Citrate transport in Salmonella typhimurium: studies with 2-fluoro-L-erythro-citrate as a substrate

1980 ◽  
Vol 58 (10) ◽  
pp. 797-803 ◽  
Author(s):  
D. M. Ashton ◽  
G. D. Sweet ◽  
J. M. Somers ◽  
W. W. Kay
Keyword(s):  
Salmonella Typhimurium ◽  
Transport System ◽  
Protein C ◽  
Competitive Inhibitor ◽  
C Protein ◽  
High Affinity ◽  
Citrate Transport ◽  
Resistant Mutants ◽  
Dependent Transport ◽  
Energy Dependent

The citrate analogue, 2-fluoro-L-erythro-[3,4,5,6-14C]citrate was synthesized as a probe for the citrate transport system of Salmonella typhimurium. This analogue was actively transported by an inducible energy-dependent transport system with high affinity for fluorocitrate (Km = 3.3 μM), and this transport system was inhibited competitively by citrate and isocitrate. Fluorocitrate was shown to be a competitive inhibitor of the citrate-binding protein (C protein) of this organism (Ki = 4–5 μM). Analogue resistant mutants were simultaneously defective in fluorocitrate transport as well as the C protein and the affected allele, tctC, was located at 59 units on the S. typhimurium chromosome map. These tctC mutants were shown to be specifically defective in K+-dependent fluorocitrate transport but still retained another system capable of transporting fluorocitrate in the presence of both Na+ and K+.

scholarly journals ATP-dependent transport of glutathione conjugate of 7β,8α-dihydroxy-9α,10α-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles

1998 ◽  
Vol 332 (3) ◽  
pp. 799-805 ◽  
Author(s):  
Sanjay K. SRIVASTAVA ◽  
Xun HU ◽  
Hong XIA ◽  
Richard J. BLEICHER ◽  
Howard A. ZAREN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Competitive Inhibitor ◽  
Dependent Manner ◽  
Plasma Membrane Vesicles ◽  
High Affinity ◽  
Dependent Transport ◽  
Detoxification Pathway ◽  
Stimulation Of

Glutathione (GSH) S-transferases (GSTs) have an important role in the detoxification of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogen of benzo[a]pyrene. However, the fate and/or biological activity of the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is not known. We now report that (-)-anti-BPD-SG is a competitive inhibitor (Ki 19 µM) of Pi-class isoenzyme mGSTP1-1, which among murine hepatic GSTs is most efficient in the GSH conjugation of (+)-anti-BPDE. Thus the inhibition of mGSTP1-1 activity by (-)-anti-BPD-SG might interfere with the GST-catalysed GSH conjugation of (+)-anti-BPDE unless one or more mechanisms exist for the removal of the conjugate. The results of the present study indicate that (-)-anti-BPD-SG is transported across canalicular liver plasma membrane (cLPM) in an ATP-dependent manner. The ATP-dependent transport of (-)-anti-[3H]BPD-SG followed Michaelis–Menten kinetics (Km 46 µM). The ATP dependence of the (-)-anti-BPD-SG transport was confirmed by measuring the stimulation of ATP hydrolysis (ATPase activity) by the conjugate in the presence of cLPM protein, which also followed Michaelis–Menten kinetics. In contrast, a kinetic analysis of ATP-dependent uptake of the model conjugate S-[3H](2,4-dinitrophenyl)-glutathione ([3H]DNP-SG) revealed the presence of a high-affinity and a low-affinity transport system in mouse cLPM, with apparent Km values of 18 and 500 µM respectively. The ATP-dependent transport of (-)-anti-BPD-SG was inhibited competitively by DNP-SG (Ki 1.65 µM). Likewise, (-)-anti-BPD-SG was found to be a potent competitive inhibitor of the high-affinity component of DNP-SG transport (Ki 6.3 µM). Our results suggest that GST-catalysed conjugation of (+)-anti-BPDE with GSH, coupled with ATP-dependent transport of the resultant conjugate across cLPM, might be the ultimate detoxification pathway for this carcinogen.

scholarly journals The ß-Glucosidase of the Yeast Cell Surface

1966 ◽  
Vol 50 (1) ◽  
pp. 9-24 ◽  
Author(s):  
J. Gordin Kaplan ◽  
Wanda Tacreiter
Keyword(s):  
Transport System ◽  
Metabolic Energy ◽  
Uptake System ◽  
Entire System ◽  
Membrane Component ◽  
Intact Cells ◽  
Yeast Cell Surface ◽  
Dependent Transport ◽  
Energy Dependent ◽  
Rate Limiting

There are two distinct components of the system which limits the rate at which intact cells of S. cerevisiae C hydrolyze external ß-glucosides; one component requires metabolic energy and the other is stereospecific for ß-glucosides. The stereospecific component is localized at the cell membrane, as shown by its sensitivity to heavy metal inhibitors which did not penetrate the cell under the conditions used. It was shown that cellobiose-grown cells were able to remove cellobiose from the medium in which they were incubated, and that the cellobiose uptake system was identical to that which limits the patent ß-glucosidase activity. In order to test the hypothesis that the system in question was a transport system, for ß-glucosides the ability of cellobiose-grown cells to take up 14C-labeled methyl-ß-glucoside (MBG) was studied. The induced cells were able to take up MBG-14C and the label could be partially chased out by cold MBG and cellobiose; glucose-grown cells could not incorporate label. However, induced cells could not take up label when incubated with 14C-MBG, thus excluding the hypothesis of transport of intact ß-glucosides. It was concluded that the stereospecific membrane component was actually a ß-glucosidase, coupled to an energy-dependent transport system for the glucose moiety; the function of the latter was rate-limiting in the over-all activity of the entire system.

scholarly journals Salmonella typhimurium HfrA, a mutant in which adenosine triphosphate can drive amino acid transport but not energy-dependent nicotinamide nucleotide transhydrogenation

1975 ◽  
Vol 150 (1) ◽  
pp. 21-29 ◽  
Author(s):  
W W Kay ◽  
P D Bragg
Keyword(s):  
Amino Acid ◽  
Salmonella Typhimurium ◽  
Energy Coupling ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
Dependent Transport ◽  
Energy Dependent ◽  
Aerobic And Anaerobic

In contrast with wild-type Salmonella typhimurium LT2, strain HfrA did not have ATP-driven energy-dependent transhydrogenase activity, although ATP-dependent quenching of atebrin fluorescence was normal. Respiration-dependent and energy-independent transhydrogenase, and Ca2+-activated ATPase (adenosine triphosphatase) activities were similar in both strains. Purified ATPases from the two strains had similar specific activities, similar subunit polypeptides, and were equally effective in restoring energy-dependent transhydrogenase activities to membrane particles of strain LT2 from which the ATPase had been stripped. The purified ATPases from both strains could restore respiration-dependent but not ATP-dependent transhydrogenation to stripped particles of strain HfrA. Both strains grew aerobically equally well on salts media containing glucose, malate, succinate, citrate, acetate, pyruvate, fumarate, lactate or aspartate as substrates. Growth on glucose under anaerobic conditions was similar. Strains LT2 and HfrA were equally effective in the accumulation under both aerobic and anaerobic conditions of the amino acids proline, phenylalanine, histidine, lysine, isoleucine and aspartic acid. Inhibition of amino acid accumulation by KCN and dicyclohexylcarbodi-imide occurred to the same extent in both strains. The complete inhibition by dicyclohexylcarbodi-imide of amino acid uptake under anaerobic conditions suggested that ATP could drive amino acid uptake in both strains. The ability of strain HfrA to carry out ATP-dependent transport or quenching of atebrin fluorescence but not ATP-dependent transhydrogenation is different from the wild-type strain and from any previously described energy-coupling mutant. It is difficult to reconcile the properties of this mutant with the chemiosmotic hypothesis.

scholarly journals Cellobiose Uptake in the Hyperthermophilic ArchaeonPyrococcus furiosus Is Mediated by an Inducible, High-Affinity ABC Transporter

2001 ◽  
Vol 183 (17) ◽  
pp. 4979-4984 ◽  
Author(s):  
Sonja M. Koning ◽  
Marieke G. L. Elferink ◽  
Wil N. Konings ◽  
Arnold J. M. Driessen
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Abc Transporter ◽  
Transport System ◽  
Abc Transporters ◽  
Pyrococcus Furiosus ◽  
Binding Protein ◽  
Hyperthermophilic Archaeon ◽  
High Affinity ◽  
Dependent Transport

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosuscan utilize different β-glucosides, like cellobiose and laminarin. Cellobiose uptake occurs with high affinity (K m = 175 nM) and involves an inducible binding protein-dependent transport system. The cellobiose binding protein (CbtA) was purified from P. furiosusmembranes to homogeneity as a 70-kDa glycoprotein. CbtA not only binds cellobiose but also cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, and sophorose. The cbtAgene was cloned and functionally expressed in Escherichia coli. cbtA belongs to a gene cluster that encodes a transporter that belongs to the Opp family of ABC transporters.

scholarly journals MOAT4, a novel multispecific organic-anion transporter for glucuronides and mercapturates in mouse L1210 cells and human erythrocytes

1996 ◽  
Vol 320 (1) ◽  
pp. 273-281 ◽  
Author(s):  
Manju SAXENA ◽  
Gary B HENDERSON
Keyword(s):  
Transport System ◽  
Organic Anion ◽  
High Sensitivity ◽  
Human Erythrocytes ◽  
Organic Anion Transporter ◽  
High Affinity ◽  
L1210 Cells ◽  
Anion Transporter ◽  
Dependent Transport ◽  
Inside Out

Glucuronides and mercapturates were examined as possible high-affinity substrates for a low-affinity ATP-dependent transport system for 2,4-dinitrophenyl S-glutathione (DNP-SG) in mouse L1210 cells. Initial inhibitor studies with inside-out vesicles revealed that the low-affinity transport of [3H]DNP-SG (Km 450 µM) exhibits a high sensitivity to N-acetyl 2,4-dinitrophenyl cysteine (NAc-DNP-Cys) (Ki 5.0 µM) and α-naphthyl β-D-glucuronide (naphthyl glucuronide) (Ki 8.5 µM). Direct transport measurements showed the presence of ATP-dependent uptake activities for NAc-DNP-[35S]Cys and naphthyl [14C] glucuronide, and Km values for half-maximal transport were comparable to the Ki values of these compounds for inhibition of [3H]DNP-SG transport. Transport of [3H]DNP-SG, NAc-DNP-[35S]Cys and naphthyl [14 C]glucuronide each showed the same sensitivity to various anions and anion conjugates. Inhibition was competitive and was most potent for bilirubin ditaurate, indoprofen, 4-biphenylacetic acid, 4-acridine 4β-D-glucuronide, N-acetyl leukotriene E4, 17β-oestradiol 3β-D-glucuronide and taurolithocholate 3-sulphate. Inside-out vesicles from human erythrocytes contain a comparable ATP-dependent transport system. These results show that NAc-DNP-Cys and naphthyl glucuronide are high-affinity substrates for a single system identified previously as a low-affinity transporter of DNP-SG. Substrate and inhibitor studies identify this system as a novel multispecific organic-anion transport system (MOAT4) that accommodates glucuronides and mercapturates and is distinct from other MOAT transporters. Human erythrocytes contain an additional ATP-dependent system for NAc-DNP-Cys (Km 33 µM) that does not transport monoglucuronides.

Purification and properties of a citrate-binding transport component the C protein of Salmonella typhimurium

1979 ◽  
Vol 57 (6) ◽  
pp. 710-715 ◽  
Author(s):  
G. D. Sweet ◽  
J. M. Somers ◽  
W. W. Kay
Keyword(s):  
Salmonella Typhimurium ◽  
Binding Protein ◽  
Divalent Cations ◽  
Osmotic Shock ◽  
Protein A ◽  
Apparent Molecular Weight ◽  
C Protein ◽  
Citrate Transport ◽  
Defective Mutant ◽  
Concomitant Reduction

Salmonella typhimurium was shown to contain a citrate-binding protein (C protein) which was purified to homogeneity from the periplasmic fraction released by cold osmotic shock. The protein is dimeric, has an apparent molecular weight of 28 000 and an isoelectric point of 6.1. Sodium ions were required for optimum substrate binding, however, the divalent cations Zn2+, Mg2+, and Co2+ were inhibitory. The C protein was relatively stable but sensitive to various detergents and chaotropic agents. Approximately one citrate molecule was bound per molecule of protein and citrate binding (Kd = 1–2.6 μM) was strongly competitively inhibited by DL-isocitrate and DL-fluorocitrate but not by other carboxylates. Neither succinate, glutamate, nor acetate were bound to the C protein. No apparent enzyme activity was associated with this protein. A concomitant reduction in the level of binding protein and in citrate transport activity occurred in osmotically shocked cells as well as with L-malate- or succinate-grown cells. Fluorocitrate-resistant mutants were simultaneously defective in citrate transport, citrate binding, and production of cross-reacting material. One transport-defective mutant did produce citrate binding protein.

scholarly journals myo-inositol transport and metabolism in fetal-bovine aortic endothelial cells

1993 ◽  
Vol 295 (3) ◽  
pp. 863-869 ◽  
Author(s):  
G T Berry ◽  
R A Johanson ◽  
J E Prantner ◽  
B States ◽  
J R Yandrasitz
Keyword(s):  
Endothelial Cells ◽  
Kinetic Analysis ◽  
Transport System ◽  
Bovine Serum ◽  
Competitive Inhibitor ◽  
Aortic Endothelial Cells ◽  
High Affinity ◽  
Bovine Aortic Endothelial Cells ◽  
Fetal Bovine ◽  
Aortic Endothelial

The myo-inositol transport system in confluent fetal-bovine aortic endothelial cells was characterized after 7-10 days in subculture, at which time the myo-inositol levels and rates of myo-[2-3H]-inositol uptake and incorporation into phospholipid had reached steady state. Kinetic analysis indicated that the uptake occurred by both a high-affinity transport system with an apparent Kt of 31 microM and Vmax. of 45 pmol/min per mg of protein, and a non-saturable low-affinity system. Uptake was competitively inhibited by phlorhizin, with a Ki of 50 microM; phloretin was a non-competitive inhibitor, with half-maximal inhibition between 0.2 and 0.5 mM. Glucose was a weak competitive inhibitor, with a Ki of 37 mM; galactose failed to inhibit uptake. A weak dependence on Na+ for the initial rate of uptake was observed at 11 microM myo-inositol. When fetal-bovine-serum (FBS)-supplemented medium, which contained 225 microM myo-inositol, was used, the cells contained about 200 nmol of myo-inositol/mg of DNA. With adult-bovine-serum (ABS)-supplemented medium, which contained 13 microM myo-inositol, the cells contained about 110 nmol/mg of DNA. Transport of 11 microM myo-[2-3]inositol was 18 and 125 pmol/min per mg of DNA for cells grown in FBS and ABS respectively. Kinetic analysis showed that for the cells grown in FBS the Vmax. of the high-affinity system was decreased by 64%, whereas the Kt remained essentially unchanged. Increased cell myo-inositol levels were not associated with an increased rate of phosphatidylinositol synthesis. After prolonged exposure of fetal endothelial cells to a myo-inositol concentration which approximated to a high fetal as opposed to a low adult blood level, cell myo-inositol levels doubled and high-affinity transport underwent down-regulation.

Uptake of methylamine via an inducible, energy-dependent transport system in the facultative methylotroph Arthrobacter P1

1982 ◽  
Vol 133 (4) ◽  
pp. 261-266 ◽  
Author(s):  
Lubbert Dijkhuizen ◽  
Lex de Boer ◽  
Roelof H. Boers ◽  
Wim Harder ◽  
Wil N. Konings
Keyword(s):  
Transport System ◽  
Arthrobacter P1 ◽  
Facultative Methylotroph ◽  
Dependent Transport ◽  
Energy Dependent
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