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1.
Cleide G da Silva César A J Ribeiro Guilhian Leipnitz Carlos S Dutra-Filho Angela T S Wyse AT Clóvis M D Wannmacher Jo?o J F Sarkis Cornelis Jakobs Moacir Wajner 《Biochimica et biophysica acta》2002,1586(1):81-91
L-2-Hydroxyglutaric (LGA) and D-2-hydroxyglutaric (DGA) acids are the characteristic metabolites accumulating in the neurometabolic disorders known as L-2-hydroxyglutaric aciduria and D-2-hydroxyglutaric aciduria, respectively. Although these disorders are predominantly characterized by severe neurological symptoms, the neurotoxic mechanisms of brain damage are virtually unknown. In this study we have evaluated the role of LGA and DGA at concentrations ranging from 0.01 to 5.0 mM on various parameters of energy metabolism in cerebral cortex slices and homogenates of 30-day-old Wistar rats, namely glucose uptake, CO(2) production and the respiratory chain enzyme activities of complexes I to IV. DGA significantly decreased glucose utilization (2.5 and 5.0 mM) by brain homogenates and CO(2) production (5 mM) by brain homogenates and slices, whereas LGA had no effect on either measurement. Furthermore, DGA significantly inhibited cytochrome c oxidase activity (complex IV) (EC 1.9.3.1) in a dose-dependent manner (35-95%) at doses as low as 0.5 mM, without compromising the other respiratory chain enzyme activities. In contrast, LGA did not interfere with these activities. Our results suggest that the strong inhibition of cytochrome c oxidase activity by increased levels of DGA could be related to the neurodegeneration of patients affected by D-2-hydroxyglutaric aciduria. 相似文献
2.
Inhibition of creatine kinase activity from rat cerebral cortex by D-2-hydroxyglutaric acid in vitro
da Silva CG Bueno AR Schuck PF Leipnitz G Ribeiro CA Rosa RB Dutra Filho CS Wyse AT Wannmacher CM Wajner M 《Neurochemistry international》2004,44(1):45-52
D-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as D-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11-34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA. 相似文献
3.
Inhibition of mitochondrial creatine kinase activity by D-2-hydroxyglutaric acid in cerebellum of young rats 总被引:2,自引:0,他引:2
da Silva CG Bueno AR Rosa RB Dutra Filho CS Wannmacher CM Wyse AT Wajner M 《Neurochemical research》2003,28(9):1329-1337
D-2-Hydroxyglutaric aciduria (DHGA) is a neurometabolic disorder biochemically characterized by tissue accumulation and excretion of high amounts of D-2-hydroxyglutaric acid (DGA). Although the affected patients have predominantly severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In previous studies we have demonstrated that DGA, at concentrations as low as 0.25 mM, significantly decreased creatine kinase activity and other parameters of energy metabolism in cerebral cortex of young rats. In the present study, we investigated the effect of DGA (0.25-5 mM) on total creatine kinase (tCK) activity, as well as on CK activity in cytosolic (Cy-CK) and mitochondrial (Mi-CK) preparations from cerebellum of 30-day-old Wistar rats in order to test whether the inhibitory effect of DGA on CK was tissue specific. We verified that tCK (22% inhibition) and Mi-CK (40% inhibition) activities were moderately inhibited by DGA at concentrations of 2.5 mM and higher, in contrast to Cy-CK, which was not affected by the acid. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by preincubation of the homogenates with reduced glutathione, suggesting that the inhibition of CK activity by DGA is possibly mediated by modification of essential thiol groups of the enzyme. Our present results therefore demonstrate a relatively weak inhibitory effect of DGA on cerebellum Mi-CK activity, as compared to that provoked in cerebral cortex, and may possibly be related to the neuropathology of DHGA, characterized by cerebral cortex abnormalities. 相似文献
4.
Mutations in the D-2-hydroxyglutarate dehydrogenase gene cause D-2-hydroxyglutaric aciduria
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Struys EA Salomons GS Achouri Y Van Schaftingen E Grosso S Craigen WJ Verhoeven NM Jakobs C 《American journal of human genetics》2005,76(2):358-360
d-2-hydroxyglutaric aciduria is a neurometabolic disorder with both a mild and a severe phenotype and with unknown etiology. Recently, a novel enzyme, d-2-hydroxyglutarate dehydrogenase, which converts d-2-hydroxyglutarate into 2-ketoglutarate, and its gene were identified. In the genes of two unrelated patients affected with d-2-hydroxyglutaric aciduria, we identified disease-causing mutations. One patient was homozygous for a missense mutation (c.1331T-->C; p.Val444Ala). The other patient was compound heterozygous for a missense mutation (c.440T-->G; p.Ile147Ser) and a splice-site mutation (IVS1-23A-->G) that resulted in a null allele. Overexpression studies in HEK-293 cells of proteins containing the missense mutations showed a marked reduction of d-2-hydroxyglutarate dehydrogenase activity, proving that mutations in the d-2-hydroxyglutarate dehydrogenase gene cause d-2-hydroxyglutaric aciduria. 相似文献
5.
《Journal of Fermentation and Bioengineering》1991,71(2):83-88
Diglycolic acid (DGA) oxidizing activity was found in crude extracts of Rhodococcus sp. no. 432 grown in DGA. Glycolic acid (GA) oxidase was purified approximately 80 times by treatment with streptomycin sulfate, precipitation with (NH4)2SO4, chromatographies with DEAE-cellulose, DEAE-Toyopearl and Butyl-Toyopearl, and gel filtration on Toyopearl HW-55. The purified GA oxidase was almost homogeneous on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The purity was calculated to be more than 95%. The molecular weight of the enzyme, which appeared to consist of three identical units, was 158,000. Each subunit of GA oxidase included one molecule of FAD as a cofactor. The isoelectric point of the enzyme was around 5.3. GA oxidase was stable below 30°C and at the pH range of 6.0–8.5. The optimum pH and temperature were around 7.5 and 40°C, respectively. Oxygen, cytochrome c, ferricyanide and 2,6-dichlorophenol indophenol (DCIP) acted as an electron acceptor. The activity of GA oxidase was strongly inhibited by potassium cyanide, quinine, quinacrine, monoiodoacetate, 1,4-benzoquinone and some heavy metal ions. GA oxidase also had activity in DGA, GA, glyoxylic acid (GOA), methoxy acetate, ethoxy acetate and l-malate. Alcohols and other organic acids were not oxidized by the enzyme. The apparent Km values for DGA, GA and GOA were about 26.7, 0.5 and 4.4 mM, respectively. The reaction products from DGA were supposed to be GOA and GA by the enzymatic assays. The reaction mechanism of GA oxidase in oxidation of DGA was supposed to be as follows: HOOCH2COCH2COOH+H2O+acceptor→HOOCCHO+HOOCCH2OH+ reduced acceptor. 相似文献
6.
Short-term effects of carbon dioxide on carnation callus cell respiration 总被引:10,自引:6,他引:4
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The addition of potassium bicarbonate to the electrode cuvette immediately stimulated the rate of dark O2 uptake of photomixotrophic and heterotrophic carnation (Dianthus caryophyllus L.) callus, of Elodea canadensis (Michx) leaves, and of other plant tissues. This phenomenon occurred at pH values lower than 7.2 to 7.8, and the stimulation depended on the concentration of gaseous CO2 in the solution. These stimulatory responses lasted several minutes and then decreased, but additional bicarbonate or gaseous CO2 again stimulated respiration, suggesting a reversible effect. Carbonic anhydrase in the solution increased the stimulatory effect of potassium bicarbonate. The CO2/bicarbonate dependent stimulation of respiration did not occur in animal tissues such as rat diaphragm and isolated hepatocytes, and was inhibited by salicylhydroxamic acid in carnation callus cells and E. canadensis leaves. This suggested that the alternative oxidase was engaged during the stimulation in plant tissues. The cytochrome pathway was severely inhibited by CO2/bicarbonate either in the absence or in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone. The activity of cytochrome c oxidase of callus tissue homogenates was also inhibited by CO2/bicarbonate. The results suggested that high carbon dioxide levels (mainly free CO2) partially inhibited the cytochrome pathway (apparently at the oxidase level), and this block in electron transport elicited a large transient engagement of the alternative oxidase when present uninhibited. 相似文献
7.
Both the seed and the leaf α-oxidation systems of higher plants require for activity a reductant and molecular oxygen. The reductant may be NADH or a coupled glucose + glucose oxidase system. d-2-Hydroxypalmitate is the only 2-hydroxy acid which is formed from palmitic acid under a variety of conditions; l-2-Hydroxypalmitate is not formed, but when added does inhibit the initial attack of the α-oxidative enzyme (s) on palmitate. Since the addition of glutathione + gluthathione peroxidase greatly increases the formation of d-2-hydroxypalmitate from palmitate, with a concomitant decrease in CO2, it is strongly suggested that the intermediate in α-oxidation is d-2-hydroperoxypalmitate, which may then undergo decarboxylation to a pentadecanal and CO2 or be reduced to d-2-hydroxypalmitate. Evidence in support of this hypothesis is presented. 相似文献
8.
The enzymatic decarboxylations of l-DOPA and l-5-hydroxytryptophan (l-5-HTP) by aromatic l-amino acid decarboxylase (AADC) were measured with homogenates from human brain regions, caduate nucleus and hypothalamus, using our new and highly sensitive methods for l-DOPA decarboxylase and l-5-HTP decarboxylase by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Dopamine formed from l-DOPA as substrate was measured for DOPA decarboxylase activity using d-DOPA for the blank. For 5-HTP decarboxylase activity, serotonin (5-HT) formed from l-5-HTP was measured, and the blank value in presence of NSD-1055 was subtracted. NSD-1055 inhibited 5-HTP decarboxylase activity completely at a concentration of 0.2 mM. In this study, the properties of l-5-HTP decarboxylase activity in human caudate nucleus were first examined. AADC activities in human brains were found to be widely variable for both l-DOPA and l-5-HTP as substrates. The ratio of the activities for l-DOPA and l-5-HTP were found to be significantly higher in hypothalamus than in caudate nucleus. AADC activity for l-DOPA in the brain was found to be linear up to 40 min of incubation, while that for l-5-HTP was found to be linear up to 240 min of incubation. The optimum pyridoxal phosphate concentration was found to be similar for both substrates and was between 0.01 and 0.1 mM. The optimum pH values were found to be 7.2 and 8.2 for l-DOPA decarboxylase and l-5-HTP decarboxylase, respectively. Km and Vmax values for a human caudate nucleus l-DOPA decarboxylase were found to be 414 μM and 482 pmol/min/g wet weight, respectively, while those for l-5-HTP decarboxylase were found to be 90 μM and 71 pmol/min/g wet weight, respectively. 相似文献
9.
Silva AR Silva CG Ruschel C Helegda C Wyse AT Wannmacher CM Wajner M Dutra-Filho CS 《Neurochemical research》2001,26(12):1277-1283
In the present study we investigated the effects of L-pyroglutamic acid (PGA), which predominantly accumulates in the inherited metabolic diseases glutathione synthetase deficiency (GSD) and -glutamylcysteine synthetase deficiency (GCSD), on some in vitro parameters of energy metabolism and lipid biosynthesis. We evaluated the rates of CO2 production and lipid synthesis from [U-14C]acetate, as well as ATP levels and the activities of creatine kinase and of the respiratory chain complexes I-IV in cerebral cortex of young rats in the presence of PGA at final concentrations ranging from 0.5 to 3 mM. PGA significantly reduced brain CO2 production by 50% at the concentrations of 0.5 to 3 mM, lipid biosynthesis by 20% at concentrations of 0.5 to 3 mM and ATP levels by 52% at the concentration of 3 mM. Regarding the enzyme activities, PGA significantly decreased NADH:cytochrome c oxireductase (complex I plus CoQ plus complex III) by 40% at concentrations of 0.5–3.0 mM and cytochrome c oxidase activity by 22–30% at the concentration of 3.0 mM, without affecting the activities of succinate dehydrogenase, succinate:DCPIP oxireductase (complex II), succinate:cytochrome c oxireductase (complex II plus CoQ plus complex III) or creatine kinase. The results strongly indicate that PGA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by these diseases. 相似文献
10.
Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid 总被引:13,自引:0,他引:13
Brusque AM Borba Rosa R Schuck PF Dalcin KB Ribeiro CA Silva CG Wannmacher CM Dutra-Filho CS Wyse AT Briones P Wajner M 《Neurochemistry international》2002,40(7):593-601
Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5 mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32–46%), complex I (61–72%), and complex II+III (15–26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1 mM succinate in the assay instead of the usual 16 mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11–27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them. 相似文献
11.
《Biochemical medicine》1981,25(2):160-167
The metabolism of d- and l-3-hydroxybutyrate by neonatal and suckling rats was investigated. Both isomers of 3-hydroxybutyrate were incorporated into hepatic lipid, amino acids, and protein throughout the developmental period. The enzyme activities of liver 3-oxo-CoA transferase and brain 3-hydroxybutyrate dehydrogenase were compared during the first 3 postnatal weeks. The results suggest that the enzymatic activity of liver 3-oxo-CoA transferase is sufficient to account for a major portion of the d-isomer incorporation. The production of CO2 by rat liver was greater from d-3-hydroxybutyrate than that measured from the l-isomer. The in vitro oxidation of both the d- and l-isomers by rat liver was stimulated by ATP + CoA or by GTP + CoA which suggests that their utilization may also be mediated by acyl-CoA synthetase enzymes. 相似文献
12.
INTRACELLULAR LOCALIZATION OF ENZYMES IN SPLEEN : I. REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE CYTOCHROMEc REDUCTASE, CYTOCHROMEc OXIDASE, AND SUCCINIC DEHYDROGENASE IN THE RAT AND GUINEA PIG
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Herbert J. Eichel With the Technical Assistance of Jeanne Hagan 《The Journal of cell biology》1957,3(3):397-412
1. The intracellular distribution of nitrogen, DPNH cytochrome c reductase, succinic dehydrogenase, and cytochrome c oxidase has been studied in fractions derived by differential centrifugation from rat and guinea pig spleen homogenates. 2. In the spleens of each species, the nuclear fraction accounted for 40 to 50 per cent of the total nitrogen content of the homogenate, and the mitochondrial, microsome, and supernatant fractions contained about 8, 12, and 30 per cent of the total nitrogen, respectively. 3. Per mg. of nitrogen, DPNH cytochrome c reductase was concentrated in the mitochondria and microsomes of both rat and guinea pig spleens. Seventy per cent of the total DPNH cytochrome c reductase activity was recovered in these two fractions. The reductase activity associated with the nuclear fraction was lowered markedly by isolating nuclei from rat spleens with the sucrose-CaCl2 layering technique. The lowered activity was accompanied by the recovery of about 90 per cent of the homogenate DNA in the isolated nuclei, indicating that little, if any, of the reductase is present in spleen cell nuclei. 4. Per mg. of nitrogen, succinic dehydrogenase was concentrated about 10-fold in the mitochondria of rat spleen, and 65 per cent of the total activity was recovered in this fraction. 5. Cytochrome c oxidase was concentrated, per mg. of nitrogen, in the mitochondria of both rat and guinea pig spleens. The activity associated with the nuclear fraction was greatly diminished when this fraction was isolated from rat spleens by the sucrose-CaCl2 layering technique. Only 50 to 70 per cent of the total cytochrome c oxidase activity of the original homogenates was recovered among the four fractions from both rat and guinea pig spleens, while the specific activities of reconstructed homogenates were only 55 to 75 per cent of those of the original whole homogenates. This was in contrast to the results with DPNH cytochrome c reductase and succinic dehydrogenase where the recovery of total enzyme activity approached 100 per cent, and the specific activities of reconstructed homogenates equalled those of the original homogenates. The recovery of cytochrome c oxidase was greatly improved when only the nuclei were separated from rat spleen homogenates. 6. Data were presented comparing the concentrations (ratio of activity per mg. of nitrogen of the fraction to activity per mg. of nitrogen of the homogenate) of DPNH cytochrome c reductase in mitochondria and microsomes derived from different organs of different animals. 7. Data were presented comparing the activities per mg. of nitrogen of DPNH cytochrome c reductase in homogenates from several organs of various animals. 相似文献
13.
M L Vorbeck A P Martin J K Park J F Townsend 《Archives of biochemistry and biophysics》1982,214(1):67-79
The effect of aging on the hepatic mitochondrial population has been determined using a rigorously defined group of Fischer 344 rats with known survivorship data. The age groups studied included mature adult controls (8.5 months; 100% survivorship), an intermediate aged group (17.5 months; 90% survivorship), and an aged group (29 months; 20% survivorship). Cytochrome oxidase activity and content were determined in homogenates and mitochondrial fractions. The mitochondrial fractions were characterized by determination of respiratory activity, and monoamine oxidase activity as well as evaluation of the polypeptide composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The yield of protein in the isolated mitochondrial fraction as well as the mitochondrial specific content decreased significantly as a function of aging. Mitochondrial specific content was determined from the specific activities of cytochrome oxidase in the homogenate (per gram liver) and in the isolated mitochondrial fraction (per mg protein). Specific activity of hepatic cytochrome oxidase decreased approximately 15% (P = 0.035) in homogenates from the 17.5-month animals with a further, highly significant (P = 0.0002) decrease (29%) in the 29-month animals. In contrast, there was no statistically significant difference among the age groups in the cytochrome oxidase specific activity in the isolated hepatic mitochondrial fractions. However, the percentage of the total homogenate cytochrome oxidase activity recovered in the isolated mitochondrial fraction decreased significantly in the 29-month animals (P = 0.0063 vs the 8.5-month controls; P = 0.022 vs the 17.5-month group). Cytochrome aa3 content of total liver homogenates from aged animals decreased (P = 0.00064) which is in agreement with the decline in cytochrome oxidase specific activity in this age group. In the mitochondrial fraction from the aged animals, cytochrome aa3 content was essentially unchanged which is consistent with the lack of aging-related change in mitochondrial cytochrome oxidase specific activity. In freshly isolated mitochondrial fractions, no aging-related alterations were observed in respiratory control and ratios. The addition of exogenous NADH and cytochrome c did not change significantly the respiratory rate of hepatic mitochondria from control or aged animals. These results demonstrate the integrity of freshly isolated mitochondrial preparations from both control and aged Fischer 344 rats. In addition, there was no aging-related alteration in either monoamine oxidase specific activity or polypeptide composition. The similarities observed in the specific activities of cytochrome oxidase and monoamine oxidase, as well as in the cytochrome aa3 content and polypeptide composition of the isolated mitochondrial fraction, suggest a generalized decrease in hepatic mitochondrial content as a function of aging rather than a selective loss of mitochondrial components. 相似文献
14.
Robert P. Metzger Kathleen D. Edwards Cecilia C. Nixon Patrick W. Mobley 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,629(3):482-489
The production of 14CO2 from l-[1-14C]fucose and d-[1-14C]arabinose has been studied in five mammalian species.Cats, guinea pigs, mice, and rabbits respired about 22% of the label of l[1-14C]fucose or of d-[1-14C]arabinose within 6 h after intraperitoneal injection of the sugar. Rats respired only 1.5% of the l-fucose label and 5% of the d-arabinose label in the same time period.Liver homogenates from cat, guinea pig, and rabbit produced significantly more 14CO2 from l-[1-14C]fucose or d-[1-14C]arabinose than mouse or rat liver homogenates. Unlike those of the other species, guinea pig liver homogenates had very low l-fucose dehydrogenase activity.The results suggest that substantial catabolism of l-fucose and d-arabinose occurs in the tissues of some animal species. Investigators wishing to employ l-fucose as a tracer of glycoprotein metabolism must, therefore, ensure that the species that they employ does not metabolize l-fucose to products interfering with their studies. 相似文献
15.
The activities of phenolase, peroxidase, cytochrome oxidase, catalase and superoxide dismutase, as well as the levels of lipid peroxides, were measured in plerocercoids of S. solidus taken from the body cavity of the fish (unactivated) and in plerocercoids which had been cultured in vitro, either under air, or under 95% N2, 5% CO2. When cultured anaerobically, the activities of phenolase, peroxidase and cytochrome oxidase all increased dramatically. Aerobically, only phenolase activity increased. Lipid peroxide levels and superoxide dismutase activity was similar at all stages and catalase could not be detected. It is suggested that the increased activity of oxidative enzymes in anaerobically cultured worms is an attempt to compensate for the reduced environmental pO2. 相似文献
16.
J Archambault 《Enzyme and microbial technology》1991,13(11):882-892
Surface-immobilized C. roseus cell cultures were grown in a 20-l modified airlift bioreactor operated at 0.51 vvm (kLa approximately 8 h-1) under various gassing regimes [air, 2% (v/v) and 5% CO2]. Extracellular ammonium, phosphate, and nitrate ions as well as carbohydrate uptake and pH value of the medium were monitored together with on-line dissolved oxygen concentration, conductivity of the medium, and carbon dioxide production rate (CPR) of the cultures. Cultures supplemented with 2% CO2 showed higher nitrate (5.0-7.0 mM d-1) and carbohydrate (3.3 g l-1 d-1) uptake rates and biomass production (mu approximately 0.24 d-1, yield approximately 0.33 g dw g CHO-1 and 7.4 g dw L-1) as compared to air (3.6 mM d-1, 2.1 g l-1 d-1; 0.20 d-1, 0.25 g dw g CHO-1 and 5 g dw l-1) and 5% CO2 (2.0-3.6 mM d-1, 2.0 g l-1 d-1; 0.11 d-1, 0.20 g dw g CHO-1 and 5 g dw l-1) cultures and as reported previously for suspension cultures. In addition, air and 5% CO2 cultures displayed incomplete carbohydrate uptake and, more important, phosphate and ammonium ion release into the medium at the end, which was ascribed to loss of viability. This was not observed for 2% CO2 immobilized bioreactor as well as shake flask control suspension cultures, which suggests that sparged C. roseus surface-immobilized cell cultures require 2% CO2 supplementation of the gas phase for both maximum growth and retained viability. The maximum CPRs of all cultures were in the same range (2.1-2.8 mM CO2 l-1 h-1). However, the estimated maximum specific CO2 production rates of 2% CO2 and 5% CO2 immobilized cultures (0.6 mM g dw-1 h-1) were lower than those found for air-sparged immobilized cultures (1.0-1.3 mM g dw-1 h-1). These rates are significantly higher than those reported in the literature for C. roseus cell suspension cultures performed in bioreactors gassed with air (approximately 0.2-0.55 mM g dw-1 h-1). 相似文献
17.
Activities of the enzymes monoamine oxidase (EC 1.4.3.4), alpha-glycerophosphate dehydrogenase (EC 1.1.99.5) and cytochrome oxidase (EC 1.9.3.1) were determined in homogenates and in the mitochondrial fraction prepared from individual regions of pig brain. The variation in the activity of alpha-glycerophosphate dehydrogenase paralleled that of cytochrome oxidase, but this was not the case with monoamine oxidase. The differences in the activities of the enzymes among homogenates of the various regions of the brain persisted in mitochondria prepared from these homogenates. The purification of these three enzymes paralleled each other when mitochondria were prepared, suggesting that the three enzymes are bound to the same particles. 相似文献
18.
Biochemical micromethods were used for the investigation of changes in mitochondrial oxidative phosphorylation associated with cytochrome c oxidase deficiency in brain cortex from Mo(vbr) (mottled viable brindled) mice, an animal model of Menkes' copper deficiency syndrome. Enzymatic analysis of cortex homogenates from Mo(vbr) mice showed an approximately twofold decrease in cytochrome c oxidase and a 1.4-fold decrease in NADH:cytochrome c reductase activities as compared with controls. Assessment of mitochondrial respiratory function was performed using digitonin-treated homogenates of the cortex, which exhibited the main characteristics of isolated brain mitochondria. Despite the substantial changes in respiratory chain enzyme activities, no significant differences were found in maximal pyruvate or succinate oxidation rates of brain cortex homogenates from Mo(vbr) and control mice. Inhibitor titrations were used to determine flux control coefficients of NADH:CoQ oxidoreductase and cytochrome c oxidase on the rate of mitochondrial respiration. Application of amobarbital to titrate the activity of NADH:CoQ oxidoreductase showed very similar flux control coefficients for control and mutant animals. Alternately, titration of respiration with azide revealed for Mo(vbr) mice significantly sharper inhibition curves than for controls, indicating a more than twofold elevated flux control coefficient of cytochrome c oxidase. Owing to the reserve capacity of respiratory chain enzymes, the reported changes in activities do not seem to affect whole-brain high-energy phosphates, as observed in a previous study using 31P NMR. 相似文献
19.
Meningoencephalitis was produced in albino mice by intranasal inoculation of Hartmanella culbertsoni. In the infected brain phosphatidyl choline (PC), phosphatidyl ethanolamine + phosphatidyl serine (PE + PS), sphingomyelin and cholesterol registered decrease, lysophospholipids and free fatty acids accumulated, and part of the cholesterol was esterified. Phospholipase A (EC 3.1.1.4) and sphingomyelinase (EC 3.1.4.12) were elaborated in the postmitochondrial supernatant fraction. The levels of lipid peroxides, in brain as well as the capacity of brain homogenates to form lipid peroxides in vitro, was higher in the infected animals as compared to the uninfected. The activities of succinate dehydrogenase (EC 1.3.99.1) and cytochrome c oxidase (EC 1.9.3.1) in the mitochondrial fraction of the infected brain decreased while adenosine triphosphatase (EC 3.6.1.3) was stimulated. Addition of cytosol fraction (105,000g supernatant) of infected brain to the mitochondrial fraction of uninfected brain caused inhibition of succinate dehydrogenase and cytochrome c oxidase and stimulation of adenosine triphosphatase. This shows that some toxic substance(s) which inhibits mitochondrial function in brain is produced in the cytosol during infection. This inhibitor was nondialyzable and heat stable. 相似文献
20.
Oxidation rates of palmitate and activities of the mitochondrial marker enzymes cytochrome c oxidase and citrate synthase have been determined in homogenates, isolated mitochondria and slices of human and rat heart and in calcium-tolerant rat cardiac myocytes. Homogenates and mitochondria from rat heart showed a 6- and 2.5-fold higher palmitate oxidation rate than the corresponding preparations from human heart. From the palmitate oxidation rates and cytochrome c oxidase and citrate synthase activities as parameters, the mitochondrial protein contents of human and rat heart were calculated to be about 18 and 45 mg/g wet weight, respectively. Based on citrate synthase activities, the fatty acid oxidation rates were about the same in homogenates and isolated mitochondria, much lower in myocytes and lowest in slices. In the cellular systems the palmitate molecule was more completely oxidized than in homogenates or isolated mitochondria. Fatty acid oxidation rates were concentration-dependent in slices, but not with myocytes. With the cellular systems, palmitate oxidation was synergistically stimulated by the addition of carnitine, coenzyme A and ATP to the incubation medium. This stimulation could be attributed only partly to an increased oxidation in damaged cells. 相似文献