scholarly journals Differential Protein Expression during Growth of Acidithiobacillus ferrooxidans on Ferrous Iron, Sulfur Compounds, or Metal Sulfides

2004 ◽  
Vol 70 (8) ◽  
pp. 4491-4498 ◽  
Author(s):  
Pablo Ramírez ◽  
Nicolas Guiliani ◽  
Lissette Valenzuela ◽  
Simon Beard ◽  
Carlos A. Jerez
Keyword(s):  
Acidithiobacillus Ferrooxidans ◽  
Ferrous Iron ◽  
Genomic Sequence ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sulfur Compounds ◽  
Mass Spectrometry Analysis ◽  
Metal Sulfides ◽  
Phosphate Binding

ABSTRACT A set of proteins that changed their levels of synthesis during growth of Acidithiobacillus ferrooxidans ATCC 19859 on metal sulfides, thiosulfate, elemental sulfur, and ferrous iron was characterized by using two-dimensional polyacrylamide gel electrophoresis. N-terminal amino acid sequencing and mass spectrometry analysis of these proteins allowed their identification and the localization of the corresponding genes in the available genomic sequence of A. ferrooxidans ATCC 23270. The genomic context around several of these genes suggests their involvement in the energetic metabolism of A. ferrooxidans. Two groups of proteins could be distinguished. The first consisted of proteins highly upregulated by growth on sulfur compounds (and downregulated by growth on ferrous iron): a 44-kDa outer membrane protein, an exported 21-kDa putative thiosulfate sulfur transferase protein, a 33-kDa putative thiosulfate/sulfate binding protein, a 45-kDa putative capsule polysaccharide export protein, and a putative 16-kDa protein of unknown function. The second group of proteins comprised those downregulated by growth on sulfur (and upregulated by growth on ferrous iron): rusticyanin, a cytochrome c 552, a putative phosphate binding protein (PstS), the small and large subunits of ribulose biphosphate carboxylase, and a 30-kDa putative CbbQ protein, among others. The results suggest in general a separation of the iron and sulfur utilization pathways. Rusticyanin, in addition to being highly expressed on ferrous iron, was also newly synthesized, as determined by metabolic labeling, although at lower levels, during growth on sulfur compounds and iron-free metal sulfides. During growth on metal sulfides containing iron, such as pyrite and chalcopyrite, both proteins upregulated on ferrous iron and those upregulated on sulfur compounds were synthesized, indicating that the two energy-generating pathways are induced simultaneously depending on the kind and concentration of oxidizable substrates available.

scholarly journals Characterization of the structure of the erythropoietin receptor by ligand blotting

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2577-2582 ◽  
Author(s):  
HL Atkins ◽  
VC Broudy ◽  
T Papayannopoulou
Keyword(s):  
Cell Lines ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Erythropoietin Receptor ◽  
Epo Receptor ◽  
Peptide Growth Factors ◽  
Cell Membrane Proteins ◽  
Erythroleukemia Cell

Abstract Erythropoietin (Epo) regulates the growth and differentiation of erythroid cells by binding to a specific receptor. We characterized the native Epo receptor on erythroleukemia cell lines by ligand blotting. Solubilized cell membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with 125I-Epo. Specificity was demonstrated by inhibition of 125I-Epo binding by unlabeled excess Epo but not other peptide growth factors and by the cellular distribution of the Epo binding protein. A single membrane protein of 61 Kd +/- 4 Kd was sufficient to bind 125I Epo in both human (OCIM2, K562) and murine (GM979, Rauscher, DA-1) cell lines. This finding is consistent with the predicted size of the Epo receptor from the murine cDNA clone. However, chemical crosslinking of 125I-Epo to its receptor has identified two Epo binding proteins of 105 Kd and 85 Kd. This difference may occur because the receptor is size fractionated before Epo binding in the ligand blot, but after Epo binding in crosslinking studies. Ligand blotting demonstrates that the native Epo receptor is composed of a single 61-Kd Epo binding protein, and suggests the presence of additional proteins of 20 to 25 Kd that associate with the receptor after Epo binding.

scholarly journals Gene Identification and Substrate Regulation Provide Insights into Sulfur Accumulation during Bioleaching with the Psychrotolerant Acidophile Acidithiobacillus ferrivorans

2012 ◽  
Vol 79 (3) ◽  
pp. 951-957 ◽  
Author(s):  
Maria Liljeqvist ◽  
Olena I. Rzhepishevska ◽  
Mark Dopson
Keyword(s):  
Acidithiobacillus Ferrooxidans ◽  
Ferrous Iron ◽  
Sulfur Compound ◽  
Iron Oxidation ◽  
Sulfur Compounds ◽  
Content Type ◽  
Inorganic Sulfur ◽  
Cold Environments ◽  
Ferrous Iron Oxidation ◽  
Inorganic Sulfur Compound

ABSTRACTThe psychrotolerant acidophileAcidithiobacillus ferrivoranshas been identified from cold environments and has been shown to use ferrous iron and inorganic sulfur compounds as its energy sources. A bioinformatic evaluation presented in this study suggested thatAcidithiobacillus ferrivoransutilized a ferrous iron oxidation pathway similar to that of the related speciesAcidithiobacillus ferrooxidans. However, the inorganic sulfur oxidation pathway was less clear, since theAcidithiobacillus ferrivoransgenome contained genes from bothAcidithiobacillus ferrooxidansandAcidithiobacillus caldusencoding enzymes whose assigned functions are redundant. Transcriptional analysis revealed that thepetA1andpetB1genes (implicated in ferrous iron oxidation) were downregulated upon growth on the inorganic sulfur compound tetrathionate but were on average 10.5-fold upregulated in the presence of ferrous iron. In contrast, expression ofcyoB1(involved in inorganic sulfur compound oxidation) was decreased 6.6-fold upon growth on ferrous iron alone. Competition assays between ferrous iron and tetrathionate withAcidithiobacillus ferrivoransSS3 precultured on chalcopyrite mineral showed a preference for ferrous iron oxidation over tetrathionate oxidation. Also, pure and mixed cultures of psychrotolerant acidophiles were utilized for the bioleaching of metal sulfide minerals in stirred tank reactors at 5 and 25°C in order to investigate the fate of ferrous iron and inorganic sulfur compounds. Solid sulfur accumulated in bioleaching cultures growing on a chalcopyrite concentrate. Sulfur accumulation halted mineral solubilization, but sulfur was oxidized after metal release had ceased. The data indicated that ferrous iron was preferentially oxidized during growth on chalcopyrite, a finding with important implications for biomining in cold environments.

A novel D-glyceraldehyde-3-phosphate binding protein, a truncated albumin, with D-glyceraldehyde-3-phosphate dehydrogenase inhibitory property

IUBMB Life ◽  
2009 ◽  
Vol 61 (10) ◽  
pp. 995-1000
Author(s):  
Amrita Roy ◽  
Soumen Bera ◽  
Subrata Patra ◽  
Subhankar Ray ◽  
Manju Ray
Keyword(s):  
Binding Protein ◽  
Protein A ◽  
Phosphate Binding ◽  
Inhibitory Property ◽  
Phosphate Binding Protein

scholarly journals Identification and Characterization of the Entamoeba Histolytica Rab8a Binding Protein: A Cdc50 Homolog

2018 ◽  
Vol 19 (12) ◽  
pp. 3831 ◽  
Author(s):  
Yuki Hanadate ◽  
Yumiko Saito-Nakano ◽  
Kumiko Nakada-Tsukui ◽  
Tomoyoshi Nozaki
Keyword(s):  
Plasma Membrane ◽  
Entamoeba Histolytica ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Membrane Traffic ◽  
Membrane Lipid ◽  
Small Gtpase ◽  
Triton X100 ◽  
Lipid Flippase

Membrane traffic plays a pivotal role in virulence in the enteric protozoan parasite Entamoeba histolytica. EhRab8A small GTPase is a key regulator of membrane traffic at the endoplasmic reticulum (ER) of this protist and is involved in the transport of plasma membrane proteins. Here we identified the binding proteins of EhRab8A. The Cdc50 homolog, a non-catalytic subunit of lipid flippase, was identified as an EhRab8A binding protein candidate by affinity coimmunoprecipitation. Binding of EhRab8A to EhCdc50 was also confirmed by reciprocal immunoprecipitation and blue-native polyacrylamide gel electrophoresis, the latter of which revealed an 87 kDa complex. Indirect immunofluorescence imaging with and without Triton X100 showed that endogenous EhCdc50 localized on the surface in the absence of permeabilizing agent but was observed on the intracellular structures and overlapped with the ER marker Bip when Triton X100 was used. Overexpression of N-terminal HA-tagged EhCdc50 impaired its translocation to the plasma membrane and caused its accumulation in the ER. As reported previously in other organisms, overexpression and accumulation of Cdc50 in the ER likely inhibited surface transport and function of the plasma membrane lipid flippase P4-ATPase. Interestingly, HA-EhCdc50-expressing trophozoites gained resistance to miltefosine, which is consistent with the prediction that HA-EhCdc50 overexpression caused its accumulation in the ER and mislocalization of the unidentified lipid flippase. Similarly, EhRab8A gene silenced trophozoites showed increased resistance to miltefosine, supporting EhRab8A-dependent transport of EhCdc50. This study demonstrated for the first time that EhRab8A mediates the transport of EhCdc50 and lipid flippase P4-ATPase from the ER to the plasma membrane.

scholarly journals Characterization of the structure of the erythropoietin receptor by ligand blotting

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2577-2582
Author(s):  
HL Atkins ◽  
VC Broudy ◽  
T Papayannopoulou
Keyword(s):  
Cell Lines ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Erythropoietin Receptor ◽  
Epo Receptor ◽  
Peptide Growth Factors ◽  
Cell Membrane Proteins ◽  
Erythroleukemia Cell

Erythropoietin (Epo) regulates the growth and differentiation of erythroid cells by binding to a specific receptor. We characterized the native Epo receptor on erythroleukemia cell lines by ligand blotting. Solubilized cell membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with 125I-Epo. Specificity was demonstrated by inhibition of 125I-Epo binding by unlabeled excess Epo but not other peptide growth factors and by the cellular distribution of the Epo binding protein. A single membrane protein of 61 Kd +/- 4 Kd was sufficient to bind 125I Epo in both human (OCIM2, K562) and murine (GM979, Rauscher, DA-1) cell lines. This finding is consistent with the predicted size of the Epo receptor from the murine cDNA clone. However, chemical crosslinking of 125I-Epo to its receptor has identified two Epo binding proteins of 105 Kd and 85 Kd. This difference may occur because the receptor is size fractionated before Epo binding in the ligand blot, but after Epo binding in crosslinking studies. Ligand blotting demonstrates that the native Epo receptor is composed of a single 61-Kd Epo binding protein, and suggests the presence of additional proteins of 20 to 25 Kd that associate with the receptor after Epo binding.

scholarly journals The 95000-Mr gelatin-binding protein in human serum and plasma

1985 ◽  
Vol 228 (3) ◽  
pp. 605-608 ◽  
Author(s):  
T Vartio
Keyword(s):  
Human Serum ◽  
Binding Protein ◽  
Blood Clot ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Human Monocyte ◽  
Secretory Component ◽  
Serum Samples ◽  
Lower Quantity

A gelatin-binding 95000-Mr protein was detected in human serum and plasma by immunoblotting using antibodies against the 95000-Mr gelatin-binding protein, a major secretory component of cultured adherent human monocyte/macrophages. Serum and plasma were prepared by incubating blood at 4, 22 or 37 degrees C for different periods of time, and gelatin-binding proteins were isolated from 200 microliter portions by gelatin-Sepharose affinity chromatography. The bound material was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In protein-stained gels, fibronectin and some minor polypeptides were seen, but not the 95000-Mr protein. In immunoblotting of identical serum samples the antibodies detected apparently two closely spaced polypeptide bands at Mr95000, and in plasma samples a single band at the position of the faster-migrating one of the two above-mentioned bands. The immunoperoxidase reaction was stronger when serum and plasma were prepared by incubating for longer periods of time (up to 8 h) or at higher temperatures (up to 37 degrees C). In samples made from plasma, the immunoperoxidase reactions were weaker than in those from serum, indicating a lower quantity of the protein. The results suggest that the 95000-Mr protein is released from monocytes and granulocytes during the incubation of blood and, more likely, when they possibly interact with the blood clot and may become adherent.

scholarly journals Identification and Characterization of Human Surfactant Protein A Binding Protein of Mycoplasma pneumoniae

2005 ◽  
Vol 73 (5) ◽  
pp. 2828-2834 ◽  
Author(s):  
T. R. Kannan ◽  
D. Provenzano ◽  
J. R. Wright ◽  
J. B. Baseman
Keyword(s):  
Amino Acids ◽  
Mycoplasma Pneumoniae ◽  
Pertussis Toxin ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Dependent Manner ◽  
Extracellular Matrix Components

ABSTRACT Mycoplasma pneumoniae infections represent a major primary cause of human respiratory diseases, exacerbate other respiratory disorders, and are associated with extrapulmonary pathologies. Cytadherence is a critical step in mycoplasma colonization, aided by a network of mycoplasma adhesins and cytadherence accessory proteins which mediate binding to host cell receptors. Furthermore, the respiratory mucosa is enriched with extracellular matrix components, including surfactant proteins, fibronectin, and mucin, which provide additional in vivo targets for mycoplasma parasitism. In this study we describe interactions between M. pneumoniae and human surfactant protein-A (hSP-A). Initially, we found that viable M. pneumoniae cells bound to immobilized hSP-A in a dose- and calcium (Ca2+)-dependent manner. Mild trypsin treatment of intact mycoplasmas reduced binding markedly (80 to 90%) implicating a surface-associated mycoplasma protein(s). Using hSP-A-coupled Sepharose affinity chromatography and polyacrylamide gel electrophoresis, we identified a 65-kDa hSP-A binding protein of M. pneumoniae. The presence of Ca2+ enhanced binding of the 65-kDa protein to hSP-A, which was reduced by the divalent cation-chelating agent, EDTA. The 65-kDa hSP-A binding protein of M. pneumoniae was identified by sequence analysis as a novel protein (MPN372) possessing a putative S1-like subunit of pertussis toxin at the amino terminus (amino acids 1 to 226), with the remaining amino acids (227 to 591) exhibiting no homology with other subunits of pertussis toxin, other known toxins, or any reported proteins. Recombinant MPN372 (MPN372) bound to hSP-A in a dose-dependent manner, which was markedly reduced by preincubation with mouse recombinant MPN372 antisera. Also, adherence of viable M. pneumoniae cells to hSP-A was inhibited by recombinant MPN372 antisera, demonstrating that MPN372, a previously designated hypothetical protein, is surface exposed and mediates mycoplasma attachment to hSP-A.

How the RegBA Redox Responding System Controls Iron and Sulfur Oxidation in Acidithiobacillus ferrooxidans

2013 ◽  
Vol 825 ◽  
pp. 186-189 ◽  
Author(s):  
Danielle Moinier ◽  
Deborah Byrne ◽  
Agnès Amouric ◽  
Violaine Bonnefoy
Keyword(s):  
Acidithiobacillus Ferrooxidans ◽  
Ferrous Iron ◽  
Iron Oxidation ◽  
Regulatory Region ◽  
Sulfur Oxidation ◽  
Sulfur Compounds ◽  
Chemical Attack ◽  
Valuable Metals ◽  
Genes Encoding ◽  
Iron And Sulfur Oxidation

Valuable metals as well as ferrous iron and sulfur compounds are released from ore by ferric iron and sulfuric acid chemical attack. Biomining microorganisms allow the recycling of these products by oxidizing ferrous iron and/or sulfur compounds. The energy released from the oxidation of these substrates is used for the growth of the acidophilic chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans. The respiratory pathways involved in these respiratory processes have been deciphered and the expression of the genes encoding these redox proteins is dependent on the electron donor present in the medium. Furthermore, in the presence of both ferrous iron and sulfur, the genes involved in iron oxidation are expressed before those involved in sulfur oxidation. We propose that the global redox responding two component system RegBA is responsible for this regulation since (i) the redox potential increases during iron oxidation but remains stable during sulfur oxidation and (ii) the transcriptional regulator RegA binds the regulatory region of a number of genes/operons involved in iron and sulfur oxidation. To understand the mechanism of the At. ferrooxidans RegBA system, the regA gene and the DNA corresponding to the DNA binding domain of RegA were cloned in an expression plasmid in Escherichia coli. The recombinant proteins, RegA and RegA-HTH respectively, were purified. The binding of RegA-HTH, phosphorylated and unphosphorylated RegA on the regulatory region of some target operons have been compared by gel shift mobility assay.

scholarly journals Analysis of the Immunological Responses to Transferrin and Lactoferrin Receptor Proteins fromMoraxella catarrhalis

1999 ◽  
Vol 67 (8) ◽  
pp. 3793-3799 ◽  
Author(s):  
Rong-hua Yu ◽  
Robert A. Bonnah ◽  
Samuel Ainsworth ◽  
Anthony B. Schryvers
Keyword(s):  
Immune Response ◽  
Solid Phase ◽  
Binding Protein ◽  
Iron Acquisition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Receptor Proteins ◽  
Convalescent Phase ◽  
Protein B

ABSTRACT Moraxella catarrhalis expresses surface receptor proteins that specifically bind host transferrin (Tf) and lactoferrin (Lf) in the first step of the iron acquisition pathway. Acute- and convalescent-phase antisera from a series of patients with M. catarrhalis pulmonary infections were tested against Tf and Lf receptor proteins purified from the corresponding isolates. After the purified proteins had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, we observed strong reactivity against Tf-binding protein B (TbpB; also called OMP1) and Lf-binding protein B (LbpB) but little or no reactivity against Tf-binding protein A (TbpA) or Lf-binding protein A (LbpA), using the convalescent-phase antisera. Considerable antigenic heterogeneity was observed when TbpBs and LbpBs isolated from different strains were tested with the convalescent-phase antisera. Comparison to the reactivity against electroblotted total cellular proteins revealed that the immune response against LbpB and TbpB constitutes a significant portion of the total detectable immune response to M. catarrhalis proteins. Preparations of affinity-isolated TbpA and LbpA reacted with convalescent-phase antisera in a solid-phase binding assay, but blocking with soluble TbpB, soluble LbpB, or extracts from an LbpA− mutant demonstrated that this reactivity was attributed to contaminants in the TbpA and LbpA preparations. These studies demonstrate the immunogenicity of M. catarrhalisTbpB and LbpB in humans and support their potential as vaccine candidates.

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