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1.
Based on results obtained with leaf discs exposed to sulfate, leaves on cucurbit plants (Cucurbita pepo L. cv Small Sugar Pumpkin and Cucumis sativus cv Chipper) 1 to 2.5 weeks old have a low potential for H2S emission (less than 10 picomoles per min per cm2 leaf area) in response to sulfate, whereas discs from most of the leaves on plants 3 to 4 weeks old emit H2S at a higher rate (50 to 150 picomoles per min per cm2 leaf area). This difference is determined by the age of the plant, and is independent of the leaves' age or developmental stage. In response to l-cysteine, however, discs from leaves on cucurbit plants 1 to 2.5 weeks old emit H2S at higher rates (15 to 50 picomoles per min per cm2 leaf area) than in response to sulfate. Furthermore, the potential for H2S emission in response to l-cysteine decreases with increasing age of the individual leaf. Thus, most of the potential for H2S emission in response to l-cysteine is developed during germination and the early growth of cucurbit plants, but most of the potential for H2S emission in response to sulfate arises later in the development of the plants.  相似文献   

2.
Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis in bacteria and higher plants, has been purified to homogeneity from etiolated pea (Pisum sativum) seedlings using a combination of conventional and affinity chromatographic steps. This is the first report on a homogeneous preparation of native dihydrodipicolinate synthase from a plant source. The pea dihydrodipicolinate synthase has an apparent molecular weight of 127,000 and is composed of three identical subunits of 43,000 as determined by gel filtration and cross-linking experiments. The trimeric quaternary structure resembles the trimeric structure of other aldolases, such as 2-keto-3-deoxy-6-phosphogluconic acid aldolase, which catalyze similar aldol condensations. The amino acid compositions of dihydrodipicolinate synthase from pea and Escherichia coli are similar, the most significant difference concerns the methionine content: dihydrodipicolinate synthase from pea contains 22 moles of methionine residue per mole of native protein, contrary to the E. coli enzyme, which does not contain this amino acid at all. Dihydrodipicolinate synthase from pea is highly specific for the substrates pyruvate and l-aspartate-β-semialdehyde; it follows Michaelis-Menten kinetics for both substrates. The pyruvate and l-aspartate-β-semialdehyde have Michaelis constant values of 1.70 and 0.40 millimolar, respectively. l-Lysine, S-(2-aminoethyl)-l-cysteine, and l-α-(2-aminoethoxyvinyl)glycine are strong allosteric inhibitors of the enzyme with 50% inhibitory values of 20, 160, and 155 millimolar, respectively. The inhibition by l-lysine and l-α-(2-aminoethoxyvinyl)glycine is noncompetitive towards l-aspartate-β-semialdehyde, whereas S-(2-aminoethyl)-l-cysteine inhibits dihydrodipicolinate synthase competitively with respect to l-aspartate-β-semialdehyde. Furthermore, the addition of (2R,3S,6S)-2,6-diamino-3-hydroxy-heptandioic acid (1.2 millimolar) and (2S,6R/S)-2,6-diamino-6-phosphono-hexanic acid (1.2 millimolar) activates dihydrodipicolinate synthase from pea by a factor of 1.4 and 1.2, respectively. This is the first reported activation process found for dihydrodipicolinate synthase.  相似文献   

3.
The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent Km-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; Vmax app was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 × 106 vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H+-translocating enzyme activities revealed Km app values of 0.45 millimolar for ATPase (EC 3.6.1.3) and 23 micromolar for pyrophosphatase (PPase) (EC 3.6.1.1). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference Δ μH+ while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp, l-Leu, and l-Tyr was also stimulated by ATP. They seem to compete for the same carrier system. l-Ala, l-Val, d-Phe, and d-Leu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for l-amino acids having a relatively high hydrophobicity.  相似文献   

4.
Evidence for a specific glutamate/h cotransport in isolated mesophyll cells   总被引:1,自引:1,他引:0  
Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO4. Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of l-glutamate (Glu) and its analog l-methionine-d,l-sulfoximine (l-MSO). d-Glu and the l isomers of the protein amino acids did not elicit alkalinization. l-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar l-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H+/106 cells·minute. l-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of l-[U-14C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by l-MSO. l-Glu had no influence on K+ efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific l-Glu/proton uptake process is present in Asparagus mesophyll cells.  相似文献   

5.
During growth on l-cysteine ethylester, Chlorella fusca (211-8b) accumulated a substance which contained bound sulfide, which could be liberated by reduction with dithioerythritol (DTE) as inorganic sulfide. This substance was extracted with hot methanol and purified by thin layer chromatography. This substance liberated free sulfide when incubated with mono- and dithiols, and thiocyanate was formed after heating with KCN. The isolated substance cochromatographed with authentic sulfur flower using different solvent systems for thin layer chromatography, high pressure liquid chromatography, and the identical spectrum with a relative λmax at 263 nm was found. The chemical structure was confirmed by mass spectrometry showing a molecular weight of 256 m/e for the S8 configuration. No labeled elemental sulfur was detected when the cells were grown on [35S]sulfate and l-cysteine ethylester indicating the origin of elemental sulfur from l-cysteine ethylester. C. fusca seems to have enzymes for the metabolism of elemental sulfur, since it disappeared after prolonged growth into the stationary phase. Cysteine was formed from O-acetyl-l-serine and elemental sulfur in the presence of thiol groups and purified cysteine synthase from spinach or Chlorella.  相似文献   

6.
Cystine lyase degrades l-cystine by a β-elimination to form cysteine persulfide, pyruvate, and ammonia. This enzyme is common in Brassica sp. and has been purified to homogeneity from extracts of broccoli (Brassica oleracea var botrytis) buds. Two isozymes were separated on DEAE-Fractogel columns and the first peak, cystine lyase I further purified to homogeneity. The purified enzyme had a narrow range of substrate specificity with l-cystine and S-alkyl-l-cysteine sulfoxides being the primary substrates. The Km for l-cystine was 1.9 millimolar and for S-ethyl-l-cysteine sulfoxide was 15.6 millimolar, suggesting that l-cystine would be preferred in vivo. Using gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular weight of the holoenzyme was estimated as 152,000 composed of subunits of approximately 49,000. This strongly suggests the native enzyme is a trimer. The presence of carbohydrate in the native enzyme was detected at the level of 5.8% on a weight basis. Except for the ability to utilize l-cystine as a substrate there are many similarities between cystine lyase I and the alliin lyase of onion (Allium cepa).  相似文献   

7.
Human cystathionine β-synthase (CBS), a novel heme-containing pyridoxal 5′-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H2O or H2S, respectively. The presence of heme in CBS has limited spectrophotometric characterization of reaction intermediates by masking the absorption of the pyridoxal 5′-phosphate cofactor. In this study, we employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions. The reactions of l-serine and l-cysteine with CBS resulted in the formation of a common aminoacrylate intermediate (kobs = 0.96 ± 0.02 and 0.38 ± 0.01 mm−1 s−1, respectively, at 24 °C) with concomitant loss of H2O and H2S and without detectable accumulation of the external aldimine or other intermediates. Homocysteine reacted with the aminoacrylate intermediate with kobs = 40.6 ± 3.8 s−1 and re-formed the internal aldimine. In the reverse direction, CBS reacted with cystathionine, forming the aminoacrylate intermediate with kobs = 0.38 ± 0.01 mm−1 s−1. This study provides the first insights into the pre-steady-state kinetic mechanism of human CBS and indicates that the reaction is likely to be limited by a conformational change leading to product release.  相似文献   

8.
Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible NOS was inhibited by the hydrogen sulfide (H2S) donor Na2S with IC50 values of ∼2.4·10−5 and ∼7.9·10−5 m, respectively, whereas human endothelial NOS was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of l-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin, indicating that H2S does not interfere with substrate or cofactor binding. The IC50 decreased to ∼1.5·10−5 m at pH 6.0 and increased to ∼8.3·10−5 m at pH 8.0. Preincubation of concentrated nNOS with H2S under turnover conditions decreased activity after dilution by ∼70%, suggesting irreversible inhibition. However, when calmodulin was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H2S and NO. Likewise, NADPH oxidation was inhibited with an IC50 of ∼1.9·10−5 m in the presence of Arg and BH4 but exhibited much higher IC50 values (∼1.0–6.1·10−4 m) when Arg and/or BH4 was omitted. Moreover, the relatively weak inhibition of nNOS by Na2S in the absence of Arg and/or BH4 was markedly potentiated by the NO donor 1-(hydroxy-NNO-azoxy)-l-proline, disodium salt (IC50 ∼ 1.3–2.0·10−5 m). These results suggest that nNOS and inducible NOS but not endothelial NOS are irreversibly inhibited by H2S/NO at modest concentrations of H2S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H2S formation.  相似文献   

9.
The main lesion in Parkinson disease (PD) is loss of substantia nigra dopaminergic neurons. Levodopa (l-DOPA) is the most widely used therapy, but it does not arrest disease progression. Some possible contributing factors to the continuing neuronal loss are oxidative stress, including oxidation of l-DOPA, and neurotoxins generated by locally activated microglia and astrocytes. A possible method of reducing these factors is to produce l-DOPA hybrid compounds that have antioxidant and antiinflammatory properties. Here we demonstrate the properties of four such l-DOPA hybrids based on coupling l-DOPA to four different hydrogen sulfide-donating compounds. The donors themselves were shown to be capable of conversion by isolated mitochondria to H2S or equivalent SH ions. This capability was confirmed by in vivo results, showing a large increase in intracerebral dopamine and glutathione after iv administration in rats. When human microglia, astrocytes, and SH-SY5Y neuroblastoma cells were treated with these donating agents, they all accumulated H2S intracellularly as did their derivatives coupled to l-DOPA. The donating agents and the l-DOPA hybrids reduced the release of tumor necrosis factor-α, interleukin-6, and nitric oxide from stimulated microglia, astrocytes as well as the THP-1 and U373 cell lines. They also demonstrated a neuroprotective effect by reducing the toxicity of supernatants from these stimulated cells to SH-SY5Y cells. l-DOPA itself was without effect in any of these assays. The H2S-releasing l-DOPA hybrid molecules also inhibited MAO B activity. They may be useful for the treatment of PD because of their significant antiinflammatory, antioxidant, and neuroprotective properties.  相似文献   

10.
Regulation of sulfate transport in filamentous fungi   总被引:2,自引:0,他引:2       下载免费PDF全文
Inorganic sulfate enters the mycelia of Aspergillus nidulans, Penicillium chrysogenum, and Penicillium notatum by a temperature-, energy-, pH-, ionic strength-, and concentration-dependent transport system (“permease”). Transport is unidirectional. In the presence of excess external sulfate, ATP sulfurylase-negative mutants will accumulate inorganic sulfate intracellularly to a level of about 0.04 m. The intracellular sulfate can be retained against a concentration gradient. Retention is not energy-dependent, nor is there any exchange between intracellular (accumulated) and extracellular sulfate. The sulfate permease is under metabolic control. Sulfur starvation of high methionine-grown mycelia results in about a 1000-fold increase in the specific sulfate transport activity at low external sulfate concentrations. l-Methionine is a metabolic repressor of the sulfate permease, while intracellular sulfate and possibly l-cysteine (or a derivative of l-cysteine) are feedback inhibitors. Sulfate transport follows hyperbolic saturation kinetics with a Michaelis constant (Km) value of 6 × 10−5 to 10−4m and a Vmax (for maximally sulfurstarved mycelia) of about 5 micromoles per gram per minute. Refeeding sulfur-starved mycelia with sulfate or cysteine results in about a 10-fold decrease in the Vmax value with no marked change in the Km. Azide and dinitrophenol also reduce the Vmax.  相似文献   

11.
12.
Organisms that overproduced l-cysteine and l-cystine from glucose were constructed by using Escherichia coli K-12 strains. cysE genes coding for altered serine acetyltransferase, which was genetically desensitized to feedback inhibition by l-cysteine, were constructed by replacing the methionine residue at position 256 of the serine acetyltransferase protein with 19 other amino acid residues or the termination codon to truncate the carboxy terminus from amino acid residues 256 to 273 through site-directed mutagenesis by using PCR. A cysteine auxotroph, strain JM39, was transformed with plasmids having these altered cysE genes. The serine acetyltransferase activities of most of the transformants, which were selected based on restored cysteine requirements and ampicillin resistance, were less sensitive than the serine acetyltransferase activity of the wild type to feedback inhibition by l-cysteine. At the same time, these transformants produced approximately 200 mg of l-cysteine plus l-cystine per liter, whereas these amino acids were not detected in the recombinant strain carrying the wild-type serine acetyltransferase gene. However, the production of l-cysteine and l-cystine by the transformants was very unstable, presumably due to a cysteine-degrading enzyme of the host, such as cysteine desulfhydrase. Therefore, mutants that did not utilize cysteine were derived from host strain JM39 by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. When a newly derived host was transformed with plasmids having the altered cysE genes, we found that the production of l-cysteine plus l-cystine was markedly increased compared to production in JM39.l-Cysteine, one of the important amino acids used in the pharmaceutical, food, and cosmetics industries, has been obtained by extracting it from acid hydrolysates of the keratinous proteins in human hair and feathers. The first successful microbial process used for industrial production of l-cysteine involved the asymmetric conversion of dl-2-aminothiazoline-4-carboxylic acid, an intermediate compound in the chemical synthesis of dl-cysteine, to l-cysteine by enzymes from a newly isolated bacterium, Pseudomonas thiazoliniphilum (11). Yamada and Kumagai (13) also described enzymatic synthesis of l-cysteine from beta-chloroalanine and sodium sulfide in which Enterobacter cloacae cysteine desulfhydrase (CD) was used. However, high level production of l-cysteine from glucose with microorganisms has not been studied.Biosynthesis of l-cysteine in wild-type strains of Escherichia coli and Salmonella typhimurium is regulated through feedback inhibition by l-cysteine of serine acetyltransferase (SAT), a key enzyme in l-cysteine biosynthesis, and repression of expression of a series of enzymes used for sulfide reduction from sulfate by l-cysteine (4), as shown in Fig. Fig.1.1. Denk and Böck reported that a small amount of l-cysteine was excreted by a revertant of a cysteine auxotroph of E. coli. In this revertant, SAT encoded by the cysE gene was desensitized to feedback inhibition by l-cysteine, and the methionine residue at position 256 in SAT was replaced by isoleucine (2). These results indicate that it may be possible to construct organisms that produce high levels of l-cysteine by amplifying an altered cysE gene. Although the residue at position 256 is supposedly part of the allosteric site for cysteine binding, no attention has been given to the effect of an amino acid substitution at position 256 in SAT on feedback inhibition by l-cysteine and production of l-cysteine. It is also not known whether isoleucine is the best residue for desensitization to feedback inhibition. Open in a separate windowFIG. 1Biosynthesis and regulation of l-cysteine in E. coli. Abbreviations: APS, adenosine 5′-phosphosulfate; PAPS, phosphoadenosine 5′-phosphosulfate; Acetyl CoA, acetyl coenzyme A. The open arrow indicates feedback inhibition, and the dotted arrows indicate repression.On the other hand, l-cysteine appears to be degraded by E. coli cells. Therefore, in order to obtain l-cysteine producers, a host strain with a lower level of l-cysteine degradation activity must be isolated. In this paper we describe high-level production of l-cysteine plus l-cystine from glucose by E. coli resulting from construction of altered cysE genes. The methionine residue at position 256 in SAT was replaced by other amino acids or the termination codon in order to truncate the carboxy terminus from amino acid residues 256 to 273 by site-directed mutagenesis. A newly derived cysteine-nondegrading E. coli strain with plasmids having the altered cysE genes was used to investigate production of l-cysteine plus l-cystine.  相似文献   

13.
The metabolism of S-methyl-l-cysteine   总被引:1,自引:1,他引:0  
1. Methylsulphinylacetic acid, 2-hydroxy-3-methylsulphinylpropionic acid and methylmercapturic acid sulphoxide (N-acetyl-S-methyl-l-cysteine S-oxide) were isolated as their dicyclohexylammonium salts from the urine of rats after they had been dosed with S-methyl-l-cysteine. 2. A fourth sulphoxide was isolated but not identified. 3. The excretion of sulphate in the urine of rats dosed with S-methyl-l-cysteine was measured. 4. The metabolism of S-methyl-l-cysteine by the hamster and guinea pig was examined chromatographically. 5. The preparation of the following compounds is reported: (−)-dicyclohexylammonium methyl-mercapturate sulphoxide; the dicyclohexylammonium salts of the optically inactive forms of 2-hydroxy-3-methylthiopropionic acid, 2-hydroxy-3-methyl-sulphinylpropionic acid and methylsulphinylacetic acid.  相似文献   

14.
Indigenous oral bacteria in the tongue coating such as Veillonella have been identified as the main producers of hydrogen sulfide (H2S), one of the major components of oral malodor. However, there is little information on the physiological properties of H2S production by oral Veillonella such as metabolic activity and oral environmental factors which may affect H2S production. Thus, in the present study, the H2S-producing activity of growing cells, resting cells, and cell extracts of oral Veillonella species and the effects of oral environmental factors, including pH and lactate, were investigated. Type strains of Veillonella atypica, Veillonella dispar, and Veillonella parvula were used. These Veillonella species produced H2S during growth in the presence of l-cysteine. Resting cells of these bacteria produced H2S from l-cysteine, and the cell extracts showed enzymatic activity to convert l-cysteine to H2S. H2S production by resting cells was higher at pH 6 to 7 and lower at pH 5. The presence of lactate markedly increased H2S production by resting cells (4.5- to 23.7-fold), while lactate had no effect on enzymatic activity in cell extracts. In addition to H2S, ammonia was produced in cell extracts of all the strains, indicating that H2S was produced by the catalysis of cystathionine γ-lyase (EC 4.4.1.1). Serine was also produced in cell extracts of V. atypica and V. parvula, suggesting the involvement of cystathionine β-synthase lyase (EC 4.2.1.22) in these strains. This study indicates that Veillonella produce H2S from l-cysteine and that their H2S production can be regulated by oral environmental factors, namely, pH and lactate.  相似文献   

15.
We investigated the pharmacological actions of a slow-releasing H2S donor, GYY 4137; a substrate for the biosynthesis of H2S, l-cysteine and its precursor, N-acetylcysteine on potassium (K+; 50 mM)-evoked [3H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10 nM–10 µM), l-cysteine (100 nM–10 µM) and N-acetylcysteine (10 µM–1 mM) elicited a concentration-dependent decrease in K+-evoked [3H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10 µM, the rank order of activity was as follows: l-cysteine?>?GYY 4137?>?N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H2S, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3 mM) reversed the inhibitory responses caused by GYY 4137, l-cysteine and N-acetylcysteine on K+-evoked [3H]D-aspartate release. Glibenclamide (300 µM), an inhibitor of KATP channels blocked the inhibitory action of GYY 4137 and l-cysteine but not that elicited by N-acetylcysteine on K+-induced [3H]D-aspartate release. The inhibitory effect of GYY 4137 and l-cysteine on K+-evoked [3H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), l-NAME (300 µM). Furthermore, a specific inhibitor of inducible NOS (iNOS), aminoguanidine (10 µM) blocked the inhibitory action of l-cysteine on K+-evoked [3H]D-aspartate release. We conclude that both donors and substrates for H2S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of KATP channels and NO synthase.  相似文献   

16.
An NADP-dependent dehydrogenase catalyzing the conversion of l-sorbosone to l-ascorbic acid has been isolated from Phaseolus vulgaris L. and Spinacia oleracea L. and partially purified. It is stable at −20°C for up to 8 months. Molecular masses, as determined by gel filtration, were 21 and 29 kilodaltons for bean and spinach enzymes, respectively. Km for sorbosone were 12 ± 2 and 18 ± 2 millimolar and for NADP+, 0.14 ± 0.05 and 1.2 ± 0.5 millimolar, for bean and spinach, respectively. Lycorine, a purported inhibitor of l-ascorbic acid biosynthesis, had no effect on the reaction.  相似文献   

17.
The superiority of d-methionine over l-methionine for stimulation of cephalosporin C synthesis in a crude medium was confirmed. The optimal level of dl-methionine was 0.5%. Methionine stimulates growth slightly but this is not thought to be the cause of the marked stimulation of antibiotic synthesis. Of a large number of sulfur compounds tested, only dl-methionine-dl-sulfoxide and S-methyl-l-cysteine showed considerable methionine-replacing activity. Lysine and α-aminoadipic acid were inactive.  相似文献   

18.
19.
Mechanically isolated Asparagus sprengeri Regel mesophyll cells cause alkalinization of the suspension medium on the addition of l-glutamate or its analog l-methionine-d,l-sulfoximine. Using a radiolabeled pH probe, it was found that both compounds caused internal acidification whereas l-aspartate did not. Fusicoccin stimulated H+ efflux from the cells by 111% and the uptake of l-[U-14C]glutamate by 55%. Manometric experiments demonstrated that, unlike l-methionine-d,l-sulfoximine, l-glutamate stimulated CO2 evolution from nonilluminated cells. Simultaneous measurements of medium alkalinization and 14CO2 evolution upon the addition of labeled l-glutamate showed that alkalinization was immediate and reached a maximum value after 45 minutes whereas 14CO2 evolution exhibited a lag before its appearance and continued in a linear manner for at least 100 minutes. Rates of alkalinization and uptake of l-[U-14C]glutamate were higher in the light while rates of 14CO2 evolution were higher in the dark. The major labeled product of glutamate decarboxylation, γ-aminobutyric acid, was found in the cells and the suspension medium. Its addition to the cell suspension did not result in medium alkalinization and evidence indicates that it is lost from the cell to the medium. The data suggest that the origin of medium alkalinization is co-transport not metabolism, and that the loss of labeled CO2 and γ-aminobutyric acid from the cell result in an overestimation of the stoichiometry of the H+/l-glutamate uptake process.  相似文献   

20.
Renal transport of four different categories of organic solutes, namely sugars, neutral amino acids, monocarboxylic acids and dicarboxylic acids, was studied by using the potential-sensitive dye 3,3′-diethyloxadicarbocyanine iodide in purified luminal-membrane and basolateral-membrane vesicles isolated from rabbit kidney cortex. Valinomycin-induced K+ diffusion potentials resulted in concomitant changes in dye–membrane-vesicle absorption spectra. Linear relationships were obtained between these changes and depolarization and hyperpolarization of the vesicles. Addition of d-glucose, l-phenylalanine, succinate or l-lactate to luminal-membrane vesicles, in the presence of an extravesicular>intravesicular Na+ gradient, resulted in rapid transient depolarization. With basolateral-membrane vesicles no electrogenic transport of d-glucose or l-phenylalanine was observed. Spectrophotometric competition studies revealed that d-galactose is electrogenically taken up by the same transport system as that for d-glucose, whereas l-phenylalanine, succinate and l-lactate are transported by different systems in luminal-membrane vesicles. The absorbance changes associated with simultaneous addition of d-glucose and l-phenylalanine were additive. The uptake of these solutes was influenced by the presence of Na+-salt anions of different permeabilities in the order: Cl>SO42−>gluconate. Addition of valinomycin to K+-loaded vesicles enhanced uptake of d-glucose and l-phenylalanine in the presence of an extravesicular>intravesicular Na+ gradient. Gramicidin or valinomycin plus nigericin diminished/abolished electrogenic solute uptake by Na+- or Na++K+-loaded vesicles respectively. These results strongly support the presence of Na+-dependent renal electrogenic transport of d-glucose, l-phenylalanine, succinate and l-lactate in luminal-membrane vesicles.  相似文献   

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