Characterization of albumin-binding properties of Peptostreptococcus magnus

1989 ◽  
Vol 35 (6) ◽  
pp. 614-618 ◽  
Author(s):  
C. Lämmler ◽  
A. Alaboudi ◽  
A. Hildebrand
Keyword(s):  
Binding Site ◽  
Binding Proteins ◽  
Binding Activity ◽  
Albumin Binding ◽  
Binding Properties ◽  
Heat Stable ◽  
Associated Proteins ◽  
Protein Nature ◽  
Peptostreptococcus Magnus

A Peptostreptococcus magnus strain demonstrated binding activity for albumin preparations from humans, mice, and dogs, but not for rabbit or bovine albumin. The albumin binding site appeared to be heat stable and of protein nature. Treatment of P. magnus cells with trypsin under specified conditions enhanced this albumin binding. Electron micrographs and kinetic analyses revealed that this enhancement was the result of the removal of some cell wall associated proteins leading to a higher binding affinity without significant changes in binding site numbers. The albumin-binding proteins could be readily solu-bilized and purified by affinity chromatography. Upon gel electrophoresis the molecular mass of the albumin-binding proteins was estimated as 130 kilodaltons.Key words: Peptostreptococcus magnus, albumin binding, trypsin enhancement.

scholarly journals Partial characterization of a binding site for von Willebrand factor on glycocalicin

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 19-26 ◽  
Author(s):  
AD Michelson ◽  
J Loscalzo ◽  
B Melnick ◽  
BS Coller ◽  
RI Handin
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Binding Activity ◽  
Proteolytic Fragment ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

The binding of von Willebrand factor (vWF) to platelet membrane glycoprotein Ib (GpIb) facilitates platelet adhesion to vascular subendothelium. In this study, we provide evidence that the vWF binding site is on glycocalicin (GC), a proteolytic fragment of GpIb, and we examine the role of the carbohydrate portion of GC on that binding. The binding to platelets of 6D1, a monoclonal antibody that recognizes an epitope on GpIb and blocks ristocetin-induced vWF binding to platelets, was inhibited by purified GC. In addition, purified GC inhibited ristocetin-dependent binding of 125I-labeled vWF to platelets. Since GC contains 60% carbohydrate by weight, we assessed the role of carbohydrate sequences on its interaction with antibody 6D1 and vWF. Based on the known sequence of the major oligosaccharide chain of GC--N- acetyl neuraminic acid, galactose, N-acetyl glucosamine, N-acetyl galactosamine--we treated GC sequentially with neuraminidase, beta- galactosidase, and beta-N-acetylglucosaminidase. Removal of sialic acid and galactose residues did not affect GC binding. Removal of N-acetyl glucosamine residues did not affect GC binding to 6D1 but did decrease the ability of GC to inhibit vWF binding to platelets, increasing the concentration needed to inhibit binding by 50% (IC50) 40-fold. This suggests that a portion of the oligosaccharide chains on GC contributes to the vWF binding activity of this molecule.

scholarly journals Characterization of a telomere-binding protein from Physarum polycephalum.

1991 ◽  
Vol 11 (4) ◽  
pp. 2282-2290 ◽  
Author(s):  
J S Coren ◽  
E M Epstein ◽  
V M Vogt
Keyword(s):  
Physarum Polycephalum ◽  
Nuclear Protein ◽  
Binding Activity ◽  
Binding Assays ◽  
Mobility Shift ◽  
Heat Stable ◽  
Dnase I Footprinting ◽  
Telomere Binding Protein ◽  
Gel Mobility Shift

We have partially purified a nuclear protein (PPT) from Physarum polycephalum that binds to the extrachromosomal ribosomal DNA telomeres of this acellular slime mold. Binding is specific for the (T2AG3)n telomere repeats, as evidenced by nitrocellulose filter binding assays, by gel mobility shift assays with both DNA fragments and double-stranded oligonucleotides, and by DNase I footprinting. PPT is remarkably heat stable, showing undiminished binding activity after incubation at 90 degrees C. It sediments at 1.2S, corresponding to a molecular weight of about 10,000 (for a globular protein), and its binding activity is undiminished by incubation with RNase, suggesting that it is not a ribonucleoprotein. We hypothesize that PPT plays a structural role in telomeres, perhaps preventing nucleolytic degradation or promoting telomere extension by a telomere-specific terminal transferase.

scholarly journals Partial characterization of a binding site for von Willebrand factor on glycocalicin

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 19-26 ◽  
Author(s):  
AD Michelson ◽  
J Loscalzo ◽  
B Melnick ◽  
BS Coller ◽  
RI Handin
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Binding Activity ◽  
Proteolytic Fragment ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

Abstract The binding of von Willebrand factor (vWF) to platelet membrane glycoprotein Ib (GpIb) facilitates platelet adhesion to vascular subendothelium. In this study, we provide evidence that the vWF binding site is on glycocalicin (GC), a proteolytic fragment of GpIb, and we examine the role of the carbohydrate portion of GC on that binding. The binding to platelets of 6D1, a monoclonal antibody that recognizes an epitope on GpIb and blocks ristocetin-induced vWF binding to platelets, was inhibited by purified GC. In addition, purified GC inhibited ristocetin-dependent binding of 125I-labeled vWF to platelets. Since GC contains 60% carbohydrate by weight, we assessed the role of carbohydrate sequences on its interaction with antibody 6D1 and vWF. Based on the known sequence of the major oligosaccharide chain of GC--N- acetyl neuraminic acid, galactose, N-acetyl glucosamine, N-acetyl galactosamine--we treated GC sequentially with neuraminidase, beta- galactosidase, and beta-N-acetylglucosaminidase. Removal of sialic acid and galactose residues did not affect GC binding. Removal of N-acetyl glucosamine residues did not affect GC binding to 6D1 but did decrease the ability of GC to inhibit vWF binding to platelets, increasing the concentration needed to inhibit binding by 50% (IC50) 40-fold. This suggests that a portion of the oligosaccharide chains on GC contributes to the vWF binding activity of this molecule.

scholarly journals Phenotypical Characterization of the Nuclear Egress of Recombinant Cytomegaloviruses Reveals Defective Replication upon ORF-UL50 Deletion but Not pUL50 Phosphosite Mutation

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 165
Author(s):  
Sigrun Häge ◽  
Eric Sonntag ◽  
Adriana Svrlanska ◽  
Eva Maria Borst ◽  
Anne-Charlotte Stilp ◽  
...  
Keyword(s):  
Protein Production ◽  
Confocal Imaging ◽  
Wild Type ◽  
Binding Properties ◽  
Nuclear Egress ◽  
Associated Proteins ◽  
Binding Groove ◽  
Rate Limiting ◽  
Late Stages

Nuclear egress is a common herpesviral process regulating nucleocytoplasmic capsid release. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that regulates multicomponent assembly with NEC-associated proteins and capsids. Recently, NEC crystal structures were resolved for α-, β- and γ-herpesviruses, revealing profound structural conservation, which was not mirrored, however, by primary sequence and binding properties. The NEC binding principle is based on hook-into-groove interaction through an N-terminal hook-like pUL53 protrusion that embraces an α-helical pUL50 binding groove. So far, pUL50 has been considered as the major kinase-interacting determinant and massive phosphorylation of pUL50-pUL53 was assigned to NEC formation and functionality. Here, we addressed the question of phenotypical changes of ORF-UL50-mutated HCMVs. Surprisingly, our analyses did not detect a predominant replication defect for most of these viral mutants, concerning parameters of replication kinetics (qPCR), viral protein production (Western blot/CoIP) and capsid egress (confocal imaging/EM). Specifically, only the ORF-UL50 deletion rescue virus showed a block of genome synthesis during late stages of infection, whereas all phosphosite mutants exhibited marginal differences compared to wild-type or revertants. These results (i) emphasize a rate-limiting function of pUL50 for nuclear egress, and (ii) demonstrate that mutations in all mapped pUL50 phosphosites may be largely compensated. A refined mechanistic concept points to a multifaceted nuclear egress regulation, for which the dependence on the expression and phosphorylation of pUL50 is discussed.

Characterization of a telomere-binding protein from Physarum polycephalum

1991 ◽  
Vol 11 (4) ◽  
pp. 2282-2290
Author(s):  
J S Coren ◽  
E M Epstein ◽  
V M Vogt
Keyword(s):  
Physarum Polycephalum ◽  
Nuclear Protein ◽  
Binding Activity ◽  
Binding Assays ◽  
Mobility Shift ◽  
Heat Stable ◽  
Dnase I Footprinting ◽  
Telomere Binding Protein ◽  
Gel Mobility Shift

We have partially purified a nuclear protein (PPT) from Physarum polycephalum that binds to the extrachromosomal ribosomal DNA telomeres of this acellular slime mold. Binding is specific for the (T2AG3)n telomere repeats, as evidenced by nitrocellulose filter binding assays, by gel mobility shift assays with both DNA fragments and double-stranded oligonucleotides, and by DNase I footprinting. PPT is remarkably heat stable, showing undiminished binding activity after incubation at 90 degrees C. It sediments at 1.2S, corresponding to a molecular weight of about 10,000 (for a globular protein), and its binding activity is undiminished by incubation with RNase, suggesting that it is not a ribonucleoprotein. We hypothesize that PPT plays a structural role in telomeres, perhaps preventing nucleolytic degradation or promoting telomere extension by a telomere-specific terminal transferase.

scholarly journals A Plant snoRNP Complex Containing snoRNAs, Fibrillarin, and Nucleolin-Like Proteins Is Competent for both rRNA Gene Binding and Pre-rRNA Processing In Vitro

2004 ◽  
Vol 24 (16) ◽  
pp. 7284-7297 ◽  
Author(s):  
Julio Sáez-Vasquez ◽  
David Caparros-Ruiz ◽  
Fredy Barneche ◽  
Manuel Echeverría
Keyword(s):  
Cleavage Site ◽  
Functional Characterization ◽  
Binding Activity ◽  
Rrna Gene ◽  
External Transcribed Spacer ◽  
Associated Proteins ◽  
Primary Cleavage

ABSTRACT In eukaryotes the primary cleavage of the precursor rRNA (pre-rRNA) occurs in the 5′ external transcribed spacer (5′ETS). In Saccharomyces cerevisiae and animals this cleavage depends on a conserved U3 small nucleolar ribonucleoprotein particle (snoRNP), including fibrillarin, and on other transiently associated proteins such as nucleolin. This large complex can be visualized by electron microscopy bound to the nascent pre-rRNA soon after initiation of transcription. Our group previously described a radish rRNA gene binding activity, NF D, that specifically binds to a cluster of conserved motifs preceding the primary cleavage site in the 5′ETS of crucifer plants including radish, cauliflower, and Arabidopsis thaliana (D. Caparros-Ruiz, S. Lahmy, S. Piersanti, and M. Echeverria, Eur. J. Biochem. 247:981-989, 1997). Here we report the purification and functional characterization of NF D from cauliflower inflorescences. Remarkably NF D also binds to 5′ETS RNA and accurately cleaves it at the primary cleavage site mapped in vivo. NF D is a multiprotein factor of 600 kDa that dissociates into smaller complexes. Two polypeptides of NF D identified by microsequencing are homologues of nucleolin and fibrillarin. The conserved U3 and U14 snoRNAs associated with fibrillarin and required for early pre-rRNA cleavages are also found in NF D. Based on this it is proposed that NF D is a processing complex that assembles on the rDNA prior to its interaction with the nascent pre-rRNA.

Characterization of Tescalcin, a Novel EF-Hand Protein with a Single Ca2+-Binding Site:  Metal-Binding Properties, Localization in Tissues and Cells, and Effect on Calcineurin†

Biochemistry ◽  
2003 ◽  
Vol 42 (49) ◽  
pp. 14553-14565 ◽  
Author(s):  
Christina Gutierrez-Ford ◽  
Konstantin Levay ◽  
Aldrin V. Gomes ◽  
Erasmo M. Perera ◽  
Tapan Som ◽  
...  
Keyword(s):  
Binding Site ◽  
Metal Binding ◽  
Binding Properties ◽  
Ef Hand
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