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1.
Kinetics of the sodium-dependent glutamine transporter in human intestinal cell confluent monolayers. 总被引:1,自引:0,他引:1
W W Souba M Pan B R Stevens 《Biochemical and biophysical research communications》1992,188(2):746-753
The intestinal epithelium metabolism of glutamine plays a critical role in inter-organ nitrogen flow. Although it is known that glutamine is the primary oxidative energy source and nucleotide precursor in intestinal cells, the luminal uptake of glutamine by the apical surface of enterocytes is poorly understood. In this study we have uncovered the sodium-dependent transporter system responsible for L-glutamine uptake by the apical membrane of a human intestinal epithelial cell line. The sodium-dependent Michaelis constant (Km) = 247 +/- 45 microM glutamine, and Jmax = 4.44 +/- 0.65 x 10(-9) mole min-1(mg protein)-1 (37 degrees C). Glutamine shares the transporter with alanine, as demonstrated by unlabeled glutamine inhibition of [3H]alanine uptake kinetics with a purely competitive-type inhibition pattern, and glutamine inhibition Ki = 205 +/- 18 microM by Dixon analysis. The inhibition pattern for a series of amino acid analogs indicated that this intestinal apical membrane sodium-dependent transporter for glutamine is distinct from any other transport system found in membranes of non-intestinal cells. 相似文献
2.
P A Henricks M E Van der Tol J H Van Kats-Renaud F P Nijkamp J Verhoef 《Biochimica et biophysica acta》1984,801(2):206-214
Incubation of human polymorphonuclear leukocytes with arachidonic acid resulted in a stimulation of the oxidative metabolism of the cells. Upon stimulation with 80 microM arachidonic acid, neutrophils (5 X 10(6) cells/ml) produced superoxide (53 +/- 8 nmol/5 X 10(6) cells per 15 min), generated chemiluminescence (1211 100 +/- 157 000 cpm) and consumed oxygen (20 +/- 1 nmol/10(6) cells per 5 min). The stimulation of the cell metabolism could be reduced 40-60% by prior incubation of the cells with 10 microM indomethacin. Incubating polymorphonuclear leukocytes with arachidonic acid also resulted in a diminished chemotaxis towards an attractant, a decreased uptake of opsonized staphylococci and aggregation of the cells. This may be due to inhibitory products of arachidonic acid metabolism and toxic oxygen species produced during stimulated oxidative metabolism. The effects of arachidonic acid are specific for neutrophils, as mononuclear phagocytes only produced 17 +/- 8 nmol superoxide/5 X 10(6) cells per 15 min and generated 27 000 +/- 15 000 cpm chemiluminescence when stimulated with 80 microM arachidonic acid. When monocytes and neutrophils were stimulated with particles such as opsonized staphylococci, the amount of superoxide produced, oxygen consumed and chemiluminescence generated were similar. The phagocytic activity of the monocytes was also not affected by prior incubation with arachidonic acid. We conclude that in contrast to monocytes, neutrophil metabolism can be stimulated with arachidonic acid and this stimulation resulted in a decreased phagocytic activity of these cells. 相似文献
3.
Kinetic properties of a phosphate-bond-driven glutamate-glutamine transport system in Streptococcus lactis and Streptococcus cremoris. 总被引:24,自引:18,他引:6
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In Streptococcus lactis ML3 and Streptococcus cremoris Wg2 the uptake of glutamate and glutamine is mediated by the same transport system, which has a 30-fold higher affinity for glutamine than for glutamate at pH 6.0. The apparent affinity constant for transport (KT) of glutamine is 2.5 +/- 0.3 microM, independent of the extracellular pH. The KTS for glutamate uptake are 3.5, 11.2, 77, and 1200 microM at pH 4.0, 5.1, 6.0, and 7.0, respectively. Recalculation of the affinity constants based on the concentration of glutamic acid in the solution yield KTS of 1.8 +/- 0.5 microM independent of the external pH, indicating that the protonated form of glutamate, i.e., glutamic acid, and glutamine are the transported species. The maximal rates of glutamate and glutamine uptake are independent of the extracellular pH as long as the intracellular pH is kept constant, despite large differences in the magnitude and composition of the components of the proton motive force. Uptake of glutamate and glutamine requires the synthesis of ATP either from glycolysis or from arginine metabolism and appears to be essentially unidirectional. Cells are able to maintain glutamate concentration gradients exceeding 4 X 10(3) for several hours even in the absence of metabolic energy. The t1/2s of glutamate efflux are 2, 12, and greater than 30 h at pH 5.0, 6.0, and 7.0, respectively. After the addition of lactose as energy source, the rate of glutamine uptake and the level of ATP are both very sensitive to arsenate. When the intracellular pH is kept constant, both parameters decrease approximately in parallel (between 0.2 and 1.0 mM ATP) with increasing concentrations of the inhibitor. These results suggest that the accumulation of glutamate and glutamine is energized by ATP or an equivalent energy-rich phosphorylated intermediate and not by the the proton motive force. 相似文献
4.
Serotonin uptake and metabolism was studied in ependymal primary cultures. Serotonin uptake was facilitated by two different systems, one of which was the neuronal serotonin transporter SERT, exhibiting a Vmax value of 3.8+/-0.1 pmol x min(-1) x (mg protein)(-1) and an apparent Michaelis-Menten constant of 0.41+/-0.03 microM. The main product of metabolism was 5-hydroxyindole acetic acid, which resulted from the action of monoamine oxidase A. This enzyme showed a maximal rate of 0.85+/-0.02 nmol x min(-1) x (mg protein)(-1) and a Michaelis-Menten constant of 78+/-5 microM. Ependymal cells were able to dispose of extracellular serotonin with initial rates of approximately 600 pmol x min(-1) x (mg protein)(-1) and of 4 pmol x min(-1) x (mg protein)(-1) when challenged with 500 microM and 1 microM extracellular serotonin, respectively. Ependymal cells are concluded to facilitate the "sink" action of the CSF by removing waste compounds upon passing of the fluid from the parenchymal extracellular space into the ventricular system. 相似文献
5.
L Shoemaker C V Greenway J Peeling G Sutherland G Tomlinson 《Canadian journal of physiology and pharmacology》1991,69(5):570-575
The effects of increasing blood ethanol levels on hepatic metabolism were studied in anesthetized cats whose prior fluid intake contained ethanol for 24 days. A hepatic venous long-circuit technique with an extracorporeal reservoir was used to allow hemodynamic measurements and repeated sampling of arterial, portal, and hepatic venous blood without depletion of blood volume. For ethanol, Vmax was 106 +/- 15 mumol.min-1.100 g-1 liver and Km was 164 +/- 31 microM. A previous study showed that there were no changes in O2 uptake by the liver, suggesting other oxidative processes were suppressed during ethanol metabolism. In this study, proton nuclear magnetic resonance spectroscopy was used to simultaneously screen several plasma metabolites to elucidate other metabolic processes that may be perturbed in the liver during ethanol infusion. Hepatic lactate uptake remained unaltered when ethanol metabolism was less than 0.5 Vmax but was suppressed on an equimolar basis with ethanol metabolism when ethanol metabolism rose above 0.5 Vmax. Thus, lactate oxidation is one process that can be suppressed to allow ethanol oxidation without additional O2 uptake by the liver. In addition, no release of acetate from the liver occurred during ethanol metabolism in these experiments. This surprising finding suggests ethanol metabolism may, under some conditions or in some species, result in fatty acid synthesis rather than acetate release. Eight other major metabolites remained unchanged during ethanol infusion. 相似文献
6.
There was net uptake of branched-chain keto acids by the fetus from the umbilical circulation. Mean fetal uptake of the 3 keto acids 2-keto isovalerate, 2-keto isocaproate and 2-keto methylvalerate was 1.8 mumol/min per kg of fetus. The concentrations in the umbilical vein for these keto acids were 10.9 +/- 3.8 microM (mean +/- SD: 2-keto isovalerate), 19.7 +/- 6.1 microM (2-keto isocaproate) and 14.8 +/- 5.3 microM (2-keto methylvalerate) respectively. The coefficients of extraction for the same keto acids were 17.2%, 16.8% and 11.9% respectively. Fetal uptakes (both mumol/min and mumol/min per kg fetus) were positively correlated with umbilical supply. There were concentration gradients across the placenta, with fetal concentration: maternal concentration ratios of 3.3 +/- 1.5 for 2-keto isovalerate, 2.1 +/- 0.8 for 2-keto isocaproate and 1.3 +/- 0.6 for 2-keto methylvalerate. The net release of 2-keto acids into the umbilical circulation may conserve the carbon skeleton of branched-chain amino acids for fetal metabolism and growth. In the uterine circulation there was not a consistent pattern of release from or uptake by the uteroplacental tissues. It is suggested that branched-chain keto acids may contribute to fetal growth or energy metabolism. 相似文献
7.
The object of this work was to find out whether Candida tropicalis can be used for monochlorophenol degradation. Phenol monooxygenase and pyrocatechase, enzymes involved in preparatory phenol metabolism were shown to catalyse transformation of 3- and 4-chlorophenols. Phenol monooxygenase catalyses hydroxylation of 3- and 4-chlorophenols to 4-chloropyrocatechol which yields beta-chloromuconic acid under the action of pyrocatechase. Synthesis of phenol monooxygenase is induced by 3- and 4-chlorophenols. beta-Chloromuconic acid is a terminal product of 3- and 4-chlorophenol transformation under neutral conditions. In a weakly acid medium (the Rieder medium, phosphate buffer, pH 5.5), transformation of these chlorophenols terminates with spontaneous lactonization of beta-chloromuconic acid and its dehalogenation. C. tropicalis hardly transforms 2-chlorophenol although certain oxygen uptake occurs in its presence. 3- and 4-chlorophenols are not nutrient sources for C. tropicalis. The yeast has not been adapted to 3- and 4-chlorophenols as sole nutrient sources. 相似文献
8.
K G Rajendran T Lopez I Parikh 《Biochemical and biophysical research communications》1987,142(3):724-731
MCF-7 cells serially subcultured in media containing phenol red show poor stimulation of progesterone receptor (PR) synthesis in response to estradiol compared to cells grown in phenol red-free media. Phenol red, when added to cytosol, did not compete with [3H]estradiol for estrogen binding sites in concentrations ranging from 2 microM-1 mM. However 25 microM of the dye was sufficient to increase nuclear translocation of estrogen receptor (ER) in the intact cell. Phenol red activates cytoplasmic ER as indicated by DNA-cellulose binding studies. When cells grown in phenol red-free medium were exposed to phenol red for 48 h, PR levels increased in a dose dependent manner. From these data, it may be concluded that phenol red causes estrogenic effect in MCF-7 cells through activation of cytoplasmic receptor by interacting at a site distinct from the steroid binding site. 相似文献
9.
The presence of an uptake mechanism for uracil in procyclic forms of the protozoan parasite Trypanosoma brucei brucei was investigated. Uptake of [3H]uracil at 22 degrees C was rapid and saturable and appeared to be mediated by a single high-affinity transporter, designated U1, with an apparent Km of 0.46 +/- 0.09 microM and a Vmax of 0.65 +/- 0.08 pmol x (10(7) cells)(-1) x s(-1). [3H]Uracil uptake was not inhibited by a broad range of purine and pyrimidine nucleosides and nucleobases (concentrations up to 1 mM), with the exception of uridine, which acted as an apparent weak inhibitor (Ki value of 48 +/- 15 microM). Similarly, most chemical analogues of uracil, such as 5-chlorouracil, 3-deazauracil, and 2-thiouracil, had little or no affinity for the U1 carrier. Only 5-fluorouracil was found to be a relatively potent inhibitor of uracil uptake (Ki = 3.2 +/- 0.4 microM). Transport of uracil was independent of extracellular sodium and potassium gradients, as replacement of NaCl in the assay buffer by N-methyl-D-glucamine, KCl, LiCl, CsCl, or RbCl did not affect initial rates of transport. However, the proton ionophore carbonyl cyanide chlorophenylhydrazone inhibited up to 70% of [3H]uracil flux. These data show that uracil uptake in T. b. brucei procyclics is mediated by a single high-affinity transporter with high substrate selectivity and are consistent with a nucleobase-H+-symporter model for this carrier. 相似文献
10.
Inward fluxes of adenosine in erythrocytes and cultured cells measured by a quenched-flow method. 总被引:4,自引:2,他引:2
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Dilazep, a vasodilator previously recognized as an inhibitor of adenosine permeation, very rapidly blocked the uptake of adenosine by cultured L5178Y cells, and accordingly was used as a quencher in a simple quenched-flow system for measuring cellular uptake of nucleosides during very short intervals. Time courses of cellular uptake of adenosine, assayed during intervals between 0.05 and 0.5s with the quenched-flow system, were linear and defined initial rates of adenosine uptake. The latter are rates of inward transport of adenosine. Kinetic constants for that process in cultured S49 cells determined with the quenched-flow procedure were similar to those determined with an assay dependent on manual timing. In studies of adenosine uptake kinetics in human erythrocytes at 22 degrees C and 37 degrees C in which the quenched-flow procedure was used, time courses of adenosine uptake were linear at both temperatures and defined initial uptake rates; kinetic constants (means +/- S.E.M.) at 22 degrees C (n = 8) were Km 25 +/- 14 microM and Vmax. 15 +/- 5 pmol/s per microliter of cell water and at 37 degrees C (n = 3) were Km 98 +/- 17 microM and Vmax. 80 +/- 9 pmol/s per microliter of cell water. 相似文献
11.
Makoto Shoda Kiminari Maruta Shigezo Udaka 《Bioscience, biotechnology, and biochemistry》2013,77(8):1841-1846
Phenol utilizing yeasts were isolated from soil. The relationship were examined between distribution of phenol uptake rate using intact cells and distribution of the activities of catechol 1,2-oxygenase which is one of the key enzymes in phenol metabolism. Two of the isolates showed catechol 1,2-oxygenase activity even when grown in glucose medium, though the enzyme activity was about 1% of the full activity induced by phenol. Partially constitutive mutants for catechol 1,2-oxygenase were obtained by mutagenesis of an inducible strain. The level of mutant enzyme activity was close to that of the isolated constitutive strain. One isolate, Trichosporon cutaneum, preferentially utilized phenol to glucose in medium containing both phenol (200 ppm) and glucose (0.1%), until the concentration of phenol decreased to 10–20 ppm. 相似文献
12.
Isolated rat lungs, pretreated with 100 microM pargyline and 100 microM U-0521 (3',4'-dihydroxy-2-methylpropriophenone) to block metabolism of norepinephrine (NE), were perfused with 0.3 microM 3H-labeled l-norepinephrine (1-[3H]-NE) for 30 min. Efflux samples were then collected for 30 min during washout of the tissue with amine-free Krebs solution. Compartmental analysis (nonlinear least-squares regression) of the efflux of tissue l-[3H]NE content vs. time indicates that NE is accumulated in a large slowly equilibrating compartment (t 1/2 = 58.15 +/- 6.84 min) in addition to distribution in the vascular (blue dextran tracer) and extracellular ([3H]sorbitol tracer) fluid compartments of the lung. Pretreatment of the lungs with 100 microM cocaine hydrochloride reduces the total l-[3H]NE space from 7.44 +/- 1.91 to 2.48 +/- 0.23 ml/g (P less than 0.05) by selectively decreasing the size of the slow NE compartment from 6.99 +/- 1.97 to 1.67 +/- 0.14 ml/g (P less than 0.05). The large size, cocaine sensitivity, and long efflux half time of this compartment suggest that neuronal uptake contributes to the pulmonary vascular inactivation of l-[3H]NE. 相似文献
13.
The vanilloid receptor VR1 is a nonspecific Ca(2+) channel, expressed in sensory neurons in the peripheral nervous system and in various brain regions, which is believed to be an important molecular integrator of several chemical (acid, vanilloids) and physical stimuli (heat) that cause pain. Recently, several endogenous ligands for VR1 have been identified such as arachidonyl ethanolamide (anandamide) and the more potent arachidonyl dopamine (AA-DO). Here, we further characterize AA-DO as a ligand for rat VR1, heterologously expressed in CHO and HEK293 cells. AA-DO inhibited the binding of [3H]RTX to VR1 with a K(d) value of 5.49 +/- 0.68 microM and with positive cooperativity (p = 1.89 +/- 0.27), indicating that AA-DO was about 5-fold more potent than anandamide in this system. The K(d) (9.7 +/- 3.3 microM), and p values (1.54 +/- 0.04) for the binding of AA-DO to spinal cord membranes are in good correlation with the CHO-VR1 data. AA-DO stimulated 45Ca(2+) uptake on CHO-VR1 and HEK-VR1 cells with EC(50) values of 4.76 +/- 1.43 and 7.17 +/- 1.64 microM and Hill coefficients of 1.28 +/- 0.11 and 1.13 +/- 0.13, respectively, consistent with the binding measurements. In contrast to anandamide, AA-DO induced virtually the same level of 45Ca(2+) uptake as did capsaicin (90 +/- 6.6% in the CHO cells expressing VR1 and 89.3 +/- 9.4% in HEK293 cells expressing VR1). In a time dependent fashion following activation, AA-DO partially desensitized VR1 both in 45Ca(2+) uptake assays (IC(50) = 3.24 +/- 0.84 microM, inhibition is 68.5 +/- 6.85%) as well as in Ca(2+) imaging experiments (35.8 +/- 5.1% inhibition) using the CHO-VR1 system. The extent of desensitization was similar to that caused by capsaicin itself. We conclude that AA-DO is a full agonist for VR1 with a potency in the low micromolar range and is able to significantly desensitize the cells in a time and dose dependent manner. 相似文献
14.
Na+/H+ antiporter is the primary proton transport system used by osteoclasts during bone resorption 总被引:2,自引:0,他引:2
We have examined the effects of inhibitors of proton transport systems on osteoclastic bone resorption using an in vitro bone slice assay, where osteoclasts (OCs) are free from the influence of other bone cells. Amiloride (AM) and dimethylamiloride (DMA), inhibitors of the Na+/H+ antiporter, were potent inhibitors of bone resorption (IC50 approximately 9 and 0.7 microM for AM and DMA, respectively). Omeprazole (OM), a potent inhibitor of parietal cell K+/H+(-)ATPase, was a poor inhibitor of OC bone resorption (IC50 approximately 100 microM). These results strongly suggest that the Na+/H+ antiporter is the primary proton system used by OCs during bone resorption. 相似文献
15.
Hydroquinone, a metabolite of benzene, is converted by human myeloperoxidase to 1,4-benzoquinone, a highly toxic species. This conversion is stimulated by phenol, another metabolite of benzene. Here we report that peroxidase-dependent hydroquinone metabolism is also stimulated by catechol, resorcinol, o-cresol, m-cresol, p-cresol, guaiacol, histidine, and imidazole. In order to gain insights into the mechanisms of this stimulation, we have compared the kinetics of human myeloperoxidase-dependent phenol, hydroquinone, and catechol metabolism. The specificity (Vmax/Km) of hydroquinone for myeloperoxidase was found to be 5-fold greater than that of catechol and 16-fold greater than that of phenol. These specificities for myeloperoxidase-dependent metabolism inversely correlated with the respective one-electron oxidation potentials of hydroquinone, catechol, and phenol and suggested that phenol- and catechol-induced stimulation of myeloperoxidase-dependent hydroquinone metabolism cannot simply be explained by interaction of hydroquinone with stimulant-derived radicals. Phenol (100 microM), catechol (20 microM), and imidazole (50 mM) did, however, all increase the specificity (Vmax/Km) of hydroquinone for myeloperoxidase, indicating that these three compounds may be stimulating hydroquinone metabolism by a common mechanism. Interestingly, the stimulation of peroxidase-dependent hydroquinone metabolism by other phenolic compounds was pH-dependent, with the stimulating effect being higher under alkaline conditions. These results therefore suggest that the interaction of phenolic compounds, presumably by hydrogen-bonding, with the activity limiting distal amino acid residue(s) or with the ferryl oxygen of peroxidase may be an important contributing factor in the enhanced myeloperoxidase-dependent metabolism of hydroquinone in the presence of other phenolic compounds. 相似文献
16.
Mandal PK Mandal A Ahearn GA 《Journal of experimental zoology. Part A, Comparative experimental biology》2006,305(3):203-214
In crustaceans, the hepatopancreas is the major organ system responsible for heavy metal detoxification, and within this structure the lysosomes and the endoplasmic reticulum are two organelles that regulate cytoplasmic metal concentrations by selective sequestration processes. This study characterized the transport processes responsible for zinc uptake into hepatopancreatic lysosomal membrane vesicles (LMV) and the interactions between the transport of this metal and those of calcium, copper, and cadmium in the same preparation. Standard centrifugation methods were used to prepare purified hepatopancreatic LMV and a rapid filtration procedure, to quantify 65Zn2+ transfer across this organellar membrane. LMV were osmotically reactive and exhibited a time course of uptake that was linear for 15-30 sec and approached equilibrium by 300 sec. 65Zn2+ influx was a hyperbolic function of external zinc concentration and followed Michaelis-Menten kinetics for carrier transport (Km = 32.3 +/- 10.8 microM; Jmax = 20.7 +/- 2.6 pmol/mg protein x sec). This carrier transport was stimulated by the addition of 1 mM ATP (Km = 35.89 +/- 10.58 microM; Jmax = 31.94+/-3.72 pmol/mg protein/sec) and replaced by an apparent slow diffusional process by the simultaneous presence of 1 mM ATP+250 microM vanadate. Thapsigargin (10 microM) was also a significant inhibitor of zinc influx (Km = 72.87 +/- 42.75 microM; Jmax =22.86 +/- 4.03 pmol/mg protein/sec), but not as effective in this regard as was vanadate. Using Dixon analysis, cadmium and copper were shown to be competitive inhibitors of lysosomal membrane vesicle 65Zn2+ influx by the ATP-dependent transport process (cadmium Ki = 68.1 +/- 3.2 microM; copper Ki = 32.7 +/- 1.9 microM). In the absence of ATP, an outwardly directed H+ gradient stimulated 65Zn2+ uptake, while a proton gradient in the opposite direction inhibited metal influx. The present investigation showed that 65Zn2+ was transported by hepatopancreatic lysosomal vesicles by ATP-dependent, vanadate-, thapsigargin-, and divalent cation-inhibited, carrier processes that illustrated Michaelis-Menten influx kinetics and was stimulated by an outwardly directed proton gradient. These transport properties as a whole suggest that this transporter may be a lysosomal isoform of the ER Sarco-Endoplasmic Reticulum Calcium ATPase. 相似文献
17.
The goal of this study was to determine the mechanisms involved in the transport of the organic cation, tetraethylammonium (TEA), across the apical membrane of OK cells. [14C]TEA accumulated in OK cell monolayers reaching equilibrium in 2 h. The uptake of [14C]TEA at equilibrium was dependent upon temperature and was inhibited by sodium azide and by various organic cations, including N1-methylnicotinamide (NMN), mepiperphenidol, and cimetidine but not by the organic anion, p-aminohippuric acid. The initial uptake of [14C]TEA was characterized by a saturable process. The mean +/- S.D. Km was 27.8 +/- 2.6 microM and the Vmax was 414 +/- 26.5 pmol/mg protein/min. Both an accelerated efflux and influx of [14C]TEA in the presence of a trans-gradient of unlabeled TEA and NMN was observed, whereas a deaccelerated influx and efflux was observed in the presence of a trans-gradient of mepiperphenidol. The mechanism of interaction between NMN and TEA was examined. NMN significantly increased the apparent Km (mean +/- S.D.) of TEA to 82.8 +/- 16.4 microM (p less than 0.001), whereas the Vmax (mean +/- S.D.) was only slightly affected (478 +/- 72 pmol/mg protein/min) suggesting a competitive inhibition. The stimulatory effect of trans-gradients of NMN on TEA transport was due to an increase in the Vmax of TEA suggesting that NMN trans-stimulates TEA transport by increasing the turnover rate of the exchanger. In the presence of an inwardly directed proton gradient, the efflux at 30 s of [14C]TEA from the OK cell monolayers was significantly accelerated (p less than 0.05). Studies with the pH-sensitive fluorescent probe, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, suggested that TEA could drive the countertransport of protons. In apical membrane vesicles prepared from OK cells, the uptake of [3H]NMN exhibited an apparent "overshoot phenomenon" in the presence of an initial outwardly directed proton gradient. Protons competitively inhibited TEA uptake suggesting that the proton/organic cation and the organic cation/organic cation self exchange mechanism are the same mechanism. This is the first report describing both TEA self-exchange and proton/TEA exchange in the apical membrane of a continuous cell line. OK cells are an excellent model for the study of organic cation transport across the apical membrane. 相似文献
18.
Transport of aspartic acid, arginine, and tyrosine by the opportunistic protist Pneumocystis carinii
In order to improve culture media and to discover potential drug targets, uptake of an acidic, a basic, and an aromatic amino acid were investigated. Current culture systems, axenic or co-cultivation with mammalian cells, do not provide either the quantity or quality of cells needed for biochemical studies of this organism. Insight into nutrient acquisition can be expected to lead to improved culture media and improved culture growth. Aspartic acid uptake was directly related to substrate concentration, Q(10) was 1.10 at pH 7.4. Hence the organism acquired this acidic amino acid by simple diffusion. Uptake of the basic amino acid arginine and the aromatic amino acid tyrosine exhibited saturation kinetics consistent with carrier-mediated mechanisms. Kinetic parameters indicated two carriers (K(m)=22.8+/-2.5 microM and K(m)=3.6+/-0.3 mM) for arginine and a single carrier for tyrosine (K(m)=284+/-23 microM). The effects of other L-amino acids showed that the tyrosine carrier was distinct from the arginine carriers. Tyrosine and arginine transport were independent of sodium and potassium ions, and did not appear to require energy from ATP or a proton motive force. Thus facilitated diffusion was identified as the mechanism of uptake. After 30 min of incubation, these amino acids were incorporated into total lipids and the sedimentable material following lipid extraction; more than 90% was in the cellular soluble fraction. 相似文献
19.
Starvation-induced modulations in binding protein-dependent glucose transport by the marine Vibrio sp. S14 总被引:3,自引:0,他引:3
The uptake kinetics of D-glucose were examined in the marine Vibrio sp. S14 during a period of 168 h of complete energy and nutrient starvation. Two glucose transport systems were distinguished in Vibrio sp. S14: a low affinity system (Km = 4.6 +/- 0.9 microM) at the onset of starvation, and a high affinity system (Km = 0.55 +/- 0.15 microM) after 168 h of starvation. Both systems had a narrow substrate specificity, and both were osmotic shock-sensitive. 相似文献
20.
Fusarium graminearum A 3/5 possesses a high affinity system (Km = 32 +/- 8 microM; mean +/- SE) for uptake of choline, which was shown to be energy-dependent and constitutive. The maximum rate of choline uptake by this system was repressed by ammonia and glucose, showing a three-fold increase in maximum activity after nitrogen (2 h) or carbon (4 h) starvation. The system was highly specific for choline with only dimethylethanolamine (Ki = 198 +/- 29 microM), betaine aldehyde (Ki = 95 +/- 14 microM) and chlorocholine (Ki = 352 +/- 40 microM) acting as competitive inhibitors. Hemicholinium-3 acted as a mixed (non-competitive) inhibitor (KIES = 1.9 +/- 0.6 microM; KIE = 3.6 +/- 1.9 microM). 相似文献