RESUMEN
Structually diverse peroxisome-proliferators (PPs) were investigated regarding their effects on NAD+ level and two key enzyme activities in the tryptophan (Trp)-NAD+ pathway in the liver of rats (Sprague-Dawley male) fed PP-containing diets freely for 2 weeks. All PPs, except for thyroxine, significantly increased hepatic NAD+ level in concert with hepatic hypertrophy. Activity of quinolinate phosphoribosyltransferase (QAPRTase), one of the key enzymes in the Trp-NAD+ pathway, was increased by the PPs which caused significant increase in the hepatic NAD+. On the other hand, alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSDase), another key enzyme in the Trp-NAD+ pathway, was drastically inhibited by all PPs except for linolenic acid, which was only slightly inhibitory. Most PPs investigated activated peroxisomal marker enzymes such as palmitoyl-CoA oxidase, catalase, and PPAR-alpha(peroxisome-proliferator activated receptor-alpha)-dependent enzymes, such as malic enzyme and l-3-glycerophosphate dehydrogenase. NAD+ was also increased in the rat hepatocytes cultured in the medium supplemented with PPs. These data suggested that regulation of the key enzymes in the Trp-NAD+ pathway was associated with PPAR-alpha directly or indirectly, and as a consequence the hepatic NAD+ was increased by PPs.
Asunto(s)
Hipertrofia/inducido químicamente , Hígado/enzimología , NAD/metabolismo , Proliferadores de Peroxisomas/farmacología , Triptófano/metabolismo , Animales , Biomarcadores , Peso Corporal/efectos de los fármacos , Células Cultivadas , Ingestión de Alimentos/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Organismos Libres de Patógenos EspecíficosRESUMEN
Some pseudo-peptide analogs of thiol proteinase inhibitors were synthesized by a conventional solution method. Among them, Suc-Ala-Val-Val-Ala-psi-(CH2-NH)-Ala-pNA (peptide 1) and Suc-Ala-Val-Val-psi-(CH2-NH)-Ala-Ala-pNA (peptide 2) showed a stronger inhibitory activity compared with parent peptide such as Suc-Ala-Val-Val-Ala-Ala-pNA. In particular, peptide 2 was about 10-fold as active as the parent peptide (IC50 = 8 microM). Inserting psi-(CH2-NH) possibly makes the inhibitor less susceptible to papain and, as a result, produces more potent inhibition.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Oligopéptidos/síntesis química , Inhibidores de Proteasas/síntesis química , Tampones (Química) , Cinética , Oligopéptidos/farmacología , Papaína/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Relación Estructura-ActividadRESUMEN
The mechanism of the elevation of hepatic NAD level in the rats administered clofibrate and other peroxisome-proliferators were investigated. In the hepatocytes from the clofibrate-fed rats, NAD biosynthesis from Trp, but not from nicotinic acid, was specifically stimulated. The activities of peroxisomal marker enzymes, the peroxisome-proliferator activated receptors (PPAR)-dependent enzymes and key enzymes in the Trp-NAD pathway changed in parallel with the hepatic NAD increase; the activity of quinolinate phosphori-bosyltransferase (QPRT) was increased whereas that of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) was drastically reduced. The results strongly suggest that the hepatic NAD increase might be caused by transcription of genes coding the key enzymes of the Trp-NAD pathway via PPAR.
Asunto(s)
Hígado/metabolismo , NAD/metabolismo , Proliferadores de Peroxisomas/farmacología , Triptófano/metabolismo , Animales , Carboxiliasas/metabolismo , Células Cultivadas , Clofibrato/farmacología , Femenino , Hígado/efectos de los fármacos , Masculino , NADP/metabolismo , Niacina/metabolismo , Oxidación-Reducción , Pentosiltransferasa/metabolismo , Peroxisomas/enzimología , Fenotiazinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiologíaRESUMEN
OBJECTIVE: To survey the present condition of community pharmacies as future sites for pharmacy students' externship in Japan. METHOD: A questionnaire consisting of 55 questions was sent to 425 graduates from Kobe Gakuin University, School of Pharmacy, who owned or worked in community pharmacies. RESULTS: Of the 85 responders, about half were owners and half employees of pharmacies. Ninety per cent of pharmacy owners operated three and fewer pharmacies. Fifty per cent of pharmacies only dispensed drugs, 32% handled both OTC drugs and dispensing, and 18% handled only OTC drugs. Among the 44 dispensing pharmacies, 16 were one-to-one type pharmacies, 13 were located in front of the big medical institutions, nine dispensed prescriptions from various medical institutions and five were hospital-owned pharmacies. Forty-five per cent of pharmacies employed 1-4 part-time pharmacists and 52% employed 1-4 pharmacist assistants. Thirty-one per cent of prescriptions came from internal medicine departments and the daily number of prescriptions dispensed by each pharmacy was in the range 10-99 for 41% of the pharmacies and 100-199 for 36% of the pharmacies. The average daily number of prescriptions dispersed by each pharmacist was in the range 30-39 for 29% of pharmacies and in the range 20-29 for 22% of pharmacies. Pharmaceutical information was provided at 73% of pharmacies and patients were counselled orally on their medication at 80% of pharmacies. Patients' medication histories were recorded at 88% pharmacies. Only 15% of pharmacies conducted patients' medication counselling at their home, but 34% of pharmacies were planning to start this service. Community pharmacists attended very few professional meetings or continuing education programmes and only 20% of them obtained information through computers. Forty-seven pharmacists out of the 85 obtained their information from medical representatives of pharmaceutical companies and 32 pharmacists through marketing specialists of wholesalers. Ninety per cent of community pharmacists who responded thought that separation of prescribing and dispensing functions would progress further in the future and 50% of them thought positively about the future social status of pharmacists. Most of our graduates who responded were willing to accept pharmacy students from Kobe Gakuin University as externs at their pharmacies. CONCLUSION: Despite the low response rate, our survey suggests that community pharmacy in Japan requires substantial improvement. This is to ensure that pharmacists working in that sector can provide the quality information that is required for the optimum management of patients and that the environment is suitable for pharmacy externships.
Asunto(s)
Educación en Farmacia/métodos , Farmacias , Recolección de Datos , Prescripciones de Medicamentos/estadística & datos numéricos , Femenino , Humanos , Japón , Masculino , Farmacias/normas , Farmacia , Recursos HumanosRESUMEN
The gene for the major spore-specific protein, termed protein W, was cloned, and it was found that protein W is composed of 426 amino acid residues including 31% charged (133 residues) and 39% hydrophobic (166 residues) amino acids. In the protein, a motif consisting of five amino acid residues [(V/L/I)-R-E-R-(V/L/I)] is repeated 28 times, and another motif [M-M-(P/G)-Q-G] five times. Protein W is synthesized during a very late stage of development, forming a single, large electron-dense particle (200-400 nm in diameter) inside a spore. X-ray microanalysis of the particle revealed that it contains a high amount of phosphate in addition to calcium and magnesium. It is proposed that protein W consisting of highly charged repetitive sequences is a polyphosphate storage protein to store energy in spores. The disruption of the gene for protein W resulted in delayed fruiting body formation and a lower spore yield.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Myxococcus xanthus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Microanálisis por Sonda Electrónica , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Myxococcus xanthus/genética , Myxococcus xanthus/crecimiento & desarrollo , Mapeo Restrictivo , Análisis de Secuencia de ADN , Esporas Bacterianas/metabolismoRESUMEN
The effect of the phenothiazines, thioridazine and chlorpromazine, on the increased hepatic NAD+ level of rats fed clofibrate, a hypolipidaemic drug, has been investigated. Short-term (6 days) addition of phenothiazines to the diet negatively affected diet intake and body-weight gain, but increased liver weight and hepatic NAD+ levels, which was synergistic to clofibrate. The phenothiazines were shown to inhibit hepatic peroxisomal fatty acid oxidation in-vivo, as determined by the increased residual catalase activity. In hepatocytes prepared from clofibrate-fed rats, phenothiazines inhibited not only peroxisomal but also mitochondrial fatty acid oxidation to the same extent. In the hepatocytes, NAD+ was maintained at the high level until the phenothiazine concentration was increased to 0.2 mM. The result suggests that the increase of hepatic NAD+ in rats fed clofibrate is not related to peroxisomal fatty acid oxidation.
Asunto(s)
Antipsicóticos/farmacología , Clorpromazina/farmacología , Clofibrato/farmacocinética , Hipolipemiantes/farmacocinética , Hígado/enzimología , NAD/metabolismo , Tioridazina/farmacología , Animales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
NAD+ biosynthesis from tryptophan in the presence of nicotinic acid or vice versa by rat hepatocytes was investigated. In the control hepatocytes, NAD+ synthesis from tryptophan was not affected by nicotinic acid from 0.026 to 0.26 mM. NAD+ synthesis from nicotinic acid was slightly inhibited with varying concentrations of tryptophan from 0.1 to 1.0 mM. In the clofibrate-treated hepatocytes, NAD+ synthesis from tryptophan was greatly increased (234% of the control), while that from nicotinic acid was decreased (71.2% of the control). Both, NAD+ synthesis from tryptophan and that from nicotinic acid were decreased by the coexisting nicotinic acid or tryptophan. Total amount of NAD+ synthesized from tryptophan and nicotinic acid at their physiological concentrations was significantly higher than that in the control hepatocytes as a result of a large increase of NAD+ synthesized from tryptophan. When the metabolic flux of 0.1 or 0.5mM tryptophan was investigated, the glutarate pathway was suppressed in the clofibrate-treated hepatocytes, the quinolinic acid-NAD+ flux being elevated. Similarly to clofibrate, DEHP and CPP revealed an increase in NAD+ synthesis from tryptophan. Mutual relationship of NAD+ biosyntheses from tryptophan and nicotinic acid in rat hepatocytes is discussed and the relevance with peroxisomal proliferation is suggested.
Asunto(s)
Clofibrato/farmacología , Hígado/metabolismo , NAD/biosíntesis , Niacina/metabolismo , Triptófano/metabolismo , Animales , Transporte Biológico , Glutaratos/metabolismo , Hidrolasas/metabolismo , Hígado/efectos de los fármacos , Masculino , Microcuerpos/efectos de los fármacos , Niacina/farmacología , Ratas , Ratas Sprague-Dawley , Triptófano/farmacologíaRESUMEN
Hepatic NAD+, NADH, and NADPH were increased significantly 3 days after feeding rats with a 0.25% clofibrate diet, increased further after 8 days, and stayed at the same levels 14 days after feeding the diet. The NAD+/NADH ratio was decreased significantly by feeding the clofibrate diet for 8 days, while the ratio remained unchanged with a 1% pyrazinamide diet. Hepatic quinolinate phosphoribosyltransferase (QAPRTase) (EC 2.4.2.19) activity was increased to 1.8 and 1.3 times that of the control animals in the clofibrate- and the pyrazinamide-fed rats, respectively, while hepatic aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase) (EC 4.1.1.45) activity was decreased to 0 and 19% of that of the control animals. The heat-treated liver homogenate from the pyrazinamide-fed rats contained inhibitory activity toward ACMSDase, while no inhibitory activity was found in the liver homogenate of the clofibrate-fed animals. We conclude that these changes of enzyme activities, which seem due to different mechanisms, may contribute to the increase of pyridine nucleotides in the liver of rats fed clofibrate or pyrazinamide.
Asunto(s)
Clofibrato/administración & dosificación , Hipolipemiantes/administración & dosificación , Hígado/metabolismo , NADP/metabolismo , NAD/metabolismo , Pirazinamida/administración & dosificación , Animales , Dieta , Femenino , Hígado/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The effects of preparations of Chinese medicinal prescriptions on the activities of digestive enzymes were investigated. The starch dextrinizing activity of pancreatin was inhibited by Keishi-bukuryo-gan, Sho-seiryu-to, Hachimi-jio-gan, Unsei-in and Keishi-ka-shakuyaku-to to below 40% of the control activity. Hachimi-jio-gan and Sho-seiryu-to, in particular, lowered the activity to 4% and 24% of the control activity, respectively. The protein digestive activity of pancreatin was lowered by Keishi-bukuryo-gan, Oren-gedoku-to, Ryutan-shakan-to, Tokaku-joki-to, Yokuinin-to and Hange-shashin-to, to 56 to 70% of the control activity. To investigate the effects of the preparations of Chinese medicinal prescriptions on digestive enzymes in vivo, 600 mg (185 kBq) of 125I-egg white albumin were administered orally to rats 30 min before the administration of 100 mg of Tokaku-joki-to or Oren-gedoku-to. The radioactivity which transferred to the blood was less in the animals pre-fed these prescriptions than in the control animals, indicating that the digestion of egg white albumin was delayed in the presence of the prescriptions.
Asunto(s)
Amilasas/metabolismo , Sistema Digestivo/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Pancreatina/metabolismo , Pepsina A/metabolismo , Albúminas/metabolismo , Animales , Sistema Digestivo/enzimología , Humanos , Radioisótopos de Yodo , Cinética , Masculino , Ratas , Ratas WistarRESUMEN
The complete nucleotide sequence of plasmid pLA106 (2862 bp) from Lactobacillus acidophilus TK8912 was determined. Based on this sequence, the location of the genes for mobilization-plasmid recombination protein (mob), replication origin (ori), transcriptional repressor protein (repA) and replication initiation protein (repB) were predicted. Deletion analysis showed that the 1.4-kb PstI-EcoRV fragment carrying the ori, repA and repB genes is able to replicate in Lactobacillus and Escherichia coli cells. The plasmid pLA106 appears to have most features of the pLS1/pE194 plasmid family.
Asunto(s)
Replicación del ADN , Lactobacillus acidophilus/genética , Plásmidos , Secuencia de Aminoácidos , Escherichia coli/genética , Datos de Secuencia MolecularRESUMEN
The effect of clofibrate, a hypolipidemic drug and also known as a peroxisomal proliferator, on the conversion ratio of tryptophan to niacin was investigated by using rats. The rats were fed with a nicotinic acid-free, 20% casein diet (control group) or the same diet + 0.25% clofibrate group) for 19 days. The conversion ratio gradually increased with increasing number of days. Around day 8, the ratio was about 10-times higher in the clofibrate group than in the control group, and the value remained almost constant after that day. The content of liver total nicotinamide was higher in the clofibrate group than in the control group. Among the enzymes involved in the conversion of tryptophan to niacin, the aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase) activity, which is critical in the conversion, was lower in the clofibrate group than in the control group. As the change in ACMSDase activity took several days, there is a possibility that clofibrate decreased the biosynthesis of ACMSDase protein and/or mRNA. To learn whether the increase in the conversion ratio by clofibrate would be nutritionally meaningful or not, the growth-promoting activity of clofibrate was determined by using weanling rats fed with a nicotinic acid-free, tryptophan-limiting diet (basal diet). As a result, the body weight gain was higher in the clofibrate group than in the basal group. This result shows that clofibrate enhanced the conversion ratio without any side-effects under the conditions used and supports again the claim that the activity of ACMSDase exerts a critical influence on the tryptophan-NAD conversion.
Asunto(s)
Clofibrato/farmacología , Hipolipemiantes/farmacología , Niacina/metabolismo , Triptófano/metabolismo , Animales , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , NAD/sangre , Niacina/farmacología , Niacinamida/metabolismo , Ratas , Ratas Wistar , Triptófano/orina , Destete , Aumento de Peso/efectos de los fármacosRESUMEN
Hepatocytes were isolated from rats fed a diet with or without 0.25% clofibrate, and NAD+ synthesis by the hepatocytes was determined using either [carboxyl-14C]nicotinic acid or [5-3H]tryptophan. NAD+ and total pyridine nucleotides synthesized from [14C]nicotinic acid by the clofibrate-treated cells were not significantly different from those synthesized by the control cells when expressed on the basis of nanomoles per hour per milligram of DNA. On the contrary, NAD+ synthesized from [3H]tryptophan was significantly higher in the clofibrate-treated cells (158% of the control cells) on the basis of nanomoles per hour per milligram of DNA. Clofibrate was inhibitory to tryptophan metabolism as a whole, affecting the glutarate pathway more (decreased to 37% of control) than the kynureninase flux (decreased to 64% of control). As a result, the quinolinate-NAD flux, estimated as the difference in the amounts of tryptophan metabolized by the two metabolic pathways, increased in the clofibrate-treated hepatocytes. The increase in quinolinate during the incubation was 8 times more in the clofibrate-treated cells than in the control cells, which confirmed alteration in the metabolic fluxes of tryptophan in the clofibrate-treated cells. Hepatic quinolinate phosphoribosyltransferase (EC 2.4.2.19) activity increased with dietary clofibrate and returned to the control level 1 week after removing clofibrate from the diet. Nicotinate phosphoribosyltransferase (EC 2.4.2.11) and NAD+ glycohydrolase (EC 3.2.2.5) activities remained unchanged with dietary clofibrate.
Asunto(s)
Clofibrato/administración & dosificación , Hígado/metabolismo , NAD/biosíntesis , Triptófano/metabolismo , Animales , Células Cultivadas , Dieta , Masculino , Niacina/metabolismo , Pentosiltransferasa/biosíntesis , Ratas , Ratas Sprague-DawleyRESUMEN
Lactobacillus acidophilus JCM 1132 produces a heat-stable, two-component bacteriocin designated acidocin J1132 that has a narrow inhibitory spectrum. Maximum production of acidocin J1132 in MRS broth was detected at pH 5.0. Acidocin J1132 was purified by ammonium sulfate precipitation and sequential cation exchange and reversed-phase chromatographies. Acidocin J1132 activity was associated with two components, termed alpha and beta. On the basis of N-terminal amino acid sequencing and the molecular masses of the alpha and beta components, it is interpreted that the compounds differ by an additional glycine residue in the beta component. Both alpha and beta had inhibitory activity, and an increase in activity by the complementary action of the two components was observed. Acidocin J1132 is bactericidal and dissipates the membrane potential and the pH gradient in sensitive cells, which affect such proton motive force-dependent processes as amino acid transport. Acidocin J1132 also caused efflux of preaccumulated amino acid taken up via a unidirectional ATP-driven transport system. Secondary structure prediction revealed the presence of an amphiphilic alpha-helix region that could form hydrophilic pores. These results suggest that acidocin J1132 is a pore-forming bacteriocin that creates cell membrane channels through the "barrel-stave" mechanism.
Asunto(s)
Bacteriocinas/aislamiento & purificación , Lactobacillus acidophilus/química , Secuencia de Aminoácidos , Bacteriocinas/química , Bacteriocinas/farmacología , Concentración de Iones de Hidrógeno , Lactobacillus acidophilus/metabolismo , Datos de Secuencia Molecular , Fuerza Protón-Motriz/efectos de los fármacosRESUMEN
Acidocin 8912 is a bacteriocin produced by Lactobacillus acidophilus TK8912. The acidocin 8912 structural gene, acdT, was cloned and determined. It was located on the 14-kb plasmid pL103 and encoded a 46 amino acid precursor including a 20 amino acid N-terminal extension. The precursor sequence of the acdT gene shows a conservation of the general structural characteristics of the bacteriocin precursors from some lactic acid bacteria.
Asunto(s)
Bacteriocinas/genética , Genes Bacterianos/genética , Lactobacillus acidophilus/genética , Secuencia de Aminoácidos , Bacteriocinas/química , Secuencia de Bases , Clonación Molecular , Lactobacillus acidophilus/química , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The structure of the region necessary for replication of the plasmid pLA103 from Lactobacillus acidophilus TK8912 has been characterized. Sequence analysis revealed that the replication region contained an open reading frame (OrfA) encoding a 282-amino acid peptide preceded by a 22-bp tandem repeat sequence region. The predicted OrfA protein showed homology to the replication protein of a plasmid from Pediococcus halophilus. The plasmid containing the repeat sequence region preceding OrfA was able to replicate in the Lactobacillus host when provided with OrfA in trans, suggesting that the repeat sequence region contains the origin sequence essential for the pLA103 replication.
Asunto(s)
Lactobacillus acidophilus/genética , Plásmidos/genética , Replicón/genética , Secuencia de Aminoácidos , ADN Bacteriano/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Análisis de Secuencia de ADNRESUMEN
Feeding rats with a diet containing 0.25% clofibrate for 2 weeks elevated the hepatic NAD+ and total nicotinate levels significantly. Other peroxisome proliferators, such as 2-(4-chlorophenoxy)propionic acid and di(2-ethylhexyl)phthalate, had similar effects. When rats were fed the control diet without clofibrate for 1 week after 2 weeks of the clofibrate diet, the hepatic NAD+ level returned to the control value. Muscular NAD+ content was not affected by the peroxisome proliferators. The results were discussed in relation to induction of peroxisomal beta-oxidation enzymes by the peroxisome proliferators.
Asunto(s)
Clofibrato/administración & dosificación , Dieta , Hipolipemiantes/administración & dosificación , Hígado/metabolismo , NAD/metabolismo , Animales , Clofibrato/farmacología , Dietilhexil Ftalato/administración & dosificación , Dietilhexil Ftalato/farmacología , Fenofibrato/administración & dosificación , Fenofibrato/análogos & derivados , Fenofibrato/farmacología , Hipolipemiantes/farmacología , Hígado/efectos de los fármacos , Hígado/ultraestructura , Masculino , Microcuerpos/efectos de los fármacos , Microcuerpos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Hepatocytes were prepared from rats 3 and 7 days after a single injection of streptozotocin (50 mg/kg) or alloxan (40 mg/kg). Using the hepatocytes, NAD-and total pyridine nucleotide-syntheses from [carboxyl-14C]-nicotinic acid were investigated. The NAD content of streptozotocin- or alloxan-treated hepatocytes was not significantly different from that of the control hepatocytes. Syntheses of NAD and total pyridine nucleotides from nicotinic acid were significantly elevated 3 days after streptozotocin-injection but only total pyridine nucleotide synthesis was significantly higher 7 days after streptozotocin. Only total pyridine nucleotide synthesis was elevated 3 days after alloxan. Differences in the effects of the two diabetogenic agents and the physiological meaning of an increased NAD synthesis from nicotinic acid in the liver of diabetic animals were discussed.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Hígado/citología , Hígado/metabolismo , NAD/biosíntesis , Niacina/metabolismo , Ácido 3-Hidroxibutírico , Acetoacetatos/farmacología , Animales , Glucemia/análisis , Radioisótopos de Carbono , Células Cultivadas , Hidroxibutiratos/sangre , Hidroxibutiratos/farmacología , Cuerpos Cetónicos/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , EstreptozocinaRESUMEN
The metabolism of L-tryptophan by Saccharomyces uvarum (carlsbergensis) was investigated by simultaneous measuring of fluxes through kynureninase, through transaminases and into protein using L-[methylene-14C] and L-[side chain-2,3-3H]tryptophan. In yeasts cultivated in synthetic medium (S medium), the flux into protein was predominant, closely followed by the flux leading to 2-3H liberation. The proportion of L-tryptophan metabolized via the latter flux increased over 10-fold (75% of total tryptophan metabolized) as the concentration of L-tryptophan was raised from 5 x 10(-5) to 5 x 10(-4) M. L-Tryptophan metabolized via the kynureninase flux was less than 5% of total tryptophan metabolized. In yeast extract-polypepton-glucose medium (YPG medium), more tryptophan was incorporated into protein than in the S medium. Contribution of the kynureninase flux remained very low. Tryptophan metabolism via each flux changed depending on the growth phase. 2-3H liberation was shown to be primarily due to tryptophol synthesis by high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR), indole-3-acetic acid and kynurenic acid also contributing to 2-3H liberation but to a much lesser extent. 2-3H liberation increased dose-dependently at tryptophan concentration higher than 10(-5)M, while the kynureninase flux reached its plateau at 10(-5)M. Formation of tryptophol and indole-3-acetic acid via indole-3-pyruvic acid and indole-3-acetaldehyde with indole aldehyde as a by-product was confirmed using exogenous tryptophan metabolites with indole rings.
Asunto(s)
Saccharomyces/metabolismo , Triptófano/metabolismo , BiotransformaciónRESUMEN
In Saccharomyces uvarum, the effect of metabolic intermediates of the tryptophan-NAD pathway on the niacin-production was investigated. Exogenously added kynurenine and 3-hydroxyanthranilic acid raised the content of total niacin of the cells 2-fold as compared to the control cells, although anthranilic acid and tryptophan were less effective. Tryptophan was taken up into the cells faster than kynurenine, and the intracellular pool of tryptophan was larger than that of kynurenine. Of kynurenine (0.05 mM) added to the medium, 55% went through the transaminase flux (2-H liberation), 20% through the kynureninase flux, but none through the acetyl-CoA flux. As for tryptophan, only 2% went through the kynureninase flux. The products through the transaminase flux were identified as kynurenic acid (85%) and xanthurenic acid. 3-Hydroxykynurenine, 3-hydroxyanthranilic acid, quinolinic acid and niacin were also detected. The metabolism of tryptophan via the kynureninase flux reached a plateau above 0.05 mM. The production of kynurenine and kynurenic acid gradually increased above 0.05 mM. Tryptophol was formed in parallel with the amount of tryptophan consumed, while the rate of niacin production increased after glucose and tryptophan were exhausted. Based on the data obtained, a possible regulatory mechanism of the tryptophan-NAD pathway was discussed.
Asunto(s)
Niacina/biosíntesis , Saccharomyces/metabolismo , Triptófano/metabolismo , Radioisótopos de Carbono , Quinurenina/análisis , Quinurenina/metabolismo , Niacina/análisis , Transaminasas/análisis , Triptófano/análisisRESUMEN
In Saccharomyces uvarum aerobically grown in the tryptophan-added medium, the niacin content started to increase when both tryptophan and glucose in the medium had almost been exhausted. In the kynurenine-added medium, the niacin production occurred immediately after incubation started. Hemin added to the medium enhanced total niacin production by increasing the amount of tryptophan metabolized via the kynureninase flux. The results suggest that the niacin biosynthesis from tryptophan was regulated by catabolite repression, which was alleviated by exogenously added hemin.