scholarly journals A fibronectin-binding glycoprotein from human platelet membranes

1982 ◽  
Vol 201 (3) ◽  
pp. 629-633 ◽  
Author(s):  
M S Hansen ◽  
I Clemmensen
Keyword(s):  
Human Platelet ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Relative Molecular Mass ◽  
Crossed Immunoelectrophoresis ◽  
Platelet Membranes ◽  
Fibronectin Binding ◽  
Cold Insoluble Globulin ◽  
Binding Glycoprotein

Fibronectin (‘cold-insoluble globulin’) has been suggested as a possible mediator of platelet adhesion. A fibronectin-binding protein as partially purified from washed solubilized human platelet membranes by affinity chromatography on fibronectin-Sepharose. The isolated protein migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr (relative molecular mass) of approx. 125 000 under reducing conditions. The protein migrated as a dimer in non-reduced gels. The purified protein did not react with immunoglobulins against fibrinogen or fibronectin when tested in crossed immunoelectrophoresis or electroimmunoassay. The protein and purified fibronectin formed a complex that had a significantly faster mobility in crossed immunoelectrophoresis than did native fibronectin. The presence of heparin in the binding-protein-fibronectin mixture resulted in an even faster mobility of the complex, whereas the mobility of native fibronectin was unaffected. Crossed affinoimmunoelectrophoresis of the complex using different lectins suggested that the binding protein is a glycoprotein containing N-acetylglucosamine residues. The complex, but not purified fibronectin, bound to phenyl-Sepharose on crossed hydrophobic-interaction immunoelectrophoresis. The results strongly suggest the presence of a fibronectin-binding glycoprotein in the platelet membrane.

Fibronectin-Binding Glycoprotein From Human Platelet Membranes

1981 ◽  
Author(s):  
M Sandbjerg Hansen ◽  
Inge Clemmensen
Keyword(s):  
Molecular Weight ◽  
Platelet Adhesion ◽  
Human Platelet ◽  
Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Reducing Conditions ◽  
Fibronectin Binding Protein ◽  
Fibronectin Binding ◽  
Cold Insoluble Globulin ◽  
Binding Glycoprotein

Fibronectin (cold insoluble globulin) has been suggested as a possible mediator of platelet adhesion. A fibronectin-binding protein was partially purified from washed solubilized human plate letmembranes by affinity chromatography on fi- bronectin-Sepharose. The isolated protein migrated as a single band in SDS-polyacrylamidegelelectro- phoresis with a molecular weight of approximately 125000 under reducing conditions. In non-reduced gels the protein migrated as a dimer. The purified protein did not react with immunoglobulins against fibrinogen or fibronectin when tested in crossed immunoelectrophoresis or electroimmuno assay. The protein and purified fibronectin formed a complex which had a significantly faster mobility in crossed immunoelectrophoresis than did native fibronectin. The presence of heparin in the binding proteinfibronectin mixture resulted in an even faster mobility of the complex, while the mobility of native fibronectin was unaffected. Crossed affinoimmunoelectrophoresis of the complex using different lectins suggested that the binding protein is a glycoprotein containing N-acetyl-glu- cosamine residues. The complex, but not purified fibronectin, bound to phenyl-Sepharose on crossed hydrophobic interaction immunoelectrophoresis. The results strongly suggest the presence of a fibronectin binding glycoprotein in the platelet membrane.

scholarly journals Ca2+-binding protein from human kidney. Purification and properties

1984 ◽  
Vol 217 (1) ◽  
pp. 229-237 ◽  
Author(s):  
M Staun ◽  
O Norén ◽  
H Sjöström
Keyword(s):  
Specific Antibody ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Human Kidney ◽  
Fold Increase ◽  
Binding Activity ◽  
Cytosol Fraction ◽  
Crossed Immunoelectrophoresis

A Ca2+-binding protein (CaBP) from human kidney was purified by two different procedures. The first involved heat-precipitation of a kidney cytosol fraction followed by gel filtration and chromatofocusing. This resulted in a 200-fold increase in the specific Ca2+-binding activity with a yield of 10%. A specific antibody was raised against the purified CaBP, as demonstrated by one precipitate in crossed immunoelectrophoresis of a kidney cytosol fraction. The antibody was coupled to Sepharose 4B and CaBP was then purified by immunoadsorbent chromatography. Applying this technique, a 500-fold purification of CaBP with a yield of 50% was obtained. Both preparations appeared homogeneous in crossed immunoelectrophoresis against a polyvalent antiserum and migrated as a single band corresponding to a mol.wt. of 26000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In gel filtration under non-denaturing conditions CaBP was eluted corresponding to a mol.wt. of 28000. The association constant for the high-affinity Ca2+-binding sites of CaBP was estimated by gel filtration to be 0.1 × 10(6)M-1, and the protein displayed Ca2+-dependent electrophoretic mobility, with more rapid anodic migration in the presence of EDTA. The protein eluted at a position corresponding to a pI of 4.5 in chromatofocusing. Immunochemical experiments with the specific antibody showed no cross-reaction between renal and intestinal CaBP.

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 Purification of two heparin-binding proteins from porcine platelets and their homology with human secreted platelet proteins

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1072-1080 ◽  
Author(s):  
B Rucinski ◽  
A Poggi ◽  
P James ◽  
JC Holt ◽  
S Niewiarowski
Keyword(s):  
Amino Acid ◽  
Basic Protein ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Heterologous Antigens

Abstract Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6–0.9 M NaCl and at 1–1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000–7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno- precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.

scholarly journals Interleukin-1 induces interleukin-6 production in peripheral blood monocytes

Blood ◽  
1990 ◽  
Vol 75 (6) ◽  
pp. 1305-1310 ◽  
Author(s):  
G Tosato ◽  
KD Jones
Keyword(s):  
Endothelial Cells ◽  
Interleukin 6 ◽  
Peripheral Blood ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Relative Molecular Mass ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Potent Inducer ◽  
Size Heterogeneity

Abstract Interleukin-6 (IL-6), a multifunctional cytokine produced in monocytes, fibroblasts, endothelial cells, and keratinocytes, is induced by a variety of stimulating signals, including lipopolysaccharide (LPS), poly (I), poly (C), IL-1, tumor necrosis factor (TNF), and platelet- derived growth factor. Some of these signals induce IL-6 effectively only in one cell type, and this selectivity of induction may explain selectivity of biologic effects. In the present study, we show that IL- 1 beta, previously known to be a potent inducer of IL-6 in fibroblasts, endothelial cells, and keratinocytes, but not in monocytes, is also a potent inducer of IL-6 in peripheral blood monocytes. High level IL-6 activity that could be neutralized by specific antibodies to IL-6 was detected in supernatants of IL-1-stimulated monocytes. Maximal induction required IL-1 concentrations of 10 ng/mL. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, IL-6 species of relative molecular mass of 19 to 26 Kd could be specifically immunoprecipitated from supernatants of IL-1- induced monocytes. Size heterogeneity is a reported feature of IL-6 produced in a variety of cell types, and monocyte-derived IL-6 induced by either IL-1 or LPS displayed similar size heterogeneity. The highly purified recombinant IL-1 beta preparation used contained little, if any, LPS. In addition, monocyte production of IL-6, induced by IL-1 beta, was specifically neutralized by anti-IL-1 beta antibodies, demonstrating that IL-1, rather than a contaminant in the IL-1 preparation, was responsible for IL-6 induction. A number of biologic activities have been ascribed both to IL-1 and IL-6. The finding that IL-1 induced IL-6 in monocytes may help in defining the spectrum of biologic activities of each of these interactive cytokines.

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.

The isolation, purification and some properties of pheromaxein, the pheromonal steroid-binding protein, in porcine submaxillary glands and saliva

1988 ◽  
Vol 118 (1) ◽  
pp. 47-NP ◽  
Author(s):  
W. D. Booth ◽  
C. A. White
Keyword(s):  
Liquid Chromatography ◽  
Affinity Chromatography ◽  
Sodium Dodecyl Sulphate ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Submaxillary Gland ◽  
Fast Protein Liquid Chromatography ◽  
Steroid Binding ◽  
Steroid Binding Protein

ABSTRACT Pheromaxein, the 16-androstene steroid-binding protein with a relative molecular mass of 15 000 was isolated in sub-milligram quantities from the submaxillary gland and saliva of the Gottingen miniature boar, after a fourfold purification involving the following methods: ultrafiltration for submaxillary gland cytosols and ethanol precipitation for saliva, Concanavalin-A-Sepharose affinity chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, 'Extractigel-D' affinity chromatography (to remove sodium dodecyl sulphate) and fast protein-liquid chromatography. Yields of purified pheromaxein obtained after fast protein-liquid chromatography represented 10–20% of total protein present in an ultrafiltrate of a submaxillary gland cytosol. Fast protein-liquid chromatography separated the α- and β-charge isomers of pheromaxein which were shown to have isoelectric points of 4·78 and 5·35 respectively on flat-bed isoelectric focusing. Some data are provided for the variable occurrence of the isomeric forms of pheromaxein in relation to different breeds of pig. Five 16-unsaturated steroids showed the highest binding to pheromaxein. Other steroids of the 5α- and 5β-androstane series also showed some binding to pheromaxein, i.e. 17β-hydroxy-5α-androstan-3-one (19·2%), with 5α-androstan-3-one, which has a similar urinous odour to 5α-androst-16-en-3-one, showing the greatest binding (42·6%) relative to 5α-androst-16-en-3-one (100%). J. Endocr. (1988) 118, 47–57

scholarly journals Disruption of the Genes Encoding Antigen 85A and Antigen 85B ofMycobacterium tuberculosis H37Rv: Effect on Growth in Culture and in Macrophages

2000 ◽  
Vol 68 (2) ◽  
pp. 767-778 ◽  
Author(s):  
Lisa Y. Armitige ◽  
Chinnaswamy Jagannath ◽  
Audrey R. Wanger ◽  
Steven J. Norris
Keyword(s):  
Cell Lines ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasmid Vector ◽  
Kanamycin Resistance ◽  
Homologous Gene ◽  
Transferase Activity ◽  
Genes Encoding ◽  
Systematic Determination ◽  
Fibronectin Binding

ABSTRACT The mechanism of pathogenesis of Mycobacterium tuberculosis is thought to be multifactorial. Among the putative virulence factors is the antigen 85 (Ag85) complex. This family of exported fibronectin-binding proteins consists of members Ag85A, Ag85B, and Ag85C and is most prominently represented by 85A and 85B. These proteins have recently been shown to possess mycolyl transferase activity and likely play a role in cell wall synthesis. The purpose of this study was to generate strains of M. tuberculosis deficient in expression of the principal members of this complex in order to determine their role in the pathogenesis ofM. tuberculosis. Constructs of fbpA andfbpB disrupted with the kanamycin resistance marker ΩKm and containing varying amounts of flanking gene and plasmid vector sequences were then introduced as linear fragments into H37Rv by electroporation. Southern blot and PCR analyses revealed disruption of the homologous gene locus in one fbpA::ΩKm transformant and one fbpB::ΩKm transformant. The fbpA::ΩKm mutant, LAa1, resulted from a double-crossover integration event, whereas thefbpB::ΩKm variant, LAb1, was the product of a single-crossover type event that resulted in insertion of both ΩKm and plasmid sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis confirmed that expression of the disrupted gene was not detectable in the fbpA andfbpB mutants. Analysis of growth rates demonstrated that the fbpB mutant LAb1 grew at a rate similar to that of the wild-type parent in enriched and nutrient-poor laboratory media as well as in human (THP-1) and mouse (J774.1A) macrophage-like cell lines. ThefbpA mutant LAa1 grew similarly to the parent H37Rv in enriched laboratory media but exhibited little or no growth in nutrient-poor media and macrophage-like cell lines. The targeted disruption of two genes encoding mycolyl transferase and fibronectin-binding activities in M. tuberculosis will permit the systematic determination of their roles in the physiology and pathogenesis of this organism.

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