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1.
Ala-504 is a determinant of substrate binding affinity in the mouse Na(+)/dicarboxylate cotransporter 总被引:1,自引:0,他引:1
The Na(+)/dicarboxylate cotransporters from mouse (mNaDC1) and rabbit (rbNaDC1) differ in their ability to handle adipate, a six-carbon terminal dicarboxylic acid. The mNaDC1 and rbNaDC1 amino acid sequences are 75% identical. The rbNaDC1 does not transport adipate and only succinate produced inward currents under two-electrode voltage clamp. In contrast, oocytes expressing mNaDC1 had adipate-dependent inward currents that were about 60% of those induced by succinate. In order to identify domains involved in adipate transport, we examined the functional properties of a series of chimeric transporters made between mouse and rabbit NaDC1. We find that multiple transmembrane helices (TM), particularly TM 8, 9, and 10, are involved in adipate transport. In TM 10 there is only one amino acid difference between the two proteins, corresponding to Ala-504 in mouse and Ser-512 in rabbit NaDC1. The mNaDC1-A504S mutant had decreased adipate-dependent currents relative to succinate-dependent currents and an increase in the K(0.5) for both succinate and glutarate. We conclude that multiple amino acids from TM 8, 9 and 10 contribute to the transport of adipate in NaDC1. Furthermore, Ala-504 in TM 10 is an important determinant of K(0.5) for both adipate and succinate. 相似文献
2.
Naomi Oshiro 《生物化学与生物物理学报:生物膜》2006,1758(6):781-788
The Na+/dicarboxylate cotransporters from mouse (mNaDC1) and rabbit (rbNaDC1) differ in their ability to handle adipate, a six-carbon terminal dicarboxylic acid. The mNaDC1 and rbNaDC1 amino acid sequences are 75% identical. The rbNaDC1 does not transport adipate and only succinate produced inward currents under two-electrode voltage clamp. In contrast, oocytes expressing mNaDC1 had adipate-dependent inward currents that were about 60% of those induced by succinate. In order to identify domains involved in adipate transport, we examined the functional properties of a series of chimeric transporters made between mouse and rabbit NaDC1. We find that multiple transmembrane helices (TM), particularly TM 8, 9, and 10, are involved in adipate transport. In TM 10 there is only one amino acid difference between the two proteins, corresponding to Ala-504 in mouse and Ser-512 in rabbit NaDC1. The mNaDC1-A504S mutant had decreased adipate-dependent currents relative to succinate-dependent currents and an increase in the K0.5 for both succinate and glutarate. We conclude that multiple amino acids from TM 8, 9 and 10 contribute to the transport of adipate in NaDC1. Furthermore, Ala-504 in TM 10 is an important determinant of K0.5 for both adipate and succinate. 相似文献
3.
Transmembrane helices 3 and 4 are involved in substrate recognition by the Na+/dicarboxylate cotransporter, NaDC1 总被引:1,自引:0,他引:1
The Na(+)/dicarboxylate cotransporters (NaDC1) from mouse (m) and rabbit (rb) differ in their ability to handle glutarate. Substrate-dependent inward currents, measured using two-electrode voltage clamp, were similar for glutarate and succinate in Xenopus oocytes expressing mNaDC1. In contrast, currents evoked by glutarate in rbNaDC1 were only about 5% of the succinate-dependent currents. To identify domains involved in glutarate transport, we constructed a series of chimeric transporters between mouse and rabbit NaDC1. Although residues found in multiple transmembrane helices (TM) participate in glutarate transport, the most important contribution is made by TM 3 and 4 and the associated loops. The R(M3-4) chimera, consisting of rbNaDC1 with substitution of TM 3-4 from mNaDC1, had a decreased K(0.5)(glutarate) of 4 mM compared with 15 mM in wild-type rbNaDC1 without any effect on K(0.5)(succinate). The chimeras were also characterized using dual-label competitive uptakes with (14)C-glutarate and (3)H-succinate to calculate the transport specificity ratio (TSR), a measure of relative catalytic efficiency with the two substrates. The TSR analysis provides evidence for functional coupling in the transition state between TM 3 and 4. We conclude that TM 3 and 4 contain amino acid residues that are important determinants of substrate specificity and catalytic efficiency in NaDC1. 相似文献
4.
The Na+/dicarboxylate cotransporter (NaDC-1) couples the transport of sodium and tricarboxylic acid cycle intermediates, such as succinate and citrate. The rabbit and human homologues (rbNaDC-1 and hNaDC-1, respectively) are 78% identical in amino acid sequence but exhibit several differences in their functional properties. rbNaDC-1 has a greater apparent affinity for citrate and sodium than hNaDC-1. Furthermore, unlike hNaDC-1, rbNaDC-1 is inhibited by low concentrations of lithium. In this study, chimeric transporters were constructed to identify the protein domains responsible for the functional differences between rbNaDC-1 and hNaDC-1. Individual substitutions of transmembrane domain (TMD) 7, 10 or 11 produced transporters with intermediate properties. However, substitution of TMD 7, 10, and 11 together resulted in a transporter with the citrate Km of the donor, suggesting that interactions between these domains determine the differences in apparent citrate affinities. TMDs 10 and 11 are most important in determining the differences in apparent sodium affinities, and TMD 11 determines the sensitivity to lithium inhibition. We conclude that transmembrane domains 7, 10, and 11 in NaDC-1 may contain at least one of the cation binding sites in close proximity to the substrate binding domain. 相似文献
5.
Role of conserved prolines in the structure and function of the Na+/dicarboxylate cotransporter 1, NaDC1 总被引:11,自引:0,他引:11
The Na+/dicarboxylate cotransporter 1 (NaDC1) is a low-affinity transporter for citric acid cycle intermediates such as succinate and citrate. The sequence of NaDC1 contains a number of conserved proline residues in predicted transmembrane helices (TMs) 7 and 10. These transmembrane domains are of particular importance because they may be involved in determining the substrate or cation-binding affinity in NaDC1. Four conserved proline residues in TMs 7 and 10 of rabbit NaDC1 were replaced with alanine to promote ideal alpha helix or glycine to promote free conformation, and the mutant transporters were expressed in the HRPE cell line. Mutations of prolines in TM 10 produced decreased protein expression and activity, whereas mutations of prolines in TM 7 completely abolished protein expression and activity. The chemical chaperone glycerol was found to improve the expression of the Pro-351 mutants in TM 7, suggesting that these mutants had defects in trafficking. The inactive mutant transporters at position 351 could also be rescued by the addition of a proline at a second site. For example, the P351A-F347P mutant had restored activity, although its substrate specificity was altered. We conclude that, in TM 7, Pro-327 may be of particular importance in the function of the transporter, whereas Pro-351 may affect protein targeting. The prolines in TM 10, at positions 523 and 524, may not be directly involved in the transporter function but may be necessary for maintaining structure. 相似文献
6.
Hering-Smith KS Schiro FR Pajor AM Hamm LL 《American journal of physiology. Renal physiology》2011,300(2):F425-F432
Urinary citrate is an important inhibitor of calcium nephrolithiasis and is primarily determined by proximal tubule reabsorption. The major transporter to reabsorb citrate is Na(+)-dicarboxylate cotransporter (NaDC1), which transports dicarboxylates, including the divalent form of citrate. We previously found that opossum kidney (OK) proximal tubule cells variably express either divalent or trivalent citrate transport, depending on extracellular calcium. The present studies were performed to delineate the mechanism of the effect of calcium on citrate and succinate transport in these cells. Transport was measured using isotope uptake assays. In some studies, NaDC1 transport was studied in Xenopus oocytes, expressing either the rabbit or opossum ortholog. In the OK cell culture model, lowering extracellular calcium increased both citrate and succinate transport by more than twofold; the effect was specific in that glucose transport was not altered. Citrate and succinate were found to reciprocally inhibit transport at low extracellular calcium (<60 μM), but not at normal calcium (1.2 mM); this mutual inhibition is consistent with dicarboxylate transport. The inhibition varied progressively at intermediate levels of extracellular calcium. In addition to changing the relative magnitude and interaction of citrate and succinate transport, decreasing calcium also increased the affinity of the transport process for various other dicarboxylates. Also, the affinity for succinate, at low concentrations of substrate, was increased by calcium removal. In contrast, in oocytes expressing NaDC1, calcium did not have a similar effect on transport, indicating that NaDC1 could not likely account for the findings in OK cells. In summary, extracellular calcium regulates constitutive citrate and succinate transport in OK proximal tubule cells, probably via a novel transport process that is not NaDC1. The calcium effect on citrate transport parallels in vivo studies that demonstrate the regulation of urinary citrate excretion with urinary calcium excretion, a process that may be important in decreasing urinary calcium stone formation. 相似文献
7.
Kaufhold M Schulz K Breljak D Gupta S Henjakovic M Krick W Hagos Y Sabolic I Burckhardt BC Burckhardt G 《American journal of physiology. Renal physiology》2011,301(5):F1026-F1034
Organic anions are taken up from the blood into proximal tubule cells by organic anion transporters 1 and 3 (OAT1 and OAT3) in exchange for dicarboxylates. The released dicarboxylates are recycled by the sodium dicarboxylate cotransporter 3 (NaDC3). In this study, we tested the substrate specificities of human NaDC3, OAT1, and OAT3 to identify those dicarboxylates for which the three cooperating transporters have common high affinities. All transporters were stably expressed in HEK293 cells, and extracellularly added dicarboxylates were used as inhibitors of [(14)C]succinate (NaDC3), p-[(3)H]aminohippurate (OAT1), or [(3)H]estrone-3-sulfate (OAT3) uptake. Human NaDC3 was stably expressed as proven by immunochemical methods and by sodium-dependent uptake of succinate (K(0.5) for sodium activation, 44.6 mM; Hill coefficient, 2.1; K(m) for succinate, 18 μM). NaDC3 was best inhibited by succinate (IC(50) 25.5 μM) and less by α-ketoglutarate (IC(50) 69.2 μM) and fumarate (IC(50) 95.2 μM). Dicarboxylates with longer carbon backbones (adipate, pimelate, suberate) had low or no affinity for NaDC3. OAT1 exhibited the highest affinity for glutarate, α-ketoglutarate, and adipate (IC(50) between 3.3 and 6.2 μM), followed by pimelate (18.6 μM) and suberate (19.3 μM). The affinity of OAT1 to succinate and fumarate was low. OAT3 showed the same dicarboxylate selectivity with ~13-fold higher IC(50) values compared with OAT1. The data 1) reveal α-ketoglutarate as a common high-affinity substrate of NaDC3, OAT1, and OAT3 and 2) suggest potentially similar molecular structures of the binding sites in OAT1 and OAT3 for dicarboxylates. 相似文献
8.
Citric acid cycle intermediates, including succinate and citrate, are absorbed across the apical membrane by the NaDC1 Na+/dicarboxylate cotransporter located in the kidney and small intestine. The secondary structure model of NaDC1 contains 11 transmembrane helices (TM). TM7 was shown previously to contain determinants of citrate affinity, and Arg-349 at the extracellular end of the helix is required for transport. The present study involved cysteine scanning mutagenesis of 26 amino acids in TM7 and the associated loops. All of the mutants were well expressed on the plasma membrane, but many had low or no transport activity: 6 were inactive and 7 had activity less than 25% of the parental. Three of the mutants had notable changes in functional properties. F336C had increased transport activity due to an increased Vmax for succinate. The conserved residue F339C had very low transport activity and a change in substrate selectivity. G356C in the putative extracellular loop was the only cysteine mutant that was affected by the membrane-impermeant cysteine reagent, MTSET. However, direct labeling of G356C with MTSEA-biotin gave a weak signal, indicating that this residue is not readily accessible to more bulky reagents. The results suggest that the amino acids of TM7 are functionally important because their replacement by cysteine had large effects on transport activity. However, most of TM7 does not appear to be accessible to the extracellular fluid and is likely to be an outer helix in contact with the lipid bilayer. 相似文献
9.
The transport routes for threonine in a primate kidney epithelial cell line (BSC-1) grown as monolayer in continuous cell culture were studied. We discovered at least four different transport systems for threonine uptake. The Na(+)-dependent route shows biphasic kinetics with a low and high affinity parameter. The apparent kinetic constants for Km1 and Km2 were 0.3 and 36 mM with apparent Vmax values of 6.3 and 90 nmol/mg protein/min, respectively. The high affinity, low Km component resembles system ASC activity, with respect to substrate selectivity. The Na(+)-independent route also exhibits biphasic kinetics. A high affinity component (apparent Km of 1.0 mM, and apparent Vmax of 7.2 nmol/mg protein/min) is sensitive to inhibition by leucine and the aminoendolevo-rotatory isomer of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, suggesting participation by system L. The low affinity component (apparent Km of 10.2 mM, and apparent Vmax of 71 nmol/mg protein/min) was specifically inhibited by threonine, serine, and alanine and could be assigned to system asc. The discrimination between system L and asc is based upon differences in pH sensitivity, trans stimulation, and Ki values. In addition, the effects of harmaline, a suspected sodium transport site inhibitor, have been studied. Harmaline noncompetitively inhibited Na(+)-dependent threonine uptake but had no effect on Na(+)-independent transport of threonine. This report is the first to present evidence for the presence of system asc in renal epithelial cells. The physiological and biochemical significance of our findings are discussed. 相似文献
10.
Structure, function, and genomic organization of human Na(+)-dependent high-affinity dicarboxylate transporter 总被引:6,自引:0,他引:6
Wang H Fei YJ Kekuda R Yang-Feng TL Devoe LD Leibach FH Prasad PD Ganapathy V 《American journal of physiology. Cell physiology》2000,278(5):C1019-C1030
We have clonedand functionally characterized the human Na+-dependenthigh-affinity dicarboxylate transporter (hNaDC3) from placenta. ThehNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the clonedtransporter mediates the transport of succinate in the presence ofNa+ [concentration of substrate necessary for half-maximaltransport (Kt) for succinate = 20 ± 1 µM]. Dimethylsuccinate also interacts with hNaDC3. TheNa+-to-succinate stoichiometry is 3:1 and concentration ofNa+ necessary for half-maximal transport(KNa+0.5) is 49 ± 1 mM as determined by uptake studies withradiolabeled succinate. When expressed in Xenopuslaevis oocytes, hNaDC3 induces Na+-dependent inwardcurrents in the presence of succinate and dimethylsuccinate. At amembrane potential of 50 mV,KSuc0.5 is 102 ± 20 µM andKNa+0.5 is 22 ± 4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer andradiolabeled succinate uptake in hNaDC3-expressing oocytes indicate acharge-to-succinate ratio of 1:1 for the transport process, suggestinga Na+-to-succinate stoichiometry of 3:1. pH titration ofcitrate-induced currents shows that hNaDC3 accepts preferentially thedivalent anionic form of citrate as a substrate. Li+inhibits succinate-induced currents in the presence of Na+.Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The humanNaDC3 gene is located on chromosome20q12-13.1, as evidenced by fluorescent in situ hybridization. Thegene is >80 kbp long and consists of 13 exons and 12 introns. 相似文献
11.
人钠/二羧酸协同转运蛋白1基因融合表达及其抗体制备 总被引:12,自引:0,他引:12
利用DNA重组技术 ,将编码人钠 羧酸协同转运蛋白 1(hNaDC1)抗原表位区 (W138 Q2 19)的cDNA克隆至融合表达载体pGEX 5X 1,构建重组质粒pGEX hNaDCL6 .在大肠杆菌BL2 1中 ,经IPTG诱导 ,获得谷胱甘肽巯基转移酶 (GST) hNaDC1重组融合蛋白的表达 .以谷胱甘肽 Sepharose 4B亲和层析 ,获得纯化的GST hNaDC1.以此为免疫原制备的抗hNaDC1抗体可特异性识别人类和大鼠肾组织以及小肠组织中天然的钠 二羧酸协同转运蛋白 1.利用该抗体 ,首次证实了hNaDC1基因编码产物分布于人肾组织近端肾小管刷状缘 ,与大鼠钠 二羧酸协同转运蛋白 1(SDCT1)分布一致 . 相似文献
12.
Marie-Theres Schrder Gertrud Schfer Peter Schauder 《Journal of cellular physiology》1990,145(1):155-161
Characteristics of glutamine transport, its substrate specificity, and its pattern of competitive and non-competitive inhibition in response to amino acid analogues were determined in peripheral human lymphocytes, incubated with or without concanavalin A (Con A). Maximum capacity of transport (Vmax) at 37 degrees C and 136.9 mM Na+ was 30 pmol/10(6) cells/30 seconds, while the apparent Km was 142 microM. In cells exposed to 10 mM histidine, asparagine, serine, or leucine transport of glutamine declined to 28%, 15%, 17%, and 21%, respectively, of the rates in controls. Inhibition by histidine (Ki = 0.58 mM) and serine (Ki = 0.25 mM) was competitive, by leucine was non-competitive (Ki = 0.64), while alpha-methylamino-isobutyric acid and 2-amino carboxy-bicyclo (2.2.1)-heptane had no effect. In cells cultured for 24 hours with or without 10 micrograms/ml Con A, the apparent Km was 70 microM vs. 89 microM and Vmax 73 vs. 26 pmol/10(6) cells/30 seconds. Sodium depletion (9.0 mM NaCl) greatly diminished glutamine transport in resting and stimulated cells. Inhibition of glutamine transport by serine was sodium sensitive, while inhibition by histidine and asparagine was not. Serine had no competitive effect in sodium-depleted media. The data demonstrate what appear to be two carrier systems for glutamine, sodium sensitive and sodium insensitive. It is suggested that glutamine transport into lymphocytes occurs via processes similar to System N and System ASC described in other cells, with System ASC as the sodium-sensitive component. Con A augments the capacity rather than the affinity of glutamine transporting systems. 相似文献
13.
The transport of succinate was studied in an effective streptomycin-resistant strain of Rhizobium leguminosarum. High levels of succinate transport occurred when cells were grown on succinate, fumarate, or malate, whereas low activity was found when cells were grown on glucose, sucrose, arabinose, or pyruvate as the sole carbon source. Because of the rapid metabolism of succinate after transport into the cells, a succinate dehydrogenase-deficient mutant was isolated in which intracellular succinate accumulated to over 400 times the external concentration. Succinate transport was completely abolished in the presence of metabolic uncouplers but was relatively insensitive to sodium arsenate. Succinate transport was a saturable function of the succinate concentration, and the apparent Km and Vmax values for transport were determined in both the parent and the succinate dehydrogenase mutant. Malate and fumarate competitively inhibited succinate transport, whereas citrate and malonate had no effect. Succinate transport mutants were isolated by transposon (Tn5) mutagenesis. These mutants were unable to transport succinate or malate and were unable to grow on succinate, malate, or fumarate as the sole carbon source. The mutants grew normally on pyruvate, oxaloacetate, citrate, or arabinose, and revertants isolated on succinate minimal medium had regained the ability to grow on malate and fumarate. From these data, we conclude that R. leguminosarum possesses a C4-dicarboxylic acid transport system which is inducible and mediates the active transport of succinate, fumarate, and malate into the cell. 相似文献
14.
A UDP-glucuronosyltransferase (GT) enzyme was isolated from ethanol-induced male New Zealand white rabbit hepatic protein. The animals were pretreated for 2 weeks with 10% ethanol in their drinking water. The GT enzyme was purified by anion-exchange and affinity chromatography and was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The molecular mass of the ethanol-induced UDP-glucuronosyltransferase was determined to be 57,000 Da. Tryptic digests of the ethanol-induced GT and a similarly purified GT from control rabbit liver appeared to be different by HPLC analysis, even though the molecular masses of the enzymes were indistinguishable. Amino acid compositions of the two proteins were different for six amino acids. The apparent Km values for the ethanol-induced GT enzyme for 1-naphthol and morphine as substrates were 43 and 109 microM, respectively. The apparent Vmax values for the ethanol-induced GT enzyme for these substrates were 83 and 4.6 nmol/min/mg protein. The increases in catalytic efficiencies, apparent Vmax/Km for 1-naphthol and for morphine, for the ethanol-induced isozyme compared to the control isozyme activities were 2.0- and 2.4-fold. A polyclonal antibody raised in sheep to the rabbit ethanol-induced GT demonstrated a 520-fold selectivity for precipitation of the ethanol-induced protein rather than the control protein. These results demonstrate the production of an unique isozyme of UDP-glucuronosyltransferase that is produced in rabbits as a result of chronic ethanol exposure. 相似文献
15.
Reduction of ferric citrate catalyzed by NADH:nitrate reductase 总被引:1,自引:0,他引:1
M G Redinbaugh W H Campbell 《Biochemical and biophysical research communications》1983,114(3):1182-1188
We show that NADH:nitrate reductase from squash cotyledons can catalyze the reduction of ferric citrate. When nitrate reductase was purified to homogeneity using a two-step affinity chromatography procedure, an NADH:Fe(III)-citrate reductase activity copurified with it and had identical electrophoretic mobility to it. The iron reductase activity was optimum near pH 6.3, had an apparent Km for Fe(III)-citrate of 0.02 mM, and was inhibited by monospecific anti-nitrate reductase rabbit sera. Differential inhibition of the enzyme's activities indicated iron and nitrate were reduced at different sites. In addition to its role in nitrogen assimilation, nitrate reductase catalyzes ferric citrate reduction and could have a role in iron assimilation. 相似文献
16.
Fujita T Katsukawa H Yodoya E Wada M Shimada A Okada N Yamamoto A Ganapathy V 《Journal of neurochemistry》2005,93(3):706-714
We investigated in the present study the transport characteristics of N-acetyl-L-aspartate in primary cultures of astrocytes from rat cerebral cortex and the involvement of NA+-coupled high-affinity carboxylate transporter NaC3 (formerly known as NaDC3) responsible for N-acetyl-L-aspartate transport. N-acetyl-L-aspartate transport was NA+-dependent and saturable with a Michaelis-Menten constant (Km) of approximately 110 microm. NA+-activation kinetics revealed that the NA+ to-N-acetyl-L-aspartate stoichiometry was 3 : 1 and concentration of Na+ necessary for half-maximal transport (KNA m) was 70 mm. NA+-dependent N-acetyl-L-aspartate transport was competitively inhibited by succinate with an inhibitory constant (Ki) of 14.7 microm, which was comparable to the Km value of NA+-dependent succinate transport (29.4 microm). L-aspartate also inhibited NA+-dependent [14C]N-acetyl-L-aspartate transport with relatively low affinity (Ki = 2.2 mm), whereas N-acetyl-L-aspartate was not able to inhibit NA+-dependent aspartate transport in astrocytes. In addition, Li+ was found to have a significant inhibitory effect on the NA+-dependent N-acetyl-L-aspartate transport in a concentration-dependent manner. Furthermore, RT-PCR and western blot analyses revealed that NaC3 is expressed in primary cultures of astrocytes. Taken collectively, these results indicate that NaC3 expressed in rat cerebrocortical astrocytes is responsible for NA+-dependent N-acetyl-L-aspartate transport. This transporter is likely to be an essential prerequisite for the metabolic role of N-acetyl-L-aspartate in the process of myelination. 相似文献
17.
Purification and characterization of the reconstitutively active citrate carrier from maize mitochondria. 总被引:3,自引:0,他引:3
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The citrate carrier from maize (Zea mays) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and hydroxyapatite/celite in the presence of cardiolipin. SDS-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31 kD. When reconstituted into liposomes, the citrate carrier catalyzed a pyridoxal 5'-P-sensitive citrate/citrate exchange. It was purified 224-fold with a recovery of 50% and a protein yield of 0.22% with respect to the mitochondrial extract. In the reconstituted system the purified citrate carrier catalyzed a first-order reaction of citrate/citrate (0.065 min-1) or citrate/malate exchange (0.075 min-1). Among the various substrates and inhibitors tested, the reconstituted protein transported citrate, cis-aconitate, isocitrate, L-malate, succinate, malonate, glutarate, alpha-ketoglutarate, oxaloacetate, and alpha-ketoadipate and was inhibited by pyridoxal 5'-P, phenylisothiocyanate, mersalyl, and p-hydroxymercuribenzoate (but not N-ethylmaleimide), 1,2, 3-benzentricarboxylate, benzylmalonate, and butylmalonate. The activation energy of the citrate/citrate exchange was 66.5 kJ/mol between 10 degrees C and 35 degrees C; the half-saturation constant (Km) for citrate was 0.65 +/- 0.05 mM and the maximal rate (Vmax) of the citrate/citrate exchange was 13.0 +/- 1.0 micromol min-1 mg-1 protein at 25 degrees C. 相似文献
18.
The activities of the mitochondrial enzymes citrate synthase (citrate oxaloacetatelyase, EC 4.1.3.7), NADP-linked isocitrate dehydrogenase (threo-Ds-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42), and succinate dehydrogenase (succinate: FAD oxidoreductase, EC 1.3.99.1) as well as their kinetic behavior in the two developmental forms of Trypanosoma cruzi at insect vector stage, epimastigotes and infective metacyclic trypomastigotes, were studied. The results presented in this work clearly demonstrate a higher mitochondrial metabolism in the metacyclic forms as is shown by the extraordinary enhanced activities of metacyclic citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase. In epimastigotes, the specific activities of citrate synthase at variable concentrations of oxalacetate and acetyl-CoA were 24.6 and 26.6 mU/mg of protein, respectively, and the Michaelis constants were 7.88 and 6.84 microM for both substrates. The metacyclic enzyme exhibited the following kinetic parameters: a specific activity of 228.4 mU/mg and Km of 3.18 microM for oxalacetate and 248.5 mU/mg and 2.75 microM, respectively, for acetyl-CoA. NADP-linked isocitrate dehydrogenase specific activities for epimastigotes and metacyclics were 110.2 and 210.3 mU/mg, whereas the apparent Km's were 47.9 and 12.5 microM, respectively. No activity for the NAD-dependent isozyme was found in any form of T. cruzi differentiation. The particulated succinate dehydrogenase showed specific activities of 8.2 and 39.1 mU/mg for epimastigotes and metacyclic trypomastigotes, respectively, although no significant changes in the Km (0.46 and 0.48 mM) were found. The cellular role and the molecular mechanism that probably take place during this significant shift in the mitochondrial metabolism during the T. cruzi differentiation have been discussed. 相似文献
19.
Expression cloning and characterization of a novel sodium-dicarboxylate cotransporter from winter flounder kidney. 总被引:2,自引:0,他引:2
J Steffgen B C Burckhardt C Langenberg L Kühne G A Müller G Burckhardt N A Wolff 《The Journal of biological chemistry》1999,274(29):20191-20196
A cDNA coding for a Na+-dicarboxylate cotransporter, fNaDC-3, from winter flounder (Pseudopleuronectes americanus) kidney was isolated by functional expression in Xenopus laevis oocytes. The fNaDC-3 cDNA is 2384 nucleotides long and encodes a protein of 601 amino acids with a calculated molecular mass of 66.4 kDa. Secondary structure analysis predicts at least eight membrane-spanning domains. Transport of succinate by fNaDC-3 was sodium-dependent, could be inhibited by lithium, and evoked an inward current. The apparent affinity constant (Km) of fNaDC-3 for succinate of 30 microM resembles that of Na+-dicarboxylate transport in the basolateral membrane of mammalian renal proximal tubules. The substrates specific for the basolateral transporter, 2,3-dimethylsuccinate and cis-aconitate, not only inhibited succinate uptake but also evoked inward currents, proving that they are transported by fNaDC-3. Succinate transport via fNaDC-3 decreased by lowering pH, as did citrate transport, although much more moderately. These characteristics suggest that fNaDC-3 is a new type of Na+-dicarboxylate transporter that most likely corresponds to the Na+-dicarboxylate cotransporter in the basolateral membrane of mammalian renal proximal tubules. 相似文献
20.
A defined medium (XF-26) containing 3 inorganic salts, 2 tricarboxylic acids, 17 amino acids, potato starch, phenol red, and agar was used as the starting point for the study. Deletions of one or more ingredients were performed to prepare various media. A medium was considered able to support growth of Xylella fastidiosa strains responsible for Pierce's disease in grapes, only after 10 serial passages had been completed. Of 3 inorganic salts, K2HPO4 and MgSO4 x 7H2O were essential, and (NH4)2HPO4 was nonessential for growth. Of the Krebs cycle intermediates, all (citrate, alpha-ketoglutarate, succinate, fumarate, malate, and oxaloacetate) but isocitrate supported growth of cultivated strains, whereas only citrate alone or citrate plus succinate supported the primary isolation of PD bacterium. Of 17 amino acids, 6 uncharged polar R groups (asparagine, cysteine, glutamine, glycine, serine, and threonine) supported growth, whereas 8 nonpolar R groups (alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine) or 3 positively charged polar groups (arginine, histidine, and lysine) did not. Starch proved to be nonessential. 相似文献