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 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 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.

Solubilization and immunoprecipitation of 125I-labelled antigens from Plasmodium knowlesi schizont-infected erythrocytes using non-ionic, anionic and zwitterionic detergents

Parasitology ◽  
1984 ◽  
Vol 88 (1) ◽  
pp. 27-36 ◽  
Author(s):  
R. J. Howard ◽  
J. W. Barnwell
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Plasmodium Knowlesi ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Ionic Detergents ◽  
Class A ◽  
Anionic Detergents ◽  
Triton X

SUMMARYPlasmodium knowlesi malaria-infected erythrocytes were radio-iodinated and several non-ionic, anionic and zwitterionic detergents were compared in their capacity to extract the labelled membrane proteins. The use of these detergents for antigen identification was tested by immunoprecipitation, after addition of Triton X-100 to some detergent extracts, using hyperimmune monkey antiserum and protein A-Sepharose. 125I-labelled antigens were specifically immunoprecipitated with all detergents tested, including the anionic detergents sodium dodecyl sulphate (SDS), deoxycholate and cholate; the zwitterions Zwittergent-312 and -314, CHAPS and Empigen BB, as well as several non-ionic detergents. The SDS-polyacrylamide gel electrophoresis patterns of 125I-labelled antigens varied after extraction with different detergents, there being no consistent pattern for detergents of a particular class. A total of 14 125I-labelled antigens were identified, 11 of them using Triton X-100. Some minor antigens identified with Triton X-100 were immunoprecipitated in greater amount after extraction in other detergents. Most importantly, two antigens Mr 200000 and 180000 were detected only after extraction with deoxycholate or SDS.

Identification of pilin pools in the membranes of Pseudomonas aeruginosa

1982 ◽  
Vol 152 (2) ◽  
pp. 687-691
Author(s):  
T H Watts ◽  
E A Worobec ◽  
W Paranchych
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Gel Electrophoresis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Outer Membranes

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin

1993 ◽  
Vol 13 (4) ◽  
pp. 2061-2068
Author(s):  
H Shirataki ◽  
K Kaibuchi ◽  
T Sakoda ◽  
S Kishida ◽  
T Yamaguchi ◽  
...  
Keyword(s):  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Target Protein ◽  
Dependent Manner ◽  
Gtp Binding Protein ◽  
Reading Frame ◽  
Gtp Binding ◽  
Putative Target

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.

scholarly journals Protein Engineering of Wzc To Generate New Emulsan Analogs

2007 ◽  
Vol 73 (12) ◽  
pp. 4020-4028 ◽  
Author(s):  
Hanna Dams-Kozlowska ◽  
David L. Kaplan
Keyword(s):  
Molecular Weight ◽  
Chain Length ◽  
Genetic Manipulation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Point Mutations ◽  
Biological Properties ◽  
Feeding Strategies ◽  
Exogenous Fatty Acid

ABSTRACT Acinetobacter venetianus Rag1 produces an extracellular, polymeric lipoheteropolysaccharide termed apoemulsan. This polymer is putatively produced via a Wzy-dependent pathway. According to this model, the length of the polymer is regulated by polysaccharide-copolymerase (PCP) protein. A highly conserved proline and glycine motif was identified in all members of the PCP family of proteins and is involved in regulation of polymer chain length. In order to control the structure of apoemulsan, defined point mutations in the proline-glycine-rich region of the apoemulsan PCP protein (Wzc) were introduced. Modified wzc variants were introduced into the Rag1 genome via homologous recombination. Stable chromosomal mutants were confirmed by Southern blot analysis. The molecular weight of the polymer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five of the eight point mutants produced polymers having molecular weights higher than the molecular weight of the polymer produced by the wild type. Moreover, four of these five polymers had modified biological properties. Replacement of arginine by leucine (R418L) resulted in the most significant change in the molecular weight of the polymer. The R418L mutant was the most hydrophilic mutant, exhibiting decreased adherence to polystyrene, and inhibited biofilm formation. The results described in this report show the functional effect of Wzc modification on the molecular weight of a high-molecular-weight polysaccharide. Moreover, in the present study we developed a genetic system to control polymerization of apoemulsan. The use of selective exogenous fatty acid feeding strategies, as well as genetic manipulation of sugar backbone chain length, is a promising new approach for bioengineering emulsan analogs.

Receptors and cGMP signalling mechanism for E. coli enterotoxin in opossum kidney

1988 ◽  
Vol 255 (5) ◽  
pp. F1040-F1046 ◽  
Author(s):  
L. R. Forte ◽  
W. J. Krause ◽  
R. H. Freeman
Keyword(s):  
Cell Lines ◽  
Atrial Natriuretic Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Kidney Cortex ◽  
Water Excretion ◽  
Opossum Kidney ◽  
E Coli ◽  
Natriuretic Factor ◽  
Atrial Natriuretic

Receptors for the heat-stable enterotoxin produced by Escherichia coli were found in the kidney and intestine of the North American opossum and in cultured renal cell lines. The enterotoxin markedly increased guanosine 3',5'-cyclic monophosphate (cGMP) production in slices of kidney cortex and medulla, in suspensions of intestinal mucosa, and in the opossum kidney (OK) and rat kangaroo kidney (PtK-2) cell lines. In contrast, atrial natriuretic factor elicited much smaller increases in cGMP levels of kidney, intestine, or cultured kidney cell lines. The enterotoxin receptors in OK cells had a molecular mass of approximately 120 kDa when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of receptors crosslinked with 125I-enterotoxin. The occurrence of receptors for the E. coli peptide in OK implies that these receptors may be involved in the regulation of renal tubular function in the opossum. E. coli enterotoxin caused a much larger increase in urine cGMP excretion (10- to 50-fold over control) than did atrial natriuretic factor when these peptides were injected intravenously into opossums. However, atrial natriuretic factor elicited a marked diuresis, natriuresis, and increased urinary excretion of calcium, phosphate, potassium, and magnesium. In contrast, the enterotoxin did not acutely influence OK fluid and electrolyte excretion. Thus the substantial increase in cGMP synthesis produced by the bacterial peptide in OK cortex and medulla in vitro and the increased renal excretion of cGMP in vivo were not associated with changes in electrolyte or water excretion. Whether cGMP represents a second messenger molecule in the kidney is an interesting question that was raised but not answered in this series of experiments.

Identification of the Poly(C) Binding Protein in the Complex Associated With the 3′ Untranslated Region of Erythropoietin Messenger RNA

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 2111-2120 ◽  
Author(s):  
Maria F. Czyzyk-Krzeska ◽  
Amy C. Bendixen
Keyword(s):  
Mrna Stability ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mobility Shift ◽  
Protein Factor ◽  
Ribonucleoprotein Complexes ◽  
Sds Page

Hypoxia regulates expression of erythropoietin (EPO), a glycoprotein that stimulates erythrocytosis, at the level of transcription and also possibly at the level of messenger RNA (mRNA) stability. A pyrimidine-rich region within the EPO mRNA 3′ untranslated region was implicated in regulation of EPO mRNA stability element and shown to bind protein factors. In the present study we wished to identify the protein factor binding to the pyrimidine-rich sequence in the EPO mRNA stability element. Using mobility shift assays, ultraviolet light cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electroelution of protein factors from the gel slices corresponding to the ribonucleoprotein complexes, we found that two isoforms of a 40 kD poly(C) binding protein (PCBP, also known as CP or hnRNPE), PCBP1, and PCBP2 are present in that complex. In Hep3B or HepG2 cells hypoxia induces neither expression of PCBP nor formation of the ribonucleoprotein complex associated with EPO mRNA that involves PCBP.

scholarly journals mgr, a Novel Global Regulator in Staphylococcus aureus

2003 ◽  
Vol 185 (13) ◽  
pp. 3703-3710 ◽  
Author(s):  
Thanh T. Luong ◽  
Steven W. Newell ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Capsular Polysaccharide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Binding Motif ◽  
Transcriptional Level ◽  
Virulence Determinants ◽  
Alpha Toxin ◽  
Extracellular Protein Production

ABSTRACT The virulence determinants of Staphylococcus aureus are coordinately controlled by several unlinked chromosomal loci. Here, we report the identification of CYL5614, derived from strain Becker, with a mutation that affects the expression of type 8 capsular polysaccharide (CP8), nuclease, alpha-toxin, coagulase, protease, and protein A. This novel locus, named mgr, was linked by transposon Tn917 and mapped by three-factorial transduction crosses. The region containing the mgr locus was cloned and sequenced. Deletion mutagenesis and genetic complementation showed that the locus consisted of one gene, mgrA. Interestingly, mgrA-null mutants exhibited a phenotype opposite to that of CYL5614. This was due to a T-to-C mutation upstream of mgrA that resulted in a four- to eightfold increase in mgrA transcription in strain CYL5614. Thus, these results indicate that mgrA is an activator of CP8 and nuclease but a repressor of alpha-toxin, coagulase, protease, and protein A. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the mgr locus profoundly affected extracellular protein production, suggesting that the locus may regulate many other genes as well. The translated MgrA protein has a region of significant homology, which includes the helix-turn-helix DNA-binding motif, with the Escherichia coli MarR family of transcriptional regulators. Northern slot blot analyses suggested that mgr affected CP8, alpha-toxin, nuclease, and protein A at the transcriptional level.

Sign in / Sign up

Export Citation Format

Share Document