ATP-dependent Transport System Research Articles

Metabolic engineering of Escherichia coli for 1,3-propanediol biosynthesis from glycerol

In this study, the engineered E. coli was constructed for efficient transformation of glycerol to 1,3-propanediol (1,3-PDO). To regenerate NADPH, the key bottleneck in 1,3-PDO production, heterologous NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDN, encoded by gapN) pathway was introduced, and the gapN expression level was fine-tuned with specific 5′-untranslated regions (5′-UTR) to balance the carbon flux distribution between the metabolic pathways of NADPH regeneration and 1,3-PDO biosynthesis. Additionally, glucose was added to the medium to promote glycerol utilization and cell growth. To elevate the utilization of glycerol in the presence of glucose, E. coli JA11 was constructed through destroying PEP-dependent glucose transport system while strengthening the ATP-dependent transport system. Subsequent optimization of nitrogen sources further improved 1,3-PDO production. Finally, under the optimal fermentation condition, E. coli JA11 produced 13.47 g/L 1,3-PDO, with a yield of 0.64 mol/mol, increased by 325% and 100% compared with the original engineered E. coli JA03, respectively.

The Non-Canonical Substrates of Trypanosoma cruzi Tyrosine and Aspartate Aminotransferases: Branched-Chain Amino Acids.

Trypanosoma cruzi, the etiological agent of Chagas disease, lacks genes that encode canonical branched-chain aminotransferases. However, early studies showed that when epimastigotes were grown in the presence of 14 C1 -DL-leucine, the label was incorporated into various intermediates. More recently, our studies provided evidence that T.cruzi epimastigotes display a single ATP-dependent and saturable transport system that enables epimastigotes to uptake branched-chain amino acids (BCAAs) from the culture media. To extend our knowledge of the first step of BCAA catabolism, the ability of this parasite's noncanonical broad specificity aminotransferases, such as tyrosine aminotransferase (TAT) and aspartate aminotransferase (ASAT), to transaminate these amino acids was investigated. Indeed, our results show that TAT and ASAT utilize BCAAs as substrates; however, both enzymes differ in their catalytic competence in utilizing these amino donors. For instance, ASAT transaminates isoleucine nearly 10-fold more efficiently than does TAT. This unique characteristic of TAT and ASAT allows to explain how BCAAs can be oxidized in the absence of a BCAA transaminase in T.cruzi.

ATP‐binding cassette transporters in liver

The human ATP-binding cassette (ABC) superfamily consists of 48 members with 14 of them identified in normal human liver at the protein level. Most of the ABC members act as ATP dependent efflux transport systems. In the liver, ABC transporters are involved in diverse physiological processes including export of cholesterol, bile salts, and metabolic endproducts. Consequently, impaired ABC transporter function is involved in inherited diseases like sitosterolemia, hyperbilirubinemia, or cholestasis. Furthermore, altered expression of some of the hepatic ABCs have been associated with primary liver tumors. This review gives a short overview about the function of hepatic ABCs. Special focus is addressed on the localization and ontogenesis of ABC transporters in the human liver. In addition, their expression pattern in primary liver tumors is discussed.

Proteomic Analysis of Bifidobacterium longum subsp. infantis Reveals the Metabolic Insight on Consumption of Prebiotics and Host Glycans

Bifidobacterium longum subsp. infantis is a common member of the intestinal microbiota in breast-fed infants and capable of metabolizing human milk oligosaccharides (HMO). To investigate the bacterial response to different prebiotics, we analyzed both cell wall associated and whole cell proteins in B. infantis. Proteins were identified by LC-MS/MS followed by comparative proteomics to deduce the protein localization within the cell. Enzymes involved in the metabolism of lactose, glucose, galactooligosaccharides, fructooligosaccharides and HMO were constitutively expressed exhibiting less than two-fold change regardless of the sugar used. In contrast, enzymes in N-Acetylglucosamine and sucrose catabolism were induced by HMO and fructans, respectively. Galactose-metabolizing enzymes phosphoglucomutase, UDP-glucose 4-epimerase and UTP glucose-1-P uridylytransferase were expressed constitutively, while galactokinase and galactose-1-phosphate uridylyltransferase, increased their expression three fold when HMO and lactose were used as substrates for cell growth. Cell wall-associated proteomics also revealed ATP-dependent sugar transport systems associated with consumption of different prebiotics. In addition, the expression of 16 glycosyl hydrolases revealed the complete metabolic route for each substrate. Mucin, which possesses O-glycans that are structurally similar to HMO did not induced the expression of transport proteins, hydrolysis or sugar metabolic pathway indicating B. infantis do not utilize these glycoconjugates.

Detection and characterization of liver lesions using gadoxetic acid as a tissue-specific contrast agent.

The value of cross-sectional liver imaging is evaluated by the accuracy, sensitivity, and specificity of the specific imaging technique. Magnetic resonance imaging (MRI) has become a key technique for the characterization and detection of focal and diffuse liver disease. More recently, gadoxetic acid, the hepatocyte-specific MR contrast agent, was clinically approved and introduced in many countries. Gadoxetic acid may be considered a "molecular imaging" probe because the compound is actively taken into hepatocytes via the ATP-dependent organic anion transport system in the plasma membrane for the hepatic uptake. The transport of gadoxetic acid from the cytoplasm to the bile is mainly determined by the capacity of the transport protein glutathione-S-transferase. Gadoxetic acid enhances hepatocyte-containing lesions and improves detection of lesions devoid of normal hepatocytes, such as metastases. Innovative rapid MR acquisition techniques with near isotropic 3D pulse sequences with fat saturation parallel the technical progress made by multidetector computed tomography combined with an impressive improvement in tumor-liver contrast when used for gadoxetic acid-enhanced MRI. The purpose of this review is to provide an overview of the development, clinical testing, and applications of this novel MR contrast agent.

Transepithelial transport of Cerulenin across Caco-2 cell monolayers

The transepithelial transport of Cerulenin across Caco-2 cell monolayers was examined in this study. The permeated amounts of Cerulenin were measured by HPLC method to calculate the permeation rate and the apparent permeability coefficient (P(app)). The transport of Cerulenin was independent on apical pH and exhibited concentration-dependent and nonsatuable even at 10 mM Cerulenin. The permeation rate at 1 mM Cerulenin in the apical-to-basolateral direction was 0.151 ng/min/mg of protein and the P(app) was 3.76 x 10(-6) cm/second. The permeation rate of Cerulenin was affected by neither metabolic inhibitors nor inhibitors for P-glycoprotein, as was the same case in monolayers treated with cytochalasin D. All these data from experiments indicated that transport of Cerulenin across Caco-2 cell monolayers was not mediated by ATP-dependent transport systems nor via paracellular pathway, but via passive diffusion without efflux by P-glycoprotein.

Plasma Disappearance Rate of Indocyanine Green in Liver Dysfunction

The presence of hepatic dysfunction significantly affects the length of hospital stay and the outcome in critically ill patients. Considering the important partial hepatic functions of metabolism, synthesis, detoxification, and excretion, the worse clinical course of patients suffering from hepatic dysfunction is not surprising. The most often used indicator of hepatic dysfunction is bilirubin. However, bilirubin and other commonly used static laboratory tests provide only indirect measures of hepatic function. In contrast to these static tests, dynamic liver tests, such as indocyanine green (ICG) disappearance rate should provide better direct measures of the actual functional state of the liver at the time of assessment. The ICG is a water-soluble inert compound that is injected intravenously. It mainly binds to albumin in the plasma. ICG is then selectively taken up by hepatocytes, independent of adenosine triphosphate (ATP), and later excreted unchanged into the bile via an ATP-dependent transport system. The ICG is not metabolized; it does not undergo enterohepatic recirculation. Thus, ICG excretion rate in bile reflects the hepatic excretory function and hepatic energy status. Because of these features, ICG has been found to be useful to assess liver function in liver donors and transplant recipients, in patients with chronic liver failure, and as a prognostic factor in critically ill patients. Further trials concerning liver dysfunction have applied the noninvasive bedside assessment of ICG among other clinical variables to monitor the progress and/or the reversal of liver dysfunction.

Heterozygous Bile Salt Export Pump Deficiency: A Possible Genetic Predisposition to Transient Neonatal Cholestasis

Heterozygous Bile Salt Export Pump Deficiency: A Possible Genetic Predisposition to Transient Neonatal Cholestasis

ATP-dependent transport of bile acid intermediates across rat liver peroxisomal membranes.

The bile acid intermediate 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid (THCA) is converted to cholic acid exclusively in peroxisomes by the oxidative cleavage of the side chain. To investigate the mechanism by which the biosynthetic intermediates of bile acids are transported into peroxisomes, we incubated THCA or its CoA ester (THC-CoA) with isolated intact rat liver peroxisomes and analyzed their oxidation products, cholic acid and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoic acid. The oxidation of both THCA and THC-CoA was dependent on incubation time and peroxisomal proteins, and was stimulated by ATP. THC-CoA was efficiently oxidized to cholic acid and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoic acid as compared with THCA, suggesting that THC-CoA is the preferred substrate for transport into peroxisomes. The oxidation of THC-CoA was significantly inhibited by sodium azide, verapamile, and N-ethylmaleimide. Furthermore, the stimulatory effect of ATP on the oxidation was not replaced by GTP or AMP. In addition, the ATP-dependent oxidation of THC-CoA was markedly inhibited by pretreatment of peroxisomes with proteinase K when peroxisomal matrix proteins were not degraded. These results suggest that an ATP-dependent transport system for THC-CoA exists on peroxisomal membranes.

Metabolism of fructooligosaccharides by Lactobacillus paracasei 1195.

Fermentation of fructooligosaccharides (FOS) and other oligosaccharides has been suggested to be an important property for the selection of bacterial strains used as probiotics. However, little information is available on FOS transport and metabolism by lactic acid bacteria and other probiotic bacteria. The objectives of this research were to identify and characterize the FOS transport system of Lactobacillus paracasei 1195. Radiolabeled FOS was synthesized enzymatically from [(3)H]sucrose and purified by column and thin-layer chromatography, yielding three main products: glucose (G) alpha-1,2 linked to two, three, or four fructose (F) units (GF(2), GF(3), and GF(4), respectively). FOS hydrolysis activity was detected only in cell extracts prepared from FOS- or sucrose-grown cells and was absent in cell supernatants, indicating that transport must precede hydrolysis. FOS transport assays revealed that the uptake of GF(2) and GF(3) was rapid, whereas little GF(4) uptake occurred. Competition experiments showed that glucose, fructose, and sucrose reduced FOS uptake but that other mono-, di-, and trisaccharides were less inhibitory. When cells were treated with sodium fluoride, iodoacetic acid, or other metabolic inhibitors, FOS transport rates were reduced by up to 60%; however, ionophores that abolished the proton motive force only slightly decreased FOS transport. In contrast, uptake was inhibited by ortho-vanadate, an inhibitor of ATP-binding cassette transport systems. De-energized cells had low intracellular ATP concentrations and had a reduced capacity to accumulate FOS. These results suggest that FOS transport in L. paracasei 1195 is mediated by an ATP-dependent transport system having specificity for a narrow range of substrates.

Genes coding for a new pathway of aerobic benzoate metabolism in Azoarcus evansii.

A new pathway for aerobic benzoate oxidation has been postulated for Azoarcus evansii and for a Bacillus stearothermophilus-like strain. Benzoate is first transformed into benzoyl coenzyme A (benzoyl-CoA), which subsequently is oxidized to 3-hydroxyadipyl-CoA and then to 3-ketoadipyl-CoA; all intermediates are CoA thioesters. The genes coding for this benzoate-induced pathway were investigated in the beta-proteobacterium A. evansii. They were identified on the basis of N-terminal amino acid sequences of purified benzoate metabolic enzymes and of benzoate-induced proteins identified on two-dimensional gels. Fifteen genes probably coding for the benzoate pathway were found to be clustered on the chromosome. These genes code for the following functions: a putative ATP-dependent benzoate transport system, benzoate-CoA ligase, a putative benzoyl-CoA oxygenase, a putative isomerizing enzyme, a putative ring-opening enzyme, enzymes for beta-oxidation of CoA-activated intermediates, thioesterase, and lactone hydrolase, as well as completely unknown enzymes belonging to new protein families. An unusual putative regulator protein consists of a regulator protein and a shikimate kinase I-type domain. A deletion mutant with a deletion in one gene (boxA) was unable to grow with benzoate as the sole organic substrate, but it was able to grow with 3-hydroxybenzoate and adipate. The data support the proposed pathway, which postulates operation of a new type of ring-hydroxylating dioxygenase acting on benzoyl-CoA and nonoxygenolytic ring cleavage. A beta-oxidation-like metabolism of the ring cleavage product is thought to lead to 3-ketoadipyl-CoA, which finally is cleaved into succinyl-CoA and acetyl-CoA.

The cryoprotective effects of glycine betaine on bacteria

It is well known that in response to a decrease in environmental temperature, some plants and animals accumulate several cryoprotective substances inside the cell. However, the role of glycine betaine as a bacterial cryoprotectant is only beginning to be understood.The growth-enhancing effect of glycine betaine on the food-borne pathogen Listeria monocytogenes at low temperature was first reported by a laboratory at the University of California, USA. This study also revealed that the uptake of glycine betaine into the bacterial cell was stimulated at low temperature. Recently, the same laboratory demonstrated a role for an ATP-dependent transport system in the cold-induced uptake of glycine betaine. Additionally, this transporter was shown to be inhibited by the glycine betaine analogs carnitine, dimethylglycine and γ-butyrobetaine [1xCharacterization of glycine betaine porter I from Listeria monocytogenes and its roles in salt and chill tolerance. Mendum, M.L. and Smith, L.T. Appl. Environ. Microbiol. 2002; 68: 813–819Crossref | Scopus (45)See all References][1].The growth-enhancing effect of glycine betaine on L. monocytogenes at low temperature was attributed to its ability to prevent cold-induced aggregation of cellular proteins and its role in maintaining membrane fluidity at low temperature. It has been shown that following inductive synthesis of some heat shock proteins (HSPs), which are believed to protect the cell from thermal stress, the frequency of survival of an Escherichia coli strain under frozen storage conditions was remarkably increased; this was thought to result from a chaperoning effect of the HSPs preventing denaturation of the cellular proteins at low temperature. Hence, it seems possible that the cryoprotective properties of glycine betaine result from a similar effect. The fact that this osmoprotectant is already known to act as a chemical chaperone during thermal stress strengthens this suggestion.An additional role of glycine betaine in regulating membrane fluidity cannot be ruled out, as its ability to increase membrane fluidity was demonstrated earlier using bilayers of small unilamellar vesicles. It has also been observed that when two strains of L. monocytogenes were grown in the presence of glycine betaine at low temperature, the amount of a branched-chain fatty acid that was essential for survival of the strains in cold (anteiso-C 15:0) was slightly enhanced. Subsequently, a cold-sensitive mutant deficient in this branched-chain fatty acid was found to have decreased membrane fluidity [2xCorrelation of long-range membrane order with temperature-dependent growth characteristics of parent and a cold-sensitive, branched-chain-fatty-acid-deficient mutant of Listeria monocytogenes. Jones, S.L. et al. Arch. Microbiol. 2002; 177: 217–222Crossref | Scopus (26)See all References][2]. It therefore appears that glycine betaine helps to maintain membrane fluidity at low temperature by promoting the synthesis of specific fatty acids. Recent investigations on isolated thylakoid membrane demonstrate the mutifaceted nature of its cryoprotective effects. Similar studies involving liposomes made of total lipids extracted from some cold-adapted bacteria promise further insights into the mechanism responsible for the cryoprotective effects of glycine betaine.

Transport of leukotriene C4 and structurally related conjugates

Transport proteins control the release of the endogenous glutathione conjugate leukotriene C4 (LTC4) from leukotriene-synthesizing cells as well as its hepatobiliary and renal elimination. The photolabile conjugated triene structure of LTC4 has enabled direct photoaffinity labeling of the multidrug resistance protein 1 (MRP1, symbol ABC C1) in membranes from mastocytoma cells, leading to the identification of the function of this protein as an ATP-dependent export pump for LTC4 and structurally related conjugates. MRP1 is assigned to the C branch of the superfamily of ATP-binding cassette (ABC) transporters and was originally identified by virtue of its association with drug resistance in tumor cells. Besides LTC4, which is a high-affinity substrate, a variety of conjugates of hydrophobic endogenous or xenobiotic substances with glutathione, glucuronate, or sulfate are transported by MRP1. In addition, hydrophobic compounds may undergo cotransport with glutathione. Effective inhibitors of MRP1-mediated transport include structural analogs of LTC4 and of other cysteinyl leukotrienes. The ATP-dependent transport system which transports cysteinyl leukotrienes across the hepatocyte canalicular membrane into bile was cloned and characterized as the second isoform or paralog of the mammalian MRP family, MRP2 (ABC C2). MRP2 is localized to the apical membrane of polarized cells. The overall substrate specificities of MRP1 and MRP2 are similar, despite an amino acid identity of only 48%. The transport proteins mediating the uptake of LTC4 into hepatocytes across the basolateral membrane are members of the organic anion transporter (OATP) branch of the solute carrier (SLC) superfamily and are thus distinct from the ATP-dependent export pumps of the MRP family.

Transcriptional regulation of transport and utilization systems for hexuronides, hexuronates and hexonates in gamma purple bacteria.

The comparative approach is a powerful tool for the analysis of gene regulation in bacterial genomes. It can be applied to the analysis of regulons that have been studied experimentally as well as that of regulons for which no known regulatory sites are available. It is assumed that the set of co-regulated genes and the regulatory signal itself are conserved in related genomes. Here, we use genomic comparisons to study the regulation of transport and utilization systems for sugar acids in gamma purple bacteria Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Yersinia pestis, Erwinia chrysanthemi, Haemophilus influenzae and Vibrio cholerae. The variability of the operon structure and the location of the operator sites for the main transcription factors are demonstrated. The common metabolic map is combined with known and predicted regulatory interactions. It includes all known and predicted members of the GntR, UxuR/ExuR, KdgR, UidR and IdnR regulons. Moreover, most members of these regulons seem to be under catabolite repression mediated by CRP. The candidate UxuR/ExuR signal is proposed, the KdgR consensus is extended, and new operators for all transcription factors are identified in all studied genomes. Two new members of the KdgR regulon, a hypothetical ATP-dependent transport system OgtABCD and YjgK protein with unknown function, are detected. The former is likely to be the transport system for the products of pectin degradation, oligogalacturonides.

Intracellular magnesium: transport and regulation in epithelial secretory cells.

The mechanisms of magnesium (Mg2+) transport, the regulation of intracellular Mg2+ concentrations and the relationship between Mg2+ and Ca2+ signaling during the stimulus-secretion coupling process in pancreatic acinar cells and other secretory epithelia are reviewed in this article. Our results demonstrate the existence of a Na+- and ATP-dependent transport system for Mg2+ extrusion from Mg2+-loaded cells. Moreover, employing such different techniques as spectrofluorimetry and atomic absorbance spectroscopy to measure intracellular free magnesium concentration [Mg2+]i from magfura-2-loaded acini and acinar cells and Mg2+ content in effluent samples from perfused pancreatic segments, respectively, confirm that secretagogues such as acetylcholine (ACh) and cholecystokinin-octapeptide (CCK-8) can evoke marked and significant extrusion of Mg2+ which is closely associated with the mobilization of intracellular calcium. These effects may be modulated by different mediators including cAMP, Protein Kinase C and nitric oxide/cGMP. This reduction in [Mg2+]i seems to be a prerequisite for optimal generation and maintenance of the calcium signal and subsequently, the secretion of enzymes, since an increase in extracellular Mg2+ concentration, [Mg2+]o and an increase in [Mg2+]i inhibit secretagogue-induced secretory responses, an effect exerted through a derangement of the calcium signaling events. In conclusion, the evidence presented in this review strongly supports an important modulatory role of magnesium in the control of secretory epithelial cells function.

Molecular interactions in ribose transport: the binding protein module symmetrically associates with the homodimeric membrane transporter.

The Escherichia coli high-affinity ribose transporter is composed of the periplasmic ribose-binding protein (RBP or RbsB), the membrane component (RbsC) and the ATP-binding protein (RbsA). In order to dissect the molecular interactions initiating the transport process, RbsC suppressors for transport-defective rbsB mutations were isolated. These suppressors are localized in two regions of RbsC, which are allele-specific to N- or C-terminal domain mutations of RBP, suggesting that there are two distinct regions of RbsC, each interacting with one of the two domains of RBP. To demonstrate that these two regions provide a homodimeric binding surface for RBP we constructed a dimeric rbsC in which two genes are joined tandemly from head to tail with the addition of a linker. The dimeric RbsC protein is stable and functional in growth and ribose uptake. By exploiting the allele specificity between the domain-specific mutations and their suppressors, we generated all mutation-suppressor combinations in a single rbsB plus the dimeric rbsC genes. Their phenotypes are consistent with the proposal that the binding protein module interacts symmetrically with homodimeric RbsC. The mode of association proposed here for the ribose transport components could be extended to other ABC transporters with similar structural organizations.

Mechanisms of pyrazinamide resistance in mycobacteria: importance of lack of uptake in addition to lack of pyrazinamidase activity

Mycobacteria are known to acquire resistance to the antituberculous drug pyrazinamide (PZA) through mutations in the gene encoding pyrazinamidase (PZase), an enzyme that converts PZA into pyrazinoic acid, the presumed active form of PZA against bacteria. Additional mechanisms of resistance to the drug are known to exist but have not been fully investigated. Among these is the non-uptake of the pro-drug, a possibility investigated in the present study. The uptake mechanism of PZA, a requisite step for the activation of the pro-drug, was studied in Mycobacterium tuberculosis. The incorporation of [14C]PZA by the bacilli was followed in both neutral and acidic environments since PZA activity is known to be optimal at acidic pH. By using a protonophore (carbonyl cyanide m-chlorophenylhydrazone; CCCP), valinomycin, arsenate and low temperature, it was shown that an ATP-dependent transport system is involved in the uptake of PZA. Whilst the structurally analogous compound nicotinamide inhibited the transport system of PZA, other structurally related compounds such as pyrazinoic acid, isoniazid and cytosine did not. Acidic conditions were also without effect. Based on diffusion experiments in liposomes, it was found that PZA diffuses rapidly through membrane bilayers, faster than glycerol, whilst the presence of OmpATb, the porin-like protein of M. tuberculosis, in proteoliposomes slightly increased the diffusion of the drug. This finding may explain why the cell wall mycolate hydrophobic layer does not represent the limiting step in the diffusion of PZA, as judged from comparative experiments using a M. tuberculosis strain and its isogenic mutant elaborating 40% less covalently linked mycolates. PZase activity, and PZA uptake and susceptibility in different mycobacterial species were compared. M. tuberculosis, a naturally PZA-susceptible species, was the only species that exhibited both PZase activity and PZA uptake; no such correlation was observed with the four naturally resistant species examined. Mycobacterium smegmatis possessed a functional PZase but did not take up PZA; the reverse was true for the PZase-negative strain of Mycobacterium avium used, with PZA uptake comparable to that of M. tuberculosis. Mycobacterium bovis BCG and Mycobacterium kansasii exhibited neither a PZase activity nor PZA uptake. These data clearly demonstrate that one of the mechanisms of resistance to PZA resides in the failure of strains to take up the drug, indicating that susceptibility to PZA in mycobacteria requires both the presence of a functional PZase and a PZA transport system. No correlation was observed between the occurrence and cellular location of PZase and of nicotinamidase in the strains examined, suggesting that one or both amides can be hydrolysed by other mycobacterial amidases.

The effect of Mg2+, nucleotides and ATPase inhibitors on the uptake of [3H]-cGMP to inside-out vesicles from human erythrocytes.

An ATP-dependent transport system is responsible for the cellular extrusion of cGMP. The objective of the present study was to determine the effect of Mg2+, ATP and other nucleotides (2'-dATP, GTP and ADP), exogenous ATPase modulators (such as metavanadate, ouabain, EGTA, NEM, bafilomycin A1 and oligomycin A) on the cGMP transport. The uptake of [3H]-cGMP (1 microM) at 37 degrees C was studied in inside-out vesicles from human erythrocytes. Magnesium caused a maximal activation between 5 and 10 mM and the optimal ATP concentration was 1.25 mM with K50-values of 0.3-0.5 mM. Among other nucleotides tested, 2'-dATP (K50 of 0.7 mM) was nearly as effective as ATP, whereas cGMP accumulated slowly in the presence of GTP. ADP and metavanadate (P-type ATPase inhibitor) showed to be competitive inhibitors with Ki values of 0.15 mM and 10 microns, respectively. NEM (a sulphydryl agent) reduced the ATP-dependent uptake in a concentration-dependent manner with a Ki value of 10 microM. Ouabain (Na+/K(+)-ATPase inhibitor) had no effect. Bafilomycin A1 (V-type ATPase inhibitor) and oligomycin (F-type ATPase inhibitor) were the most potent inhibitors with Ki values of 0.7 and 1.8 microM, respectively. The present study suggests that the cellular cGMP extrusion is energized by an ATPase with a unique inhibitor profile, which clearly differentiates it from the other major classes of membrane-bound ATPases.

Decreased hepatobiliary secretion of inorganic mercury, its deposition and toxicity in the Eisai hyperbilirubinemic rat with no hepatic canalicular organic anion transporter

Eisai hyperbilirubinemic (EHB) rats, a new mutant strain inbred from Sprague–Dawley (SD) rats, show no inherent expression of the canalicular multidrug resistance protein (cMrp) and lack canalicular multispecific organic anion transporter (cMOAT) activity. A sample of 203Hg (40 μCi with 40 μg Hg/kg) was injected intravenously (i.v.) into four male SD and EHB rats. Biliary excretion of reduced-glutathione (GSH) was 426 and 2 μg/bile for 15 min in the SD and EHB rats, respectively. Biliary excretion of 203Hg for 45 min in EHB rats significantly decreased to 1/4 of that of the SD rats. However, there was no difference in the hepatic uptake of 203Hg between the two strains. Other rats were injected subcutaneously (s.c.) with HgCl 2 solution (at 0.2 and 1.6 mg/kg) containing 203Hg. Some 4 days after the injection of 0.2 mg/kg, about 3 and 13% of the total dose was found in the liver in SD and EHB rats, respectively. The hepatic supernatant Hg was recovered mainly in the void volume of a Sephadex column. Some 2 days after the injection of 1.6 mg/kg, these values were 3 and 23% in SD and EHB rats, respectively. The increased retention stimulated hepatic metallothionein (MT) induction and increased the proportion of Hg in the MT region on the Sephadex column. On the other hand, biliary excretion of 203Hg for 15 min in EHB rats was about 1/6–1/4 of that in SD rats. With the injection of 1.6 mg/kg, hepatic and renal functions worsened in EHB rats. In particular, severe necrosis was found in the renal tubules. Our results suggest that biliary secretion of inorganic Hg may be partly regulated by the ATP-dependent transport system, the glutathione S-conjugate export pump (GS-X pump) composed of Mrp and MOAT. Significantly decreased excretion stimulates hepatic retention of inorganic Hg. However, the hepatic lesions are less predictive. The MT induction may reduce the toxicity of metal to the liver cells.

Energized Ca2+ transport by hepatopancreatic basolateral plasma membranes of Homarus americanus.

Ca2+ transport by hepatopancreatic basolateral membrane vesicles of Atlantic lobster (Homarus americanus) occurred by at least two independent processes: (1) an ATP-dependent carrier transport system, and (2) a Na+-gradient-dependent carrier mechanism. The sensitivity of ATP-dependent Ca2+ transport to vanadate indicated that it was probably due to a P-type ATPase. This system exhibited an extremely high apparent affinity for Ca2+ (Kt=65.28+/-14.39 nmol l-1; Jmax=1. 07+/-0.06 pmol microg-1 protein 8 s-1). The Na+-gradient-dependent carrier transport system exhibited the properties of a Ca2+/Na+ antiporter capable of exchanging external Ca2+ with intravesicular Na+ or Li+. Kinetic analysis of the Na+-dependence of the antiport indicated that at least three Na+ were exchanged with each Ca2+ (n=2. 91+/-0.22). When Li+ replaced Na+ in exchange for 45Ca2+, the apparent affinity for Ca2+ influx was not significantly affected (with Na+, Kt=14.57+/-5.02 micromol l-1; with Li+, Kt=20.17+/-6.99 micromol l-1), but the maximal Ca2+ transport velocity was reduced by a factor of three (with Na+, Jmax=2.72+/-0.23 pmol microg-1 protein 8 s-1; with Li+, Jmax=1.03+/-0.10 pmol microg-1 protein 8 s-1). It is concluded that Ca2+ leaves hepatopancreatic epithelial cells across the basolateral membrane by way of a high-affinity, vanadate-sensitive Ca2+-ATPase and by way of a low-affinity Ca2+/Na+ antiporter with an apparent 3:1 exchange stoichiometry. The roles of these transporters in Ca2+ balance during the molt cycle are discussed.