Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae

The SLC13 transporter family, whose members play key physiological roles in the regulation of fatty acid synthesis, adiposity, insulin resistance, and other processes, catalyzes the transport of Krebs cycle intermediates and sulfate across the plasma membrane of mammalian cells. SLC13 transporters are part of the divalent anion:Na⁺ symporter (DASS) family that includes several well-characterized bacterial members. Despite sharing significant sequence similarity, the functional characteristics of DASS family members differ with regard to their substrate and coupling ion dependence. The publication of a high resolution structure of dimer VcINDY, a bacterial DASS family member, provides crucial structural insight into this transporter family. However, marrying this structural insight to the current functional understanding of this family also demands a comprehensive analysis of the transporter’s functional properties. To this end, we purified VcINDY, reconstituted it into liposomes, and determined its basic functional characteristics. Our data demonstrate that VcINDY is a high affinity, Na⁺-dependent transporter with a preference for C₄- and C₅-dicarboxylates. Transport of the model substrate, succinate, is highly pH dependent, consistent with VcINDY strongly preferring the substrate’s dianionic form. VcINDY transport is electrogenic with succinate coupled to the transport of three or more Na⁺ ions. In contrast to succinate, citrate, bound in the VcINDY crystal structure (in an inward-facing conformation), seems to interact only weakly with the transporter in vitro. These transport properties together provide a functional framework for future experimental and computational examinations of the VcINDY transport mechanism.     

Functional characterization of a Na(+)-coupled dicarboxylate carrier protein from Staphylococcus aureus

We have cloned and functionally characterized a Na(+)-coupled dicarboxylate transporter, SdcS, from Staphylococcus aureus. This carrier protein is a member of the divalent anion/Na(+) symporter (DASS) family and shares significant sequence homology with the mammalian Na(+)/dicarboxylate cotransporters NaDC-1 and NaDC-3. Analysis of SdcS function indicates transport properties consistent with those of its eukaryotic counterparts. Thus, SdcS facilitates the transport of the dicarboxylates fumarate, malate, and succinate across the cytoplasmic membrane in a Na(+)-dependent manner. Furthermore, kinetic work predicts an ordered reaction sequence with Na(+) (K(0.5) of 2.7 mM) binding before dicarboxylate (K(m) of 4.5 microM). Because this transporter and its mammalian homologs are functionally similar, we suggest that SdcS may serve as a useful model for DASS family structural analysis.     

Functional reconstitution of SdcS, a Na+-coupled dicarboxylate carrier protein from Staphylococcus aureus

In Staphylococcus aureus, the transport of dicarboxylates is mediated in part by the Na+-linked carrier protein SdcS. This transporter is a member of the divalent-anion/Na+ symporter (DASS) family, a group that includes the mammalian Na+/dicarboxylate cotransporters NaDC1 and NaDC3. In earlier work, we cloned and expressed SdcS in Escherichia coli and found it to have transport properties similar to those of its eukaryotic counterparts (J. A. Hall and A. M. Pajor, J. Bacteriol. 187:5189-5194, 2005). Here, we report the partial purification and subsequent reconstitution of functional SdcS into liposomes. These proteoliposomes exhibited succinate counterflow activity, as well as Na+ electrochemical-gradient-driven transport. Examination of substrate specificity indicated that the minimal requirement necessary for transport was a four-carbon terminal dicarboxylate backbone and that productive substrate-transporter interaction was sensitive to substitutions at the substrate C-2 and C-3 positions. Further analysis established that SdcS facilitates an electroneutral symport reaction having a 2:1 cation/dicarboxylate ratio. This study represents the first characterization of a reconstituted Na+-coupled DASS family member, thus providing an effective method to evaluate functional, as well as structural, aspects of DASS transporters in a system free of the complexities and constraints associated with native membrane environments.     

Regulation of the Na+-coupled glutamate transporter EAAT3 by PIKfyve

The Na⁺,glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end, EAAT3 was expressed in Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current (I_glu) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I_glu. Coexpression of either, SGK1 or PIKfyve, significantly enhanced I_glu in EAAT3 expressing oocytes. The increased I_glu was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I_glu and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressing EAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant ^K127NSGK1 did not significantly alter I_glu in EAAT3 expressing oocytes and completely reversed the stimulating effect of PIKfyve coexpression on I_glu. The stimulating effect of PIKfyve on I_glu was abolished by replacement of the serine by alanine in the SGK consensus sequence (^S318APIKfyve). Moreover, additional coexpression of ^S318APIKfyve significantly blunted I_glu in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.     

Differential regulation of Na+,K+-ATPase and the Na+-coupled glucose transporter in hypertensive rat kidney

Several Na⁺ transporters are functionally abnormal in the hypertensive rat. Here, we examined the effects of a high-salt load on renal Na⁺,K⁺-ATPase and the sodium-coupled glucose transporter (SGLT1) in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. The protein levels of Na⁺,K⁺-ATPase and SGLT1 in the DS rat were the same as those in the DR rat, and were not affected by the high-salt load. In the DS rat, a high-salt load decreased Na⁺,K⁺-ATPase activity, and this decrease coincided with a decrease in the apparent Mechaelis constant (K_m) for ATP, but not with a change of maximum velocity (V_max). On the contrary, a high-salt load increased SGLT1 activity in the DS rat, which coincided with an increase in the V_max for α-methyl glucopyranoside. The protein level of phosphorylated tyrosine residues in Na⁺,K⁺-ATPase was decreased by the high-salt load in the DS rat. The amount of phosphorylated serine was not affected by the high-salt load in DR rats, and could not be detected in DS rats. On the other hand, the amount of phosphorylated serine residues in SGLT1 was increased by the high-salt load. However, the phosphorylated tyrosine was the same for all samples. Therefore, we concluded that the high-salt load changes the protein kinase levels in DS rats, and that the regulation of Na⁺,K⁺-ATPase and SGLT1 activity occurs via protein phosphorylation.     

Human Na+ -coupled citrate transporter: primary structure,genomic organization,and transport function

This paper describes the cloning and functional characterization of the human Na(+)-coupled citrate transporter (NaCT). The cloned human NaCT shows 77% sequence identity with rat NaCT. The nact gene is located on human chromosome 17 at p12-13. NaCT mRNA is expressed most predominantly in the liver, with moderate expression detectable in the brain and testis. When functionally expressed in mammalian cells, human NaCT mediates the Na(+)-coupled transport of citrate. Studies with several monocarboxylates, dicarboxylates, and tricarboxylates show that the transporter is selective for citrate with comparatively several-fold lower affinity for other intermediates of citric acid cycle. The Michelis-Menten constant for citrate is approximately 650 microM. The activation of citrate transport by Na(+) is sigmoidal, suggesting involvement of multiple Na(+) ions in the activation process. The transport process is electrogenic. This represents the first plasma membrane transporter in humans that mediates the preferential entry of citrate into cells. Citrate occupies a pivotal position in many important biochemical pathways. Among various citric acid cycle intermediates, citrate is present at the highest concentrations in human blood. The selectivity of NaCT towards citrate and its predominant expression in the liver suggest that this transporter may facilitate the utilization of circulating citrate for the generation of metabolic energy and for the synthesis of fatty acids and cholesterol.     

Gene cloning and characterization of VcrM,a Na+-coupled multidrug efflux pump,from Vibrio cholerae non-O1

We cloned a DNA fragment responsible for drug resistance from chromosome of Vibrio cholerae non-O1. Nucleotide sequence analysis of this fragment revealed the presence of a single open reading frame encoding a protein consisting of 445 amino acid residues. We designated the gene as vcrM. Hydropathy analysis of the deduced amino acid sequence of VcrM suggests the presence of 12 trans-membrane segments. A dendrogram showed that VcrM is a member of the DinF-subfamily within the MATE family of multidrug efflux pumps. Expression of the cloned vcrM gene in drug-hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4', 6-diamidino-2-phenylindole, Hoechst 33342, rhodamine 6G, tetraphenylphosphonium chloride (TPPCl) and ethidium bromide. Efflux of acriflavine due to VcrM was dependent on Na+ or Li+. Moreover, Na+ efflux was observed with VcrM when TPPCl was added to Na+-loaded cells. Therefore, we conclude that VcrM is a Na+/drug antiporter-type multidrug efflux pump.     

Transport model of the human Na+-coupled L-ascorbic acid (vitamin C) transporter SVCT1

Vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant and as a cofactor in many enzymatic reactions. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial apical L-ascorbic acid cotransporter SVCT1 (SLC23A1). We explored the molecular mechanisms of SVCT1-mediated L-ascorbic acid transport using radiotracer and voltage-clamp techniques in RNA-injected Xenopus oocytes. L-ascorbic acid transport was saturable (K(0.5) approximately 70 microM), temperature dependent (Q(10) approximately 5), and energized by the Na(+) electrochemical potential gradient. We obtained a Na(+)-L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-ascorbic acid and Na(+) saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na(+), L-ascorbic acid, Na(+). In the absence of L-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3-15 ms. Transients were described by single Boltzmann distributions. At 100 mM Na(+), maximal charge translocation (Q(max)) was approximately 25 nC, around a midpoint (V(0.5)) at -9 mV, and with apparent valence approximately -1. Q(max) was conserved upon progressive removal of Na(+), whereas V(0.5) shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first Na(+) partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT1 to vitamin C metabolism in health and disease.     

Cloning and functional characterization of a high-affinity Na(+)/dicarboxylate cotransporter from mouse brain

Neurons contain a high-affinity Na(+)/dicarboxylate cotransporter for absorption of neurotransmitter precursor substrates, such as alpha-ketoglutarate and malate, which are subsequently metabolized to replenish pools of neurotransmitters, including glutamate. We have isolated the cDNA coding for a high-affinity Na(+)/dicarboxylate cotransporter from mouse brain, called mNaDC-3. The mRNA coding for mNaDC-3 is found in brain and choroid plexus as well as in kidney and liver. The mNaDC-3 transporter has a broad substrate specificity for dicarboxylates, including succinate, alpha-ketoglutarate, fumarate, malate, and dimethylsuccinate. The transport of citrate is relatively insensitive to pH, but the transport of succinate is inhibited by acidic pH. The Michaelis-Menten constant for succinate in mNaDC-3 is 140 microM in transport assays and 16 microM at -50 mV in two-electrode voltage clamp assays. Transport is dependent on sodium, although lithium can partially substitute for sodium. In conclusion, mNaDC-3 likely codes for the high-affinity Na(+)/dicarboxylate cotransporter in brain, and it has some unusual electrical properties compared with the other members of the family.     

Production and characterization of biosurfactants from Bacillus licheniformis F2.2

A biosurfactant-producing strain, Bacillus licheniformis F2.2, was isolated from a fermented food in Thailand. The strain was capable of producing a new biosurfactant, BL1193, as well as two kinds of popular lipopeptide biosurfactants, plipastatin and surfactin. Mass spectrometry and FT-IR analysis indicated that BL1193 had a molecular mass of 1,193 Da with no peptide portion in the molecule. While plipastatin and surfactin were abundantly produced in a nutrient YPD medium, BL1193 was produced only in a synthetic DF medium containing no amino acids. According to an oil displacement activity test, the specific activity of BL1193 (6.53 kBS units/mg) is equivalent to that of surfactin (5.78-6.83 kBS units/mg).     

Functional characterization of high-affinity Na(+)/dicarboxylate cotransporter found in Xenopus laevis kidney and heart

The SLC13 gene family includes sodium-coupled transporters for citric acid cycle intermediates and sulfate. The present study describes the sequence and functional characterization of a SLC13 family member from Xenopus laevis, the high-affinity Na⁺/dicarboxylate cotransporter xNaDC-3. The cDNA sequence of xNaDC-3 codes for a protein of 602 amino acids that is 70% identical to the sequences of mammalian NaDC-3 orthologs. The message for xNaDC-3 is found in the kidney, liver, intestine, and heart. The xNaDC-3 has a high affinity for substrate, including a K_m for succinate of 4 µM, and it is inhibited by the NaDC-3 test substrates 2,3-dimethylsuccinate and adipate. The transport of succinate by xNaDC-3 is dependent on sodium, with sigmoidal activation kinetics, and lithium can partially substitute for sodium. As with other members of the family, xNaDC-3 is electrogenic and exhibits inward substrate-dependent currents in the presence of sodium. However, other electrophysiological properties of xNaDC-3 are unique and involve large leak currents, possibly mediated by anions, that are activated by binding of sodium or lithium to a single site. SLC13 gene family; citric acid cycle intermediate; lithium     

地衣芽胞杆菌dif序列的功能鉴定

地衣芽胞杆菌是重要的工业菌株,如何为其建立一种有效的基因删除技术是对该菌株进行遗传改良的基础。枯草芽胞杆菌difB8序列已经被成功用于枯草芽胞杆菌多基因的删除。在分析地衣芽胞杆菌基因组序列并获得与枯草芽胞杆菌difB8以。序列十分相似的一段序列difBLi的基础上,构建了在庆大霉素抗性基因两侧具有difBLi的重组质粒pMD19-difGm和pHY-XI’：：difGm,通过电击转化法将质粒pHY-XI’：：difGm导入B．1icheniformis ATCC14580中,筛选获得了具有庆大霉素抗性的转化子。在转化子的传代过程中,重组质粒的庆大霉素抗性基因在体内Xer／dif／f位点特异性重组系统的介导下通过其侧翼的dif位点进行同源重组而被准确删除。确证了地衣芽胞杆菌中dif序列的功能,为地衣芽胞杆菌基因组中多基因的删除提供了一种新的实验途径。     

Purification and characterization of antimicrobial substances from Bacillus licheniformis BFP011

Bacillus licheniformis BFP011 isolated from papaya (Thailand) could produce extracellular antimicrobial substances which were active against some important phytopathogens, pathogenics and spoilage microorganisms such as Colletotrichum capsici, Escherichia coli O157: H7 and Salmonella typhi ATCC 5784. The antimicrobial substances of this bacterium showed resistance to pronase enzyme and high temperature at 100 and 121°C for 15 min. They were purified by TLC on silica gel plates F254 using the different solvent mixtures. The best solvent mixture was revealed as n-butanol: ethanol: acetic acid: water (30: 60: 5: 30, v/v). The spots F4, F5 and F6 from TLC were able to inhibit growth of S. typhi ATCC 5784 assayed in vitro by the disc diffusion method. The characterization of the active fractions F4, F5 and F6 from TLC and reversedphase HPLC indicated that the antimicrobial substances of B. licheniformis BFP011 contain peptides and unsaturated fatty acids.     

Functional characterization of the heterooligomeric EbrAB multidrug efflux transporter of Bacillus subtilis

The Bacillus subtilis genome contains two tandem genes, ebrA and ebrB, which encode two homologues of the SMR family of multidrug efflux transporters. The sequences of EbrA and EbrB are highly similar to each other and to that of EmrE, the prototypical SMR transporter of Escherichia coli. Drug resistance profiling and drug binding experiments showed that the presence of both EbrA and EbrB is required for proper transport function. EbrA and EbrB directly interact and combine to form a functional transporter. They likely form a heterodimer in analogy to the EmrE homodimer. Mutagenesis experiments indicate that the conserved membrane-embedded glutamates in the first transmembrane helices of both EbrA and EbrB are required for multidrug efflux activity. However, the two glutamates are nonequivalent since EbrA E15 is required for substrate binding while EbrB E14 is not. Our studies support a model in which functional residues in EbrAB are relegated to at least two sets that participate in distinct steps of the active drug transport process.     

Aminopeptidase from a thermophilic strain of Bacillus licheniformis

Aminopeptidase is isolated and purified from the culture liquid of the thermophilic strain of Bacillus licheniformis. The aminopeptidase predominantly splits off N-terminal leucin in short peptides and hydrolyzes leucinamide as well. The molecular weight of the enzyme is about 60 kDa. The enzyme is able to form aggregates. Optimum of aminopeptidase activity was demonstrated at pH 8.0-8.3 and temperature of 85 degrees C. The enzyme is inactivated by metal-binding reagents and reducing substances, and is activated by cobalt and PCMB ions. The EDTA-inactivated enzyme activity is reduced by cobalt and zinc ions, however the latter has no activating action. The enzyme under study is characterized by high thermostability: in the presence of the substrate at the temperature of 90 degrees C the reaction linearity is retained for not less than 2 h and without the substrate the half-life of the aminopeptidase at 90 degrees C is 145 min. Extracellular aminopeptidase of the thermophilic strain of B. licheniformis is a new enzyme differing from the aminopeptidases described by the present in high thermostability, induced, evidently, by the presence of one or several disulphide bonds in the enzyme molecule.     

Charge Compensation Mechanism of a Na+-coupled, Secondary Active Glutamate Transporter

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na(+) and one proton and the subsequent outward movement of one K(+) in a separate step. Based on indirect evidence, it was speculated that the cation binding sites bear a negative charge. However, little is known about the electrostatics of the transport process. Valences calculated using the Poisson-Boltzmann equation indicate that negative charge is transferred across the membrane when only one cation is bound. Consistently, transient currents were observed in response to voltage jumps when K(+) was the only cation on both sides of the membrane. Furthermore, rapid extracellular K(+) application to EAAC1 under single turnover conditions (K(+) inside) resulted in outward transient current. We propose a charge compensation mechanism, in which the C-terminal transport domain bears an overall negative charge of -1.23. Charge compensation, together with distribution of charge movement over many steps in the transport cycle, as well as defocusing of the membrane electric field, may be combined strategies used by Na(+)-coupled transporters to avoid prohibitive activation barriers for charge translocation.     

Purification and characterization of phosphofructokinase of Bacillus licheniformis

The phosphofructokinase (PFK) of Bacillus licheniformis was purified about 50–65-fold and examined for a number of enzymatic and physical characteristics. The enzyme is quite unstable under normal assay conditions, but Mg²⁺, K⁺, adenosine-5′-diphosphate, phosphoenolpyruvate (PEP), and fructose-6-phosphate (fru-6-P) are fairly effective stabilizing agents. Saturation functions for ATP and fru-6-P were hyperbolic. Several attempts to induce positive cooperative binding of fru-6-P were unsuccessful. However, “sigmoidal” saturation kinetics for fru-6-P could be observed under assay conditions that permitted an irreversible inactivation of the PFK during assay. Several divalent cations could support the catalysis of B. licheniformis PFK and the enzyme was activated by both NH₄⁺ and K⁺ ions. B. licheniformis PFK is inhibited by citrate, ATP, PEP, Ca²⁺, and several other metabolic intermediates, but the inhibition caused by citrate and ATP at high fru-6-P concentration and by calcium can be relieved by Mg²⁺ addition while PEP inhibition is specifically relieved by fru-6-P. There are at least three binding sites for PEP on the PFK molecule. The active form of this PFK has a molecular weight of about 134,000 daltons. In the presence of Mg²⁺, adenosine-5′-triphosphate (ATP), and PEP, at 0 °C, the PFK molecule is rapidly dissociated to an inactive form with a molecular weight of about 68,000 daltons. Association of these subunits to yield the active form of PFK occurs spontaneously, and rapidly, when the temperature is raised to 30 °C. Ninety percent of the original activity is recovered after activation. Growth of B. licheniformis on several different substrates resulted in minor variations of PFK activity. In a parallel fashion, sporulation involved no irreversible inactivation of PFK and the level of the activity was about the same throughout the life cycle. Control of this enzyme during sporulation could be affected by any or all of the cell constituents found to regulate PFK activity in vitro, but it is considered likely that the most significant in vivo negative effector is PEP, with this inhibition being reversed by fru-6-P.