scholarly journals Cysteine Metabolism in Legionella pneumophila: Characterization of an l-Cystine-Utilizing Mutant

2006 ◽  
Vol 72 (6) ◽  
pp. 3993-4000 ◽  
Author(s):  
Fanny Ewann ◽  
Paul S. Hoffman
Keyword(s):  
Legionella Pneumophila ◽  
Kinetic Studies ◽  
General View ◽  
Natural Environments ◽  
Cysteine Synthase ◽  
High Affinity ◽  
Cell Extracts ◽  
Carbonyl Cyanide ◽  
Major Facilitator ◽  
Energy Dependent

ABSTRACT Growth of Legionella pneumophila on buffered charcoal-yeast extract (BCYE) medium is dependent on l-cysteine (but not l-cystine), which is added in excess over what is required for nutrition. We investigated the biochemical and genetic bases for this unusual requirement and determined that much of the l-cysteine in BCYE medium is rapidly oxidized to l-cystine and is unavailable to the bacteria. Analysis of cysteine consumption during bacterial growth indicated that of the 11% consumed, 3.85% (∼0.1 mM) was incorporated into biomass. The activities of two key cysteine biosynthetic enzymes (serine acetyltransferase and cysteine synthase) were not detected in cell extracts of L. pneumophila, and the respective genes were not present in the genome sequences, confirming cysteine auxotrophy. Kinetic studies identified two energy-dependent cysteine transporters, one with high affinity (apparent Km , 3.29 μM) and the other with low affinity (apparent Km , 93 μM), each of which was inhibited by the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone. Cystine was not transported by L. pneumophila; however, a mutant strain capable of growth on l-cystine (CYS1 mutant) transported l-cystine with similar kinetics (Km , 4.4 μM and 90 μM). Based on the bipartite kinetics, requirement for proton motive force, and inhibitor studies, we suggest that a high-affinity periplasmic binding protein and a major facilitator/symporter (low affinity) mediate uptake. The latter most likely is functional at high cysteine concentrations and most likely displays altered substrate specificity in the CYS-1 mutant. Our studies provide biochemical evidence to support a general view that L. pneumophila is restricted to an intracellular lifestyle in natural environments by an inability to utilize cystine, which most likely ensures that the dormant cyst-like transmissible forms do not germinate outside suitable protozoan hosts.

scholarly journals Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria

1980 ◽  
Vol 191 (2) ◽  
pp. 421-427 ◽  
Author(s):  
J F Turrens ◽  
A Boveris
Keyword(s):  
Cytochrome B ◽  
Nadh Dehydrogenase ◽  
Submitochondrial Particles ◽  
Bovine Heart ◽  
Biphasic Effect ◽  
Carbonyl Cyanide ◽  
Autocatalytic Process ◽  
Ph Range ◽  
Energy Dependent ◽  
O2 Production

Submitochondrial particles from bovine heart in which NADH dehydrogenase is reduced by either addition of NADH and rotenone or by reversed electron transfer generate 0.9 +/- 0.1 nmol of O2-/min per mg of protein at pH 7.4 and at 30 degrees C. When NADH is used as substrate, rotenone, antimycin and cyanide increase O2- production. In NADH- and antimycin-supplemented submitochondrial particles, rotenone has a biphasic effect: it increases O2- production at the NADH dehydrogenase and it inhibits O2- production at the ubiquinone-cytochrome b site. The generation of O2- by the rotenone, the uncoupler carbonyl cyanide rho-trifluoromethoxyphenylhydrazone and oligomycin at concentrations similar to those required to inhibit energy-dependent succinate-NAD reductase. Cyanide did not affect O2- generation at the NADH dehydrogenase, but inhibited O2- production at the ubiquinone-cytochrome b site. Production of O2- at the NADH dehydrogenase is about 50% of the O2- generation but the ubiquinone-cytochrome b area at pH 7.4. Additivity of the two mitochondrial sites of O2- generation was observed over the pH range from 7.0 to 8.8. AN O2–dependent autocatalytic process that requires NADH, submitochondrial particles and adrenaline is described.

scholarly journals High‐affinity binding of phosphatidylinositol 4‐phosphate by Legionella pneumophila DrrA

EMBO Reports ◽  
2010 ◽  
Vol 11 (8) ◽  
pp. 598-604 ◽  
Author(s):  
Stefan Schoebel ◽  
Wulf Blankenfeldt ◽  
Roger S Goody ◽  
Aymelt Itzen
Keyword(s):  
Legionella Pneumophila ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Phosphatidylinositol 4

scholarly journals Genome-wide identification and analyses of tomato (Solanum lycopersicum L.) high-affinity nitrate transporter 2 (NRT2) family genes and their responses to drought and salinity

2021 ◽  
Author(s):  
M. AYDIN AKBUDAK ◽  
Ertugrul Filiz ◽  
Durmus Cetin
Keyword(s):  
Solanum Lycopersicum ◽  
Expression Profiles ◽  
Gene Families ◽  
Major Facilitator Superfamily ◽  
Nitrate Transporter ◽  
Tomato Genome ◽  
High Affinity ◽  
Solanum Lycopersicum L ◽  
Major Facilitator ◽  
Systematic Identification

High-affinity nitrate transporter 2 (NRT2) proteins have vital roles in nitrate (NO3-) uptake and translocation in plants. The gene families coding NRT2 proteins have been identified and functionally characterized in many plant species. However, no systematic identification of NRT2 family members have been reported in tomato (Solanum lycopersicum). There is also little known about their expression profiles under environmental stresses. Accordingly, the present study aimed to identify NRT2 gene family in the tomato genome; then, investigate them in detail through bioinformatics, physiological and expression analyses. As a result, four novel NRT2 genes were identified in the tomato genome, all of which contain the same domain belonging to the Major Facilitator Superfamily (PF07690). The co-expression network of SlNRT genes revealed that they were co-expressed with several other genes in many different molecular pathways including transport, photosynthesis, fatty acid metabolism and amino acid catabolism. Programming many crucial physiological and metabolic pathways, various numbers of phosphorylation sites were predicted in the NRT2 proteins.

scholarly journals Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation.

1988 ◽  
Vol 107 (2) ◽  
pp. 743-751 ◽  
Author(s):  
O Saksela ◽  
D Moscatelli ◽  
A Sommer ◽  
D B Rifkin
Keyword(s):  
Growth Factor ◽  
Heparan Sulfate ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Deae Cellulose ◽  
Proteolytic Degradation ◽  
Fibroblast Growth ◽  
Intact Cells ◽  
High Affinity ◽  
Cell Extracts

Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.

scholarly journals The major facilitator superfamily-type protein LbtC promotes the utilization of the legiobactin siderophore by Legionella pneumophila

Microbiology ◽  
2012 ◽  
Vol 158 (3) ◽  
pp. 721-735 ◽  
Author(s):  
Christa H. Chatfield ◽  
Brendan J. Mulhern ◽  
V. K. Viswanathan ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Major Facilitator Superfamily ◽  
Type Protein ◽  
Major Facilitator

Studies on the Mechanism of NAD-photoreduction by Chromatophores of the Facultative Phototroph, Rhodopseudomonas capsulata

1969 ◽  
Vol 24 (1) ◽  
pp. 67-76 ◽  
Author(s):  
J.-H. Klemme
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Molecular Hydrogen ◽  
Reaction System ◽  
Electron Donors ◽  
Rhodopseudomonas Capsulata ◽  
Antimycin A ◽  
Inhibitory Effects ◽  
Carbonyl Cyanide ◽  
Cyclic Photophosphorylation ◽  
Energy Dependent

The light-driven and the ATP-driven reduction of nicotinamide adenine dinucleotide (NAD) catalyzed by the chromatophore fraction of Rhodopseudomonas capsulata was investigated. Efficient electron donors for the photoreduction of NAD are molecular hydrogen and succinate. In the ATP-dependent reaction system, succinate is a more efficient electron donor than H2. The energydependent NAD-reduction is driven by ATP, but not by pyrophosphate or ADP. Oligomycin stimulates the NAD-photoreductions and completely inhibits the ATP-driven NAD-reductions. Rotenone and piericidin A are inhibitors for both the light-driven and the ATP-driven NAD-reductions. Antimycin A is an inhibitor only for the light-driven reductions. The H2-linked NAD-photoreduction is less sensitive to these inhibitors and to the uncoupler desaspidin than the succinate-linked reduction. Atebrine, carbonyl cyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol and phenazonium methosulfate are inhibitors for the light-driven and the ATP-driven reductions. Some of the compounds used as inhibitors of the NAD-reduction were also investigated with concerns to their inhibitory effects on cyclic photophosphorylation and O2-linked oxidations of reduced NAD, succinate and H2. Based on the results of these inhibitor studies, the relationships between cyclic photophosphorylation, light-induced noncyclic electron transport and energy-dependent NAD-reduction are discussed.

scholarly journals Glycine transport into plasma-membrane vesicles derived from rat brain synaptosomes

1981 ◽  
Vol 198 (3) ◽  
pp. 535-541 ◽  
Author(s):  
F Mayor ◽  
J G Marvizón ◽  
M C Aragón ◽  
C Gimenez ◽  
F Valdivieso
Keyword(s):  
Rat Brain ◽  
Membrane Vesicles ◽  
Uptake System ◽  
Plasma Membrane Vesicles ◽  
Glycine Uptake ◽  
High Affinity ◽  
Carbonyl Cyanide ◽  
Brain Synaptosomes ◽  
Glycine Transport ◽  
Sole Energy

1. Transport of glycine has been demonstrated in membrane vesicles isolated from rat brain, using artificially imposed ion gradients as the sole energy source. 2. The uptake of glycine is strictly dependent on the presence of Na+ and Cl- in the medium, and the process can be driven either by an Na+ gradient (out greater than in) or by a C1- gradient (out greater than in) when the other essential ion is present. 3. The uptake of glycine is stimulated by a membrane potential (interior negative), as demonstrated by the effects of the ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions of different permeabilities. 4. The kinetic analysis shows that glycine is accumulated by two systems with different affinities. 5. The presence of ouabain, an inhibitor (Na+ + K+)-activated ATPase, does not affect glycine transport. 6. The existence of a high-affinity, Na+-dependent glycine-uptake system in membrane vesicles derived from rat brain suggests that this amino acid may have a transmitter role in some areas of the rat brain.

scholarly journals Integrin cytoplasmic domains mediate inside-out signal transduction

1994 ◽  
Vol 124 (6) ◽  
pp. 1047-1059 ◽  
Author(s):  
TE O'Toole ◽  
Y Katagiri ◽  
RJ Faull ◽  
K Peter ◽  
R Tamura ◽  
...  
Keyword(s):  
K562 Cells ◽  
Cytoplasmic Domain ◽  
Alpha Subunit ◽  
Affinity Binding ◽  
Cell Type ◽  
Cytoplasmic Domains ◽  
High Affinity Binding ◽  
High Affinity ◽  
Energy Dependent ◽  
Inside Out

We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.

scholarly journals mmpL7 Gene of Mycobacterium tuberculosis Is Responsible for Isoniazid Efflux in Mycobacterium smegmatis

2005 ◽  
Vol 49 (11) ◽  
pp. 4775-4777 ◽  
Author(s):  
Maria R. Pasca ◽  
Paola Guglierame ◽  
Edda De Rossi ◽  
Francesca Zara ◽  
Giovanna Riccardi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Resistance ◽  
Mycobacterium Smegmatis ◽  
Efflux Pump ◽  
Gene Encoding ◽  
Resistance Level ◽  
Carbonyl Cyanide ◽  
Resistance Nodulation Division ◽  
Efflux Pump Inhibitors ◽  
Energy Dependent

ABSTRACT The Mycobacterium tuberculosis mmpL7 gene, encoding a hypothetical resistance nodulation division transporter, confers a high resistance level to isoniazid when overexpressed in Mycobacterium smegmatis. The resistance level decreased in the presence of the efflux pump inhibitors reserpine and CCCP (carbonyl cyanide m-chlorophenylhydrazone). Energy-dependent efflux of isoniazid from M. smegmatis cells expressing the mmpL7 gene was observed.

scholarly journals Modification of Outer Membrane Protein Profile and Evidence Suggesting an Active Drug Pump in Enterobacter aerogenes Clinical Strains

2003 ◽  
Vol 47 (5) ◽  
pp. 1555-1559 ◽  
Author(s):  
Stéphane Gayet ◽  
Renaud Chollet ◽  
Gérard Molle ◽  
Jean-Marie Pagès ◽  
Jacqueline Chevalier
Keyword(s):  
Outer Membrane ◽  
Protein Profile ◽  
Outer Membrane Proteins ◽  
Enterobacter Aerogenes ◽  
Normal Structure ◽  
Clinical Strains ◽  
Resistance Strategies ◽  
Carbonyl Cyanide ◽  
Energy Dependent ◽  
Drug Pump

ABSTRACT Two clinical strains of Enterobacter aerogenes that exhibited phenotypes of multiresistance to β-lactam antibiotics, fluoroquinolones, chloramphenicol, tetracycline, and kanamycin were investigated. Both strains showed a porin pattern different from that of a susceptible strain, with a drastic reduction in the amount of the major porin but with an apparently conserved normal structure (size and immunogenicity), together with overproduction of two known outer membrane proteins, OmpX and LamB. In addition, the full-length O-polysaccharide phenotype was replaced by a semirough Ra phenotype. Moreover, in one isolate the intracellular accumulation of chloramphenicol was increased in the presence of the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone, suggesting an energy-dependent efflux of chloramphenicol in this strain. The resistance strategies used by these isolates appear to be similar to that induced by stress in Escherichia coli cells.

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