activated Kupfer cells release into the intercellular space several mediators which act directly on hepatocytes as well as via stellet cells. In both cases Kupfer cells downregulate hepatocytes as a part of paracrine system. However, downregulated part of liver parenchyma might be extended by several mechanisms. The first one is release of vasoconstrictors from activated Kupfer cells which stimulate stellet cells contraction. This effect may also be achieved by formation of hypermetabolicfocuses by Kupffer cells mediators with further activation of hepatocyte-hepatocyte interactions based on the principle of cell competition for oxygen in the intercellular space. Regulatory influence of activated Kupfer cells may be spread in liver parenchyma with participation of the mechanism of intratissue hepatocyte-hepatocyte interactions which also realize tissue stress reaction.
Assuntos
Hepatócitos/citologia , Hepatócitos/fisiologia , Células de Kupffer/citologia , Células de Kupffer/fisiologia , Fígado/citologia , Animais , Cálcio/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/fisiologia , Prodigiozan/farmacologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
We studied the effect of quinocitrinines on the respiratory activity of yeasts (Yarrowia lipolytica) and bacteria (Arthrobacter globiformis). Quinocitrinines were shown to activate respiration of native cells in both types of organisms. Studies of yeast mitochondria showed that quinocitrinine exerts an uncoupling effect on oxidative phosphorylation, which activates the respiration, reduces the respiratory control, and decreases the ADP/O ratio. Experiments with intact mitochondria and native cells of Arthrobacter globiformis revealed that quinocitrinine decreases the membrane potential. The uncoupling effect likely constitutes a mechanism of the antibiotic activity of quinocitrinines.
Assuntos
Antibacterianos/farmacologia , Arthrobacter/efeitos dos fármacos , Penicillium , Quinolinas/farmacologia , Yarrowia/efeitos dos fármacos , Difosfato de Adenosina/análise , Difosfato de Adenosina/metabolismo , Antibacterianos/química , Antibacterianos/isolamento & purificação , Arthrobacter/fisiologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Oxigênio/análise , Oxigênio/metabolismo , Quinolinas/química , Quinolinas/isolamento & purificação , Yarrowia/fisiologiaRESUMO
Electron transfer via the alternative pathway in cyanide-resistant mitochondria of the yeast Candida lipolytica is not coupled with ATP synthesis, generation of membrane potential or energy-dependent reverse electron transport in the main respiratory chain. We conclude that during transfer via the alternative pathway no accumulation of energy in the form of high-energy compounds or membrane potential occurs.
Assuntos
Candida/metabolismo , Cianetos/farmacologia , Mitocôndrias/metabolismo , Metabolismo Energético , Transferência de Energia , Cinética , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacosRESUMO
We studied biosynthesis of colored naphthoquinone metabolites by Fusarium decemcellulare, F. graminearum, and F. bulbigenum fungi. F. bulbigenum and F. graminearum synthesized bikaverin and aurofusarin, respectively, which depended on the conditions of cultivation. F. decemcellulare synthesized soluble extracellular naphthoquinones of the naphthazarin structure (javanicin, anhydrojavanicin, fusarubin, anhydrofusarubin, bostricoidin, and novarubin) or extracellular dimeric naphthoquinone aurofusarin. Biosynthesis of naphthoquinone pigments was shown to be the main fungal response to stress exposure. It occurs under conditions of growth inhibition or arrest.
Assuntos
Fusarium/fisiologia , Naftoquinonas/metabolismo , Antifúngicos/farmacologia , Antimicina A/farmacologia , Meios de Cultura , Fusarium/crescimento & desenvolvimento , Fusarium/metabolismo , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Naftoquinonas/química , Nitrogênio , Oxidantes/farmacologia , Fósforo , Xantonas/metabolismoRESUMO
Structures and physico-chemical properties of 100 naphthoquinone metabolites produced by filamentous fungi are reviewed. The conditions of pigment formation, biogenesis and the mechanism of biosynthesis of pigments by fungi are described. Sixty-three fungi cultures able to produce naphthoquinone are listed. The biological activities of the main pigments and the mechanism of fungal resistance to their own metabolites are described. The physiological role of the naphthoquinones in producers is discussed.
Assuntos
Fungos/fisiologia , Naftoquinonas/química , Naftoquinonas/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Fungos/metabolismo , Estrutura Molecular , Naftoquinonas/farmacologia , Pigmentos Biológicos/biossíntese , Pigmentos Biológicos/metabolismoRESUMO
Twenty isolates of Fusarium graminearum have been isolated from infested grain. When laying hens were fed with biomass of most isolates, the quality of eggs deteriorated. It was found that all the isolated produced a yellow-orange metabolite with an antibiotic activity against mycelial fungi and yeasts. The metabolite was identified by physico-chemical methods as the dimeric naphthoquinone aurofusarin. The production of the other mycotoxins zearalenone and deoxynivalenol by the fungal isolates did not evoke the syndrome of egg quality deterioration.
Assuntos
Ovos , Fusarium/química , Micotoxinas/análise , Naftoquinonas/análise , Doenças das Aves Domésticas/induzido quimicamente , Ração Animal/análise , Animais , Galinhas , Contaminação de Alimentos/análise , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Micotoxinas/toxicidade , Naftoquinonas/toxicidade , Espectrofotometria UltravioletaRESUMO
Conditions for L-lactate oxidase synthesis by the yeast Yarrowia lpolytica were investigated. The enzyme was found to be synthesized during growth on L-lactate in the exponential growth phase. L-lactate oxidase synthesis was observed, also on glucose after adaptation to stress conditions (oxidative or thermal stress) r during the stationary growth phase after glucose consumption. The cells grown on L-lactate exhibited high levels of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase), which exceeded those of glucose-grown cells. The ultrastructure of L-lactate-grown cellsand of those grown on glucose and adapted to various stress.conditions was also found to besimilar, with increased mitochondria, elevated number and size ofperoxisomes, and formation of lipid and polyphosphate inclusions. In order to determine the intracellular localization of L-lactate oxidase, the cells were disintegrated by the lytic enzyme complex from Helix pomatia. Centrifugation of the homogenate in Percoll gradient resulted in the isolation of purified fractions of the native mitochondria and peroxisomes. L-Lactate oxidase was shown to be localized in peroxisomes.
Assuntos
Proteínas Fúngicas/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Oxigenases de Função Mista/biossíntese , Peroxidase/metabolismo , Yarrowia/enzimologiaAssuntos
Peptídeos Catiônicos Antimicrobianos , Bactérias/crescimento & desenvolvimento , Proteínas Fúngicas , Trichoderma/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/farmacologia , Peso MolecularRESUMO
During previous decade L-amino acid oxidases (LAAO) attracted the steady interest of researchers due to their poly functional effects on different biological systems. The review summarizes information concerning the sources, structure, phisico-chemical and catalytical properties of LAAO which exhibit antibacterial, antifungal, antiprotozoal, antiviral effects as well as the ambiguous action on platelet aggregation. Special attention is devoted to the elucidation of molecular mechanisms of LAAO action. It is proposed that the unique properties of LAAO) are based on their catalytic reaction, which causes the decrease of L-amino acid levels, including the essential amino acids and formation of hydrogen peroxide. The action of liberated H2O2 on cells involves the synthesis of oxygen reactive species and the development of necrotic and apoptotic pathways of cell death. The presence of carbohydrate moieties in LAAO molecules promotes their attachment to cell's surface and creation of high H2O2 local concentrations. The wide range of LAAO biological effects is undoubtedly connected with their important functional roles in the organism. In particular, it was shown that in the mice brain the LAAO-catalyzed reaction is the single pathway of L-lysine degradation, while in the mice milk LAAO carry out the antibacterial effect and in human leucocytes LAAO take part in fulfilling their defending role. Protector action may be also attributed to the oxidases from the other numerous sources: microscopic fungi, snake venoms and sea inhabitants.
Assuntos
Aminoácido Oxirredutases/metabolismo , Aminoácidos/metabolismo , Aminoácido Oxirredutases/imunologia , Aminoácidos/imunologia , Animais , Catálise , Humanos , Peróxido de Hidrogênio/imunologia , Peróxido de Hidrogênio/metabolismo , OxirreduçãoAssuntos
Antibacterianos/farmacologia , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Triazinas/farmacologia , Adenosina Trifosfatases/metabolismo , Animais , Antimicina A/farmacologia , Candida/citologia , Candida/efeitos dos fármacos , Candida/metabolismo , Carcinoma de Ehrlich/patologia , Cianetos/farmacologia , Transporte de Elétrons , Concentração de Íons de Hidrogênio , Ácidos Cetoglutáricos/metabolismo , Malonatos/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , NAD/metabolismo , Oxirredução , Fenil-Hidrazinas/farmacologia , Polarografia , Potenciometria , Pirimidinonas/farmacologia , Ratos , Rotenona/farmacologia , Especificidade da Espécie , Espectrometria de Fluorescência , Succinatos/metabolismoAssuntos
Candida/metabolismo , Citratos/metabolismo , Glucose/metabolismo , Lactatos/metabolismo , Mitocôndrias/metabolismo , Difosfato de Adenosina/metabolismo , Arsênio/farmacologia , Candida/citologia , Candida/efeitos dos fármacos , Cianetos/farmacologia , Transporte de Elétrons , Glicerofosfatos/farmacologia , Hidrazonas/farmacologia , Cinética , Mitocôndrias/efeitos dos fármacos , NAD/metabolismo , NADP/metabolismo , Oligomicinas/farmacologia , Rotenona/farmacologia , Espectrometria de Fluorescência , Succinatos/farmacologia , Fatores de TempoRESUMO
Heat shock (45 degrees C) and the effect of oxidants (H2O2) resulted in a decrease of the respiratory activity of yeast cells and their survival rate. Increased resistance to stress effects after mild heat treatment (37 degrees C) or treatment with a nonlethal dose of oxidants (0.5 mM H2O2 for 60 min) was accompanied by appearance of an alternative (cyanide-resistant) oxidative pathway in the mitochondria, which promotes survival due to retention of the capacity for ATP synthesis in the first coupling point at the level of endogenous NADH dehydrogenase. The alternative oxidative pathway is more resistant to the effect of stressors that disrupt electron transfer in the cytochrome site of the respiratory chain.
Assuntos
Temperatura Alta , Estresse Oxidativo , Oxigênio/metabolismo , Yarrowia/fisiologia , Adaptação Fisiológica , Cianetos/farmacologia , Proteínas Fúngicas/metabolismo , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais , NADH Desidrogenase/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas , Yarrowia/efeitos dos fármacosAssuntos
Aminoácido Oxirredutases/biossíntese , Proteínas Fúngicas/biossíntese , Peróxido de Hidrogênio/metabolismo , Cloreto de Sódio/farmacologia , Trichoderma/efeitos dos fármacos , Aminoácido Oxirredutases/metabolismo , Aminoácidos/metabolismo , Meios de Cultura , Fibras na Dieta/metabolismo , Ensaios Enzimáticos , Proteínas Fúngicas/metabolismo , Cinética , Salinidade , Estresse Fisiológico , Trichoderma/metabolismoRESUMO
The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45 degrees C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. The 60-min preincubation of cells at 37 degrees C or pretreatment with low concentrations of H2O2 (0.5 mM) and menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).