共查询到20条相似文献,搜索用时 15 毫秒
1.
Transport of N-acetyl-D-glucosamine and N-acetyl-D-galactosamine by rat liver lysosomes 总被引:4,自引:0,他引:4
A J Jonas R J Speller P B Conrad W P Dubinsky 《The Journal of biological chemistry》1989,264(9):4953-4956
Transport of N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, products of lysosomal glycoprotein and glycosaminoglycan degradation, was examined in Percoll gradient purified rat liver lysosomes. Uptake of these two sugars was competitive and quite specific remaining largely unaffected by the presence of L-fucose, D-glucosamine, D-glucose, D-glucuronic acid, D-mannose, or N-acetylneuraminic acid. Kinetic studies revealed a Km of 4.4 mM for both N-acetyl-D-glucosamine and N-acetyl-D-galactosamine uptake. Temperature dependence studies revealed a Q10 of 2.3. N-Acetyl-D-glucosamine uptake was not dependent upon NaCl, KCl, MgCl2, or ATP/MgCl2 and was unaffected by 5 mM dithiothreitol or variation of buffer pH between 6.0 and 8.0. Cytochalasin B at a concentration of 50 microM effectively inhibited uptake of N-acetyl-D-glucosamine by 90% and N-acetyl-D-galactosamine by 65%. Prior incubation of lysosomes in 20 mM N-acetyl-D-glucosamine stimulated uptake of both sugars 3-4-fold, although it had no effect on the uptake of D-glucose. Countertransport was unaffected by neutral and cationic amino acids demonstrating independence from these amino acid transport systems. We conclude that lysosomes possess a highly specific transport system for N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. 相似文献
2.
Characterization of a glucose transport system in Vibrio parahaemolyticus. 总被引:2,自引:1,他引:1
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Cells of a glucose-PTS (phosphoenolpyruvate:carbohydrate phosphotransferase system)-negative mutant of Vibrio parahaemolyticus transport D-glucose in the presence of Na+. Maximum stimulation of D-glucose transport was observed at 40 mM NaCl, and Na+ could be replaced partially with Li+. Addition of D-glucose to the cell suspension under anaerobic conditions elicited Na+ uptake. Thus, we conclude that glucose is transported by a Na+/glucose symport mechanism. Calculated Vmax and Km values for the Na(+)-dependent D-glucose transport were 15 nmol/min/mg of protein and 0.57 mM, respectively, when NaCl was added at 40 mM. Na+ lowered the Km value without affecting the Vmax value. D-Glucose was the best substrate for this transport system, followed by galactose, alpha-D-fucose, and methyl-alpha-glucoside, judging from the inhibition pattern of the glucose transport. D-Glucose itself partly repressed the transport system when cells were grown in its presence. 相似文献
3.
E Brot-Laroche M A Serrano B Delhomme F Alvarado 《The Journal of biological chemistry》1986,261(14):6168-6176
D-Glucose transport was studied with isolated brush border membrane vesicles from guinea pig jejunum. Saturation curves were carried out at either 25 or 35 degrees C in buffers containing Na+, Li+, K+ (100 mM chloride salt), or sorbitol (200 mM). Uncorrected uptake rates were fitted by nonlinear regression analysis to an equation involving one diffusional and two saturable terms. In the presence of Na+ at 35 degrees C, two saturable systems (Km = 0.4 and 24 mM, respectively) were evident, as well as a diffusion component quantitatively identical with that measured with L-glucose in separate experiments. In contrast, at 25 degrees C only one saturable system was apparent (Km = 1.2 mM): the second exhibited diffusion-like kinetics. In the presence of Na+ at 35 degrees C, D-glucose uptake was fully inhibited by both D-glucose and D-galactose, whereas alpha-methylglucoside gave kinetics of partial inhibition. We conclude that in the presence of Na+ there are at least two distinct D-glucose transport systems: 1) System I, a low temperature-sensitive system, fully inhibited by D-glucose, D-galactose, and alpha-methylglucoside; we identify it as the "classical" D-glucose/Na+ cotransport system, insensitive to inhibition by cytochalasin B and obligatorily dependent on Na+; and 2) System II, a high temperature-sensitive system where D-glucose and D-galactose inhibit but alpha-methylglucoside is inert. Its cation specificity is unclear but it appears to be sensitive to cytochalasin B inhibition. When Li+ or K+ substituted for Na+, only one transport system was apparent. The Li+-activated transport was: independent of the incubation temperature; inhibited by D-glucose and D-galactose but not by alpha-methylglucoside, 2-deoxy-D-glucose, D-mannose, and D-xylose; and sensitive to cytochalasin B inhibition. The exact nature of the system (or systems) involved in D-glucose transport in the absence of sodium remains to be established. 相似文献
4.
Effects of some chlorinated sugar derivatives on the hexose transport system of the blood/brain barrier.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The inhibition of D-glucose transport into brain by several hexose analogues has been investigated in adult anaesthetized rats. D-Glucose was transported with apparent Vmax. = 1.22 mumol/g per min, Km = 11.12 mM and Kd = 0.008 ml/g per min. 6-Chloro-6-deoxyglucose was transported with corresponding values of Vmax. = 1.33 mumol/g per min, Km = 5.5 mM and Kd = 0.0155 ml/g per min and inhibited D-glucose transport with apparent Ki = 3.01 mM. 6-Chloro-6-deoxymannose, 6-chloro-6-deoxygalactose and 6-tosyl-6-deoxygalactose also inhibited D-glucose transport, but 6-chloro-6-deoxyfructose was without effect. The results were consistent with a model for glucose transport at the blood/brain interface that involves a hydrophobic site on the transport protein at or near the 6-position of bound glucose. 相似文献
5.
Glucose transport in lysosomal membrane vesicles. Kinetic demonstration of a carrier for neutral hexoses 总被引:3,自引:0,他引:3
Lysosomal membrane vesicles isolated from rat liver were exploited to analyze the mechanism of glucose transport across the lysosomal membrane. Uptake kinetics of [14C]D-glucose showed a concentration-dependent saturable process, typical of carrier-mediated facilitated transport, with a Kt of about 75 mM. Uptake was unaffected by Na+ and K+ ions, membrane potentials, and proton gradients but showed an acidic pH optimum. Lowering the pH from 7.4 to 5.5 had no effect on the affinity of the carrier for the substrate but increased the maximum rate of transport about 3-fold. As inferred from the linearity of Scatchard plots, a single transport mechanism could account for the uptake of glucose under all conditions tested. As indicated by the transstimulation properties of the carrier, other neutral monohexoses, including D-galactose, D-mannose, D- and L-fucose were transported by this carrier. The transport rates and affinities of these sugars, measured by the use of their radiolabeled counterparts, were in the same range as those for D-glucose. Pentoses, sialic acid, and other acidic monosaccharides including their lactones, aminosugars, N-acetyl-hexosamines, and most L-stereoisomers, particularly those not present in mammalian tissues, were not transported by this carrier. Glucose uptake and transstimulation were inhibited by cytochalasin B and phloretin. The biochemical properties of this transporter differentiate it from other well-characterized lysosomal sugar carriers, including those for sialic acid and N-acetylhexosamines. The acidic pH optimum of this glucose transporter is a unique feature not shared with any other known glucose carrier and is consistent with its lysosomal origin. 相似文献
6.
Identification of proximal tubular transport functions in the established kidney cell line, OK 总被引:2,自引:0,他引:2
OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, L-glutamate, L-proline, L-alanine, and alpha-methyl-glucopyranoside (alpha-methyl D-glucoside) were accumulated through Na+-dependent and Na+-independent transport pathways. D-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na+. Influx of inorganic phosphate was only observed in the presence of Na+. Na+/alpha-methyl D-glucoside uptake was preferentially inhibited by phlorizin and D-glucose uptake by cytochalasin B. An amiloride-sensitive Na+-transport was also identified. In isolated apical vesicles (enriched 8-fold in gamma-glutamyltransferase), L-glutamate, L-proline, L-alanine, alpha-methyl D-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na+-gradient as compared to an inwardly directed K+-gradient. L-Glutamate transport required additionally intravesicular K+. D-Glucose transport was similar in the presence of a Na+- and a K+-gradient. Na+/alpha-methyl D-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na+/D-glucose transport. An amiloride-sensitive Na+/H+ exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na+-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. D-Glucose appears a poor substrate for the Na+/hexose transport system. 相似文献
7.
Microdissected, beta-cell-rich pancreatic islets from ob/ob mice were used in studies of 86Rb+ transport. D-Glucose (20 mM) induced a biphasic reduction in 86Rb+ efflux. The reduction stabilized within 10 min at 34% of the efflux rate at zero glucose. The initial 86Rb+ uptake (5 min) was dose-dependently reduced by ouabain with maximum inhibition at 1 mM. D-Glucose (20 mM) did not affect the ouabain-sensitive 86Rb+ influx but markedly reduced (48%) the ouabain-resistant isotope influx. The results suggest that D-glucose does not affect the Na+/K+ pump in pancreatic beta-cells and that the glucose-sensitive K+-transporting modalities (K+ channels) in the beta-cells can mediate both inward and outward K+ flux. 相似文献
8.
Sugar uptake by intestinal basolateral membrane vesicles 总被引:6,自引:0,他引:6
A high yield of membrane vesicles was prepared from the basolateral surface of rat intestinal cells using an N2 cavitation bomb and density gradient centrifugation. The membranes were enriched 10-fold and were free of significatn contamination by brush border membranes and mitochondria. The rate of D-E114C]glucose and L-E13H]glucose uptake into the vesicle was measured using a rapid filtration technique. D-Glucose equilibrated within the vesicles with a half-time 1/25th that for L-glucose. The stereospecific uptake exhibited saturation kinetics with a Km of approx. 44 mM and a V of approx. 110 nmol . mg-1 min-1 at 10 degrees C. The activation energy for the process was 14 kcal . mol-1 below 15 degrees C and it approached 3 kcal . mol-1 above 22 degrees C. Carrier-mediated uptake was eliminated in the presence of 1 mM HgCl2 and 0.5 mM phloretin. The rate of transport was unaffected by the absence or presence of sodium concentration gradients. Competition studies demonstrated that all sugars with the D-glucose pyranose ring chair conformation shared the transport system, and that, with the possible exception of the -OH group at carbon No. 1, there were no specific requirements for an equatorial -OH group at any position in the pyranose ring. In the case of alpha-methyl-D-glucoside its inability to share the D-glucose transport system may be due to steric hindrance posed by the -OCH3 group rather than by a specific requirement for a free hydroxyl group at the position in the ring. It is concluded that sugars are transported across the basolateral membrane of the intestinal epithelium by a facilitated diffusion system reminiscent of that in human red blood cells. 相似文献
9.
W. David Lust Joan P. Schwartz Janet V. Passonneau 《Molecular and cellular biochemistry》1975,8(3):169-176
The transport and metabolism of glucose was examined in monolayers of C-6 glioma cells. 1) Glucose transport appeared to have both a low (Km = 7.74 mM) and a high (Km = 1.16 mM) affinity site in C-6cells; whereas 2-deoxyglucose had only one (Km = 3.7 mM). 2) A large portion of the accumulated glucose was rapidly metabolized to the two glycolytic end products, lactate and pyruvate, and then extruded into the medium. The temperature-dependent efflux of lactate and pyruvate was linear up to 2 hrs with 6 to 10 times more lactate being extruded into the medium than pyruvate. 3) The efflux of lactate and pyruvate increased with increasing extracellular (medium) pH. The presence of 5 percent CO2 not only inhibited the acid efflux but also inhibited the short-term uptake of glucose. The CO2 effect was attributed to a lowering of the medium pH since bicarbonate alone either increased or did not inhibit efflux. 4) Valinomycin increased the levels of cellular lactate but not those of pyruvate by almost three-fold. Lactate efflux was stimulated while that of pyruvate was inhibited. The addition of 5 percent CO2 increased the cellular levels of both lactate and pyruvate, but unlike valinomycin decreased the acid efflux. Idoacetate inhibited the acid efflux by 50 percent suggesting that glycolysis is necessary for efflux. 相似文献
10.
Mark A. Yorek Joyce A. Dunlap Mark R. Stefani Eric P. Davidson 《Journal of neurochemistry》1992,58(5):1626-1636
It has been proposed that abnormal myo-inositol metabolism may be a factor in the development of diabetic complications. Studies with animal models of diabetes and cultured cells have suggested that hyperglycemia by an unknown mechanism may alter myo-inositol metabolism and content. Recently, we have shown that L-fucose, a 6-deoxy sugar whose content has been reported to be increased in diabetes, is a potent inhibitor of myo-inositol transport. To examine the effect of L-fucose on myo-inositol metabolism, neuroblastoma cells were cultured in medium supplemented with L-fucose. L-Fucose is a competitive inhibitor of Na(+)-dependent, high-affinity myo-inositol transport. The Ki for inhibition of myo-inositol transport by L-fucose is about 3 mM. L-Fucose is taken up and accumulates in neuroblastoma cells. The uptake of L-fucose is inhibited by Na+ depletion, D-glucose, glucose analogues, phloridzin, and cytochalasin B. In contrast, neither myo-inositol nor L-glucose inhibits L-fucose uptake. Chronic exposure of neuroblastoma cells to 1-30 mM L-fucose causes a decrease in myo-inositol accumulation and incorporation into inositol phospholipids, intracellular free myo-inositol content, and phosphatidylinositol levels. Na+,K(+)-ATPase transport activity is decreased by about 15% by acute or chronic exposure of neuroblastoma cells to L-fucose. Similar defects occur when neuroblastoma cells are exposed chronically to 30 mM glucose. Cell myo-inositol metabolism and Na+/K(+)-pump activity are maintained when 250 microM myo-inositol is added to the L-fucose-supplemented medium. Unlike the effect of chronic exposure of neuroblastoma cells to medium containing 30 mM glucose, the resting membrane potential of neuroblastoma cells is not altered by chronic exposure of the cells to 30 mM L-fucose. The effect of L-fucose on cultured neuroblastoma cell properties occurs at concentrations of L-fucose which may exist in the diabetic milieu. These data suggest that increased concentrations of L-fucose may have a role in myo-inositol-related defects in mammalian cells. 相似文献
11.
The kinetic parameters of D-glucose transport in liposomes reconstituted with the purified glucose transporter were determined. Net uptake and efflux both had Km values of 0.7 to 1.2 mM and Vmax values of 1.6 mumol/mg of protein/min. Equilibrium exchange had a Km of 35 mM and a Vmax of 50 mumol/mg of protein/min. By separating the liposomes from unreconstituted protein using density centrifugation, the Vmax of exchange was increased to 86 mumol/mg of protein/min, about 3 times that of the erythrocyte membrane. Trypsin, which inhibits erythrocyte glucose transport only from the cytoplasmic side, inhibited reconstituted transport activity about 40% when added externally. With internal treatment as well, the inhibition was about 80%. This suggests that the reconstituted transporter is oriented about equally in both directions. Antibody prepared against the purified transporter inhibits transport to a maximum of about 50%, also indicating a scrambled orientation. External trypsin treatment decreased the Km for uptake and increased the Km for efflux, consistent with asymmetric kinetic parameters for the two faces of the transporter. However, the calculated Km values are lower than those reported for erythrocytes. Phloretin and diethylstilbestrol inhibit the reconstituted transporter. However, they bind to liposomes, producing anomalous results under some experimental conditions. When this binding is taken into account, phloretin inhibits completely and symmetrically. The binding accounts for the apparent asymmetric effects of phloretin reported by others. The inhibitory effects of mercuric ions are consistent with action at two classes of binding sites. Treatment with trypsin increases the sensitivity to Hg2+, indicating that the more sensitive site is on the external face of the transporter. 相似文献
12.
J E Lever 《The Journal of biological chemistry》1979,254(8):2961-2967
Plasma membrane vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated D-glucose transport without detectable metabolic conversion to glucose 6-phosphate. Glucose transport activity was stereospecific, temperature-dependent, sensitive to inactivation by p-chloromercuriphenylsulfonate, and accompanied plasma membrane material during subcellular fractionation. D-Glucose efflux from vesicles was inhibited by phloretin, an inhibitor of glucose uptake in intact cells. Cytochalasin B, a potent inhibitor of glucose uptake when tested with the intact cells used for vesicle isolation did not inhibit glucose transport in vesicles despite the presence of high affinity cytochalasin binding sites in isolated membranes. The enhanced glucose uptake observed in intact cells after viral transformation was not expressed in vesicles: no significant differences in glucose transport specific activity could be detected in vesicle preparations from nontransformed and transformed mouse fibroblast cultures. These findings indicate that cellular components distinct from glucose carriers can mediate changes in glucose uptake in mouse fibroblast cultures in at least two cases: sensitivity to inhibition by cytochalasin B and the enhanced cellular sugar uptake observed after viral transformation. 相似文献
13.
Anomalous asymmetric kinetics of human red cell hexose transfer: role of cytosolic adenosine 5'-triphosphate 总被引:2,自引:0,他引:2
A Carruthers 《Biochemistry》1986,25(12):3592-3602
Cytosolic adenosine 5'-triphosphate (ATP) modifies the properties of human red cell sugar transport. This interaction has been examined by analysis of substrate-induced sugar transporter intrinsic fluorescence quenching and by determination of Michaelis and velocity constants for D-glucose transport in red cell ghosts and inside-out vesicles lacking and containing ATP. When excited at 295 nm, human erythrocyte ghosts stripped of peripheral proteins display an emission spectrum characterized by a scattering peak and a single emission peak centered at about 333 nm. Addition of sugar transport substrate or cytochalasin B and phloretin (sugar transport inhibitors) reduces emission peak height by 10% and 5%, respectively. Cytochalasin B induced quenching is a simple saturable phenomenon with an apparent Kd (app Kd) of 60 nM and a capacity of 1.4 nmol of sites/mg of membrane protein. Quenching by D-glucose (and other transported sugars) is characterized by at least two (high and low) app Kd parameters. Inhibitor studies indicate that these sites correspond to sugar efflux and influx sites, respectively, and that both sites can exist simultaneously. ATP induces quenching of stripped ghost fluorescence with half-maximal effects at 20-30 microM ATP. ATP reduces the low app Kd and increases the high app Kd for sugar-induced fluorescence quenching. D-Glucose transport in intact red cells is asymmetric (Km and Vmax for influx less than Km and Vmax for efflux). In addition, two operational Km parameters for efflux are detected in zero- and infinite-trans efflux conditions. Protein-mediated sugar transport in ghosts and inside-out vesicles (IOVs) is symmetric with respect to Km and Vmax for entry and exit, and only one Km for exit is detected. Addition of millimolar levels of ATP to the interior of ghosts or to the exterior of IOVs restores both transport asymmetry and two operational Km parameters for native efflux. A model for red cell hexose transport is proposed in which ATP modifies the catalytic properties of the transport system. This model mimics the behavior of the sugar transport systems of intact cells, ghosts, and inside-out vesicles. 相似文献
14.
Uterine plasma membrane preparations were obtained by centrifugation on discontinuous sucrose gradients. The specific activity of the plasma membrane marker 5'-nucleotidase was increased 10-fold while the specific activity of glucose-6-phosphatase was increased 3-fold. Electron microscopy showed mainly closed vesicles having diameters mainly in the range of 0.1 to 0.4 micron and an absence of other recognizable organelles such as mitochondria. D-Glucose transport was inhibited by sulfhydryl reagents, phloretin, and cytochalasin B. Uptake was prevented at high osmotic pressures. The Km of glucose transport was 12.2 +/- 1.1 mM. Studies of the inhibition of [3H]cytochalasin B binding by D-glucose indicated that the value of the Kd of the cytochalasin B-transporter complex was larger than 1 microM. These data demonstrate the potential usefulness of these preparations in the study of glucose transport in rat uterus and its control by steroid hormones. 相似文献
15.
Osamu Kuwahara Yasuhide Mitsumoto Kenzo Chiba Tetsuro Mohri 《Journal of neurochemistry》1992,59(2):616-621
The cellular uptake of D-aspartic acid (D-Asp) as a model compound for glutamic acid transport was studied in rat hippocampal slices. D-Asp is accumulated by both Na(+)-dependent and Na(+)-independent processes in hippocampal slices, and both processes are dependent on temperature. The Na(+)-dependent uptake is assumed to be high in affinity (apparent Km = 0.17 mM), but low in capacity, whereas the Na(+)-independent uptake is much lower in affinity (Km = 2.86 mM), but higher in capacity. L-Aspartic acid, L-glutamic acid, dihydrokainic acid, and threo-3-hydroxy-DL-aspartic acid markedly inhibited the uptake of D-Asp with Na+ in the medium, whereas D-glutamic acid, glycine, and L-lysine had no significant effect. The Na(+)-dependent uptake of D-Asp was significantly reduced under "hypoglycemic," "anoxic," and "ischemic" conditions, whereas the Na(+)-independent uptake was unaffected. Metabolic inhibitors such as NaCN and ICH2COOH significantly inhibited the Na(+)-dependent uptake, but not the Na(+)-independent uptake. These results suggest that the Na(+)-dependent component of D-Asp transport in rat hippocampal cells is inactivated under ischemic conditions, whereas the Na(+)-independent component is unaffected. 相似文献
16.
The activity and Km of glucose transport of rat adipocytes are quite variable in the basal state. This could be due to differing levels of highly saturable transport against a background of less saturable transport. Such heterogeneity could lead to differing conclusions as to the Km of basal cells compared to insulin-stimulated cells depending on the choice of substrate, the range of concentrations tested, and the rigor of data analysis. In the present work, we used a cell preparation which was stable and partially activated by constant agitation. We used a two-component model to fit the concentration dependence of D-glucose uptake. We defined two parallel pathways of glucose entry, a high-affinity/low-capacity pathway and a low-affinity/high-capacity pathway. Both pathways were stereospecific and were inhibited by cytochalasin B. The low-affinity pathway in basal cells had 97% of the total capacity (Vmax) with a high Km (greater than 50 mM). A second pathway had a very low Km (less than 1 mM) and only 3% of the total capacity, but contributed to 30-60% of glucose uptake at 8 mM glucose. In insulin-stimulated cells, a pathway with a Km of 4-5 mM dominated and contributed 85% of glucose transport. The low-affinity but not the very high affinity pathway persisted in stimulated cells, but its contribution was only 10-15% of transport at 8 mM glucose. These results suggest the presence of at least two functionally distinct transporters whose respective contributions can be characterized by nonlinear regression of data over a wide range of glucose concentrations.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
Sulfate transport by rat liver lysosomes 总被引:2,自引:0,他引:2
Sulfate transport was examined using membrane vesicles (pH 7.0 inside) prepared from rat liver lysosomes. Sulfate uptake was dependent upon external pH with increased uptake at lower buffer pH. The Km for uptake was 160 microM at pH 5.0 while at pH 7.0, a lower affinity system with a Km of 1.4 mM was present. The protonophore carbonyl cyanide m-chlorophenylhydrazone increased uptake at pH 5.0 while valinomycin/KCl had no effect. In contrast, at pH 7.0, valinomycin-induced changes in membrane potential stimulated uptake. Countertransport of sulfate at pH 7.0 was inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonic acid, and a variety of anions: SO4(2-) greater than MoO4(2-) greater than Cl- greater than HPO4- greater than HCO3-. Trans-stimulation of sulfate uptake at pH 7.0 was observed with MoO4(2-) and, to a lesser extent, with S2O3(2-) while Cl-, HPO4-, and HCO3- had little effect. However, chloride loading of vesicles resulted in marked stimulation of sulfate uptake at pH 5.0. It appears that sulfate and protons exit lysosomes in exchange for chloride by a specific, pH-regulated anion transport system. 相似文献
18.
Highly purified [D-glucose-1-14C]lactose has been used to study the transport of lactose by Klebsiella sp. strain CT-1. Strain CT-1 transports lactose by a lactose-inducible system that exhibited an apparent Km of 6 mM lactose and an apparent Vmax of 140 nmol/min per mg of cell protein. Lactose uptake was inhibited competitively by o-nitrophenyl-beta-D-galactoside with a Ki value of 8 mM, but was not inhibited by thio-beta-methyl-galactoside. D-Glucose, D-mannose, 2-deoxyglucose, and alpha-methyl-D-glucoside also inhibited lactose uptake. Phosphoenolpyruvate-dependent hydrolysis of o-nitrophenyl-beta-D-galactoside and lactose-dependent release of pyruvate from phosphoenolpyruvate by benzene-treated CT-1 cells showed that CT-1 transports lactose by a phosphoenolpyruvate:sugar phosphotransferase system. Correlations between the growth rate of CT-1 on lactose and properties of the transport system indicated that transport is the rate limiting step in utilization of lactose. 相似文献
19.
The energetics of amino acid uptake by the developing small intestine was investigated in vitro. L-valine, L-leucine, L-phenylalanine, L-methionine, L-lysine and L-arginine were all actively transported by the newborn rat jejunum. Metabolic inhibitors (e.g. 2,4-dinitrophenol) significantly reduced uptake of all amino acids but uptake against a concentration gradient was not totally abolished. Uptake of all amino acids was reduced at low[Na+]. Inhibition of transport of neutral amino acids by reduced luminal [Na+] was greater than that of basic amino acids, and the tissue was barely able to concentrate the neutral amino acids. [Na+] affected the Michaelis constant (Km) of neutral transport systems for their substrates; for the basic amino acids Km values were unaffected by the presence or absence of Na+. Ouabain significantly inhibited neutral amino acid uptake but had no effect on L-lysine or L-arginine uptake. These results are discussed in terms of the Na+ gradient hypothesis for amino acid transport, and the site of energy input to active transport. The role of glycolysis in providing energy for intestinal transport in the neonatal rat and the efficiency of Na+ dependent and independent transport mechanisms are considered. It is concluded that the energetics of amino acid transport systems in neonatal and adult rats are essentially similar. 相似文献
20.
The mechanism of pantothenate transport by rat liver parenchymal cells in primary culture 总被引:1,自引:0,他引:1
The mechanism of pantothenate transport across the plasma membrane was investigated with initial velocity studies of [14C]pantothenate uptake and efflux in rat liver parenchymal cells maintained in primary culture. At 116 mM sodium, double-reciprocal plots of the initial velocity of uptake versus [pantothenate] were linear from 0.3 to 36.5 microM pantothenate and gave an apparent Km,pant of 11 +/- 2 microM. The rate of pantothenate uptake at 0 [sodium] was about 14% of the rate at 116 mM sodium, and the reciprocal of the apparent Km,pant was a linear function of [sodium]. Vmax obtained by extrapolation to infinite [pantothenate] was independent of [sodium]. Ouabain, gramicidin D, cyanide, azide, and 2,4-dinitrophenol inhibited uptake, but preloading cells with pantothenate did not. Pantothenate derivatives or carboxylic acids were only weak inhibitors of uptake. Efflux was measured in cells preloaded with [14C]pantothenate. The apparent Km for efflux was 85 +/- 29 microM, and the rate of efflux was unaffected by addition of pantothenate, sodium, ouabain, gramicidin D, or 2,4-dinitrophenol to the external medium. These features are consistent with a mechanism for pantothenate transport in which sodium and pantothenate are cotransported in a 1:1 ratio on a carrier highly specific for pantothenate; sodium decreases the apparent Km for pantothenate, and a sodium-carrier complex forms only on the intracellular side of the membrane. 相似文献