scholarly journals Biotin binding to avidin. Oligosaccharide side chain not required for ligand association

1987 ◽  
Vol 248 (1) ◽  
pp. 167-171 ◽  
Author(s):  
Y Hiller ◽  
J M Gershoni ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Concanavalin A ◽  
Sugar Content ◽  
Binding Activity ◽  
Side Chain ◽  
Affinity Column ◽  
Binding Properties ◽  
Commercial Preparation ◽  
Oligosaccharide Moiety ◽  
Biotin Binding

A commercially available, purified preparation of avidin was found to comprise two polypeptide bands (Mr 18,000 and Mr 15,500 respectively). Both bands bound biotin as assessed by biotin overlays of protein blots. The Mr 15,500 polypeptide was found to differ from the Mr 18,000 polypeptide only in its sugar content. When the commercial preparation was applied to a concanavalin A affinity column, the glycosylated forms were retarded as expected, and homotypic nonglycosylated avidin tetramers which failed to bind selectively to the column were collected in the effluent. The biotin-binding properties of the nonglycosylated avidin were equivalent to those obtained for the native (glycosylated) avidin molecule, indicating that the oligosaccharide moiety is not essential for the binding activity.

scholarly journals Hepatitis B Virus X Protein and Simian Virus 5 V Protein Exhibit Similar UV-DDB1 Binding Properties To Mediate Distinct Activities

2003 ◽  
Vol 77 (11) ◽  
pp. 6274-6283 ◽  
Author(s):  
Olivier Leupin ◽  
Séverine Bontron ◽  
Michel Strubin
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Simian Virus ◽  
Binding Activity ◽  
X Protein ◽  
Binding Properties ◽  
V Protein ◽  
Dna Binding Activity ◽  
Simian Virus 5 ◽  
B Virus

ABSTRACT The UV-damaged DNA-binding activity protein (UV-DDB) consists of two subunits, DDB1 and DDB2, and functions in DNA repair and cell cycle regulation. The DDB1 subunit is a target for the hepatitis B virus X protein (HBx). Binding of HBx to DDB1 interferes with cell growth and viability in culture and has been implicated in the establishment of viral infection. DDB1 also interacts with the V proteins encoded by several paramyxoviruses including simian virus 5 (SV5), which prevent interferon signaling by targeting either STAT1 or STAT2 proteins for proteolysis. The role of V binding to DDB1, however, remains unclear. Here we show that the V protein of SV5 (SV5-V) and HBx exhibit strikingly similar DDB1 binding properties. Thus, SV5-V and HBx bind to DDB1 in a mutually exclusive manner, and SV5-V shares with HBx the ability to enhance the steady-state levels of DDB1 and to inhibit its association with DDB2. Yet only HBx induces cell death, and SV5-V can prevent HBx from doing so by blocking its interaction with DDB1. Binding of SV5-V to DDB1 may serve another function, since SV5-V shows a decreased ability to induce STAT1 degradation in cells expressing reduced amounts of DDB1. These findings demonstrate that HBx performs a unique function through its association with DDB1 for which SV5-V cannot substitute and suggest that SV5-V and HBx have evolved to bind DDB1 to achieve distinct functions, both by a mechanism that does not involve DDB2.

scholarly journals The Binding Properties of Dimeric and Tetrameric Concanavalin A

1973 ◽  
Vol 248 (2) ◽  
pp. 549-556
Author(s):  
Geoffrey H. McKenzie ◽  
William H. Sawyer
Keyword(s):  
Concanavalin A ◽  
Binding Properties

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor

1986 ◽  
Vol 6 (12) ◽  
pp. 4723-4733
Author(s):  
L A Chodosh ◽  
R W Carthew ◽  
P A Sharp
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Affinity Column ◽  
Dna Binding Activity ◽  
Dna Fragment

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

The yeast alpha 1 and MCM1 proteins bind a single strand of their duplex DNA recognition site

1992 ◽  
Vol 12 (8) ◽  
pp. 3573-3582
Author(s):  
E J Grayhack
Keyword(s):  
Yeast Cell ◽  
Binding Sites ◽  
Dna Recognition ◽  
Binding Activity ◽  
Single Strand ◽  
Duplex Dna ◽  
Cell Type ◽  
Recognition Sequence ◽  
Binding Properties ◽  
Constitutive Factor

The yeast cell type regulator alpha 1 cooperates with a constitutive factor, MCM1 protein, to recognize the promoter and activate transcription of several alpha-specific genes. I show here that the alpha 1 and MCM1 proteins bind specifically to one of the two strands of their recognition sequence. This single-strand-binding activity shares several characteristics with the duplex-binding properties of these proteins: (i) the MCM1 protein binds alone to single-stranded and duplex sequences of both the alpha-specific (P'Q) and a-specific (P) binding sites; (ii) the alpha 1 protein requires both the MCM1 protein and the Q sequence to bind either single-stranded or duplex DNA; (iii) the alpha 1 protein stimulates binding of the MCM1 protein to both single-stranded and duplex DNAs; and (iv) the affinities of the proteins for single-stranded and duplex DNAs are comparable.

Acid proteases from species of Mucor. III. Interaction with concanavalin A and concanavalin A Sepharose

1976 ◽  
Vol 54 (2) ◽  
pp. 120-129 ◽  
Author(s):  
W. S. Rickert ◽  
P. A. McBride-Warren
Keyword(s):  
Molecular Weight ◽  
Concanavalin A ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Affinity Column ◽  
Mucor Miehei ◽  
Highly Active ◽  
Elution Buffer ◽  
Turbidimetric Assay ◽  
Ph Range

The reaction of Mucor miehei protease with concanavalin A was followed by a turbidimetric assay in the pH range 5–8. At pH 4.0, no turbidity developed but binding of the enzyme to concanavalin A could be demonstrated by gel filtration. Two fractions of apparent molecular weight 65 000 and 52 000 were isolated, the 65 000 molecular weight species apparently representing a protomer of concanavalin A (24 000) bound to the enzyme. An analysis of the circular dichroism spectrum of this complex suggested that protomer binding results in a conformational change in the enzyme which is associated with a 30% increase in proteolytic activity.At pH 6.0, the enzyme was strongly bound to columns of concanavalin A Sepharose but could be removed by including α-methyl D-glucoside and NaCl in the elution buffer. Some column degradation occurred at room temperature but was not detectable at 4 °C where rapid elution of the enzyme resulted in a greater than 90% yield of highly active protein. Periodate-oxidized Mucor miehei protease and Mucor rennin did not react with concanavalin A and were not bound to the affinity column.

scholarly journals Pre-hybridisation: An efficient way of suppressing endogenous biotin-binding activity inherent to biotin–streptavidin detection system

2014 ◽  
Vol 406 ◽  
pp. 143-147 ◽  
Author(s):  
Raju Ahmed ◽  
Emma Spikings ◽  
Shaobo Zhou ◽  
Andrew Thompsett ◽  
Tiantian Zhang
Keyword(s):  
Detection System ◽  
Binding Activity ◽  
Biotin Binding ◽  
Endogenous Biotin

Autodisplay of an avidin with biotin-binding activity on the surface of Escherichia coli

2018 ◽  
Vol 40 (3) ◽  
pp. 591-600
Author(s):  
H. D. Pardavé-Alejandre ◽  
J. E. Alvarado-Yaah ◽  
E. N. Pompa-Mera ◽  
J. E. Muñoz-Medina ◽  
B. Sárquiz-Martínez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Activity ◽  
Biotin Binding

scholarly journals Human IgA as a heterovalent ligand: switching from the asialoglycoprotein receptor to secretory component during transport across the rat hepatocyte.

1986 ◽  
Vol 102 (3) ◽  
pp. 920-931 ◽  
Author(s):  
J M Schiff ◽  
M M Fisher ◽  
A L Jones ◽  
B J Underdown
Keyword(s):  
Secretory Component ◽  
Binding Activity ◽  
Asialoglycoprotein Receptor ◽  
Galactose Oxidase ◽  
Rat Hepatocyte ◽  
Binding Properties ◽  
Affinity Adsorption ◽  
Switching Event ◽  
Transit Times

Asialoglycoproteins are taken up by the rat liver for degradation; rat polymeric IgA is taken up via a separate receptor, secretory component (SC), for quantitative delivery to bile. There is negligible uptake of these ligands by the converse receptor, and only a low level of missorting of ligands to opposite destinations. The two pathways are not cross-inhibitable and operate independently (Schiff, J.M., M. M. Fisher, and B. J. Underdown, 1984, J. Cell Biol., 98:79-89). We report here that when human IgA is presented as a ligand in the rat, it is processed using elements of both pathways. To study this in detail, different IgA fractions were prepared using two radiolabeling methods that provide separate probes for degradation or re-secretion. Behavior of intravenously injected human polymeric IgA in the rat depended on its binding properties. If deprived of SC binding activity by affinity adsorption or by reduction and alkylation, greater than 80% of human IgA was degraded in hepatic lysosomes; radioactive catabolites were released into bile by a leupeptin-inhibitable process. If prevented from binding to the asialoglycoprotein receptor by competition or by treatment with galactose oxidase, human IgA was cleared and transported to bile directly via SC, but its uptake was about fivefold slower than rat IgA. Untreated human IgA was taken up rapidly by the asialoglycoprotein receptor, but depended on SC binding to get to bile: the proportion secreted correlated 1:1 with SC binding activity determined in vitro, and the IgA was released into bile with SC still attached. These results demonstrate that human IgA is normally heterovalent: it is first captured from blood by the asialoglycoprotein receptor, but escapes the usual fate of asialoglycoproteins by switching to SC during transport. Since the biliary transit times of native human and rat IgA are the same, it is probable that the receptor switching event occurs en route. This implies that the two receptors briefly share a common intracellular compartment.

Novel application of streptavidin-hapten derivatives as protein-tracer conjugate in competitive-type immunoassays involving biotinylated detection probes

1991 ◽  
Vol 37 (1) ◽  
pp. 58-63 ◽  
Author(s):  
M J Khosravi ◽  
R C Morton
Keyword(s):  
No Reduction ◽  
Solid Phase ◽  
Binding Activity ◽  
Fluorescence Signal ◽  
Antibody Immobilization ◽  
Aggregate Formation ◽  
Cross Link ◽  
Time Resolved ◽  
Biotin Binding ◽  
Detection Reagent

Abstract To investigate the use of streptavidin-hapten derivatives as potential protein-tracer conjugates for competitive-type immunoassays, we labeled streptavidin with cortisol and compared biotin-binding activity of the conjugates with that of unlabeled streptavidin. In this model system, streptavidin labeled with one to approximately 17 cortisol molecules retained its capability to cross-link a biotinylated protein on microtiter wells to a biotin-based general detection reagent developed for time-resolved fluorometry. Compared with unlabeled streptavidin, there was no reduction in the binding activity of the conjugate carrying as many as 2.6 cortisol molecules per molecule of streptavidin. Conjugation ratios greater than 4.4 showed a slight decrease in binding activity, presumably because of the aggregate formation evident at these labeling ratios. As expected, the conjugates were also capable of linking a solid-phase-bound anti-cortisol monoclonal antibody to the biotinylated detection reagent. The fluorescence signal generated increased almost linearly with increasing conjugation ratios from about three to nine cortisol molecules per molecule of streptavidin. At greater ratios, the assay response plateaued. The calibration curves obtained were typical for competitive-type immunoassays when the conjugates were incorporated in a cortisol assay based on a second-antibody immobilization approach.

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