A Monoclonal Antibody to Avidin Dissociates Quaternary Structure and Curtails Biotin Binding to Avidin and Streptavidin

Subramanian, Nithya; Subramanian, S.; Karande, AA; Adiga, P. Radhakantha

doi:10.1006/abbi.1997.0196

Cited by 6 publications

(3 citation statements)

References 39 publications

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“…A second potential mechanism for enhancing unbinding is suggested by recent studies conducted on purified proteins. When added to biotinstreptavidin complexes, two different classes of proteins have been demonstrated to have the ability to rapidly displace the streptavidin from the biotin (Subramanian et al, 1997;Morris and Raney, 1999). In both cases, the extra protein (a helicase in one study and a monoclonal antibody in the other) can be thought of as a chaperone, placing the streptavidin in a nonnative conformation that enhances unbinding.…”

Section: Discussionmentioning

confidence: 99%

Identification of Nicotinic Acetylcholine Receptor Recycling and Its Role in Maintaining Receptor Density at the Neuromuscular JunctionIn Vivo

Bruneau¹,

Sutter²,

Hume³

et al. 2005

J. Neurosci.

122

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In the CNS, receptor recycling is critical for synaptic plasticity; however, the recycling of receptors has never been observed at peripheral synapses. Using a novel imaging technique, we show here that nicotinic acetylcholine receptors (AChRs) recycle into the postsynaptic membrane of the neuromuscular junction. By sequentially labeling AChRs with biotin-bungarotoxin and streptavidin-fluorophore conjugates, we were able to distinguish recycled, preexisting, and new receptor pools at synapses in living mice. Time-lapse imaging revealed that recycled AChRs were incorporated into the synapse within hours of initial labeling, and their numbers increased with time. At fully functional synapses, AChR recycling was robust and comparable in magnitude with the insertion of newly synthesized receptors, whereas chronic synaptic activity blockade nearly abolished receptor recycling. Finally, using the same sequential labeling method, we found that acetylcholinesterase, another synaptic component, does not recycle. These results identify an activity-dependent AChRrecycling mechanism that enables the regulation of receptor density, which could lead to rapid alterations in synaptic efficacy.

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Section: Discussionmentioning

confidence: 99%

Identification of Nicotinic Acetylcholine Receptor Recycling and Its Role in Maintaining Receptor Density at the Neuromuscular JunctionIn Vivo

Bruneau¹,

Sutter²,

Hume³

et al. 2005

J. Neurosci.

122

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“…First, monoclonal antibodies are well-known to induce conformational changes in the proteins to which they bind. For instance, a monoclonal antibody to avidin is reported to not only dissociate tetrameric avidin into monomers but also to cause the avidin to release bound biotin (18). Using fluorescence resonance energy transfer (FRET), Valenzuela et al (35) report that binding of a different antibody [mAb A6 against the A site of Torpedo receptors (7)] induced the other agonist-binding site (the R-δ interface) to move about 30 Å closer to the membrane than observed for receptors without the bound antibody.…”

Section: Resultsmentioning

confidence: 99%

Novel Biotinylated Phenylarsonous Acids as Bifunctional Reagents for Spatially Close Thiols: Studies on Reduced Antibodies and the Agonist Binding Site of ReducedTorpedoNicotinic Receptors

et al. 1999

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We synthesized three novel organoarsenicals as prototype bifunctional reagents for spatially close thiols, N-(4-arsenosophenyl) hexahydro-2-oxo-(3aS,4S,6aR)-1H-thieno[3, 4-d]imidazole-4-pentamide (1), 2-[4-[(4-arsenosophenyl)amino]-1, 4-dioxobutyl] hydrazide, (3aS,4S,6aR)-hexahydro-2-oxo- 1H-thieno[3, 4-d] imidazole-4-pentanoic acid (2), and [4-[[12-[[5-[(3aS,4S, 6aR)-hexahydro-2-oxo-1H-thieno[3, 4-d]imidazol-4-yl]-1-oxopentyl]amino]-1-oxododecyl]amino]phe nyl]-arso nous acid (3) containing both biotin and arsenic with intervening varying length spacers extending from 2 to 15 A beyond biotin bound to streptavidin. Conceptually, the arsenical group can form a stable, covalent ring structure with appropriately spaced thiols and thereby anchor the reagent to a macromolecule, while biotin allows for the detection of the reagent-macromolecule complex via avidin binding. Because the alpha-subunits of all characterized nicotinic receptors contain an easily reducible disulfide bond between adjacent cysteine residues, the reduced alpha-subunit is an attractive site for labeling. Compounds 1-3 all simultaneously bound streptavidin and dithiols, and all three decreased the number of [125I]alpha-bungarotoxin-binding sites in reduced Torpedo nicotinic receptors (IC50s 10-300 nM). Moreover, arsenylation of the receptors prevented their reoxidation with dithio-bis(nitrobenzoic acid), was reversible with 2,3-dimercaptopropanesulfonic acid, and protected the receptor from irreversible alkylation by bromoacetylcholine. However, in no case did 1-3 allow simultaneous binding to reduced nicotinic receptors and to [125I]streptavidin, although 3 alone allowed simultaneous labeling of a spatially close dithiol located in reduced antibodies.

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“…314 Others already reported that special macromolecules can reduce the affinity of avidin and streptavidin towards biotin. 315,316 First unbinding rate from the biotin samples, here attributed to the entanglement unbinding of quasi 2D actin network attached to lipid membranes ((0.468 ± 0.091) s −1 , averaged: 0.41 s −1 ), N=2. Red: Second unbinding rate from the biotin samples, here attributed to the dissociation of the binding complex biotinylated lipidneutravidin-biotinylated actin ((0.028 ± 0.020) s −1 , averaged: 0.014 s −1 ), N=2.…”

Section: Modelmentioning

confidence: 99%

Active and Passive Microrheology of F-Actin Membrane Composites

Nöding¹

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The complex mechanical properties of a living cell are not only a function of its structural components but also of the organizational super-structure and the dynamic interconnection of filaments, proteins and the plasma membrane. Especially, the F-actin cortex plays a central role in cell adhesion, migration, division, growth and differentiation. Abnormalities in these essential cellular processes are tightly connected to diseases such as cancer and malaria. It is thus of pivotal interest to study the determinants of cellular mechanics.The main part of this thesis is dedicated to the investigation of a minimal cortex model of the F-actin cortex. A pre-polymerized network of semi-flexible F-actin polymers is attached to a lipid bilayer by the physiological cross-linker ezrin. A pseudophosphorylated mutant of ezrin, T567D, is used to study the impact of transient membrane linkage on the frequency dependent viscoelastic properties. Here, passive as well as active microrheological measurements are established on these thin composite materials and frequency spectra ranging from 10 -3 -10 2 Hz are measured. In an intermediate frequency regime (10 -2 -10 1 Hz) the elastic properties dominate the force response of the model system. The stiffness of the system is dominated by the mesh size of the self-organized model cortex, which is in turn a function of the availability of pinning-points in the membrane. These findings suggest the formation of an affine network. The low frequency regime (10 -3 -10 -2 Hz) of the shear modulus is dominated by the transient binding kinetic of the membrane cross-link ezrin. An apparent unbinding rate constant is determined from the microrheological data and the transient nature of the membrane attachment is supported by a low energy barrier for the unbinding. For the high frequency regime (10 1 -10 2 Hz) deviations from the typically found power law scaling of ¾ are observed and discussed in the context of increased inertia upon F-actin attachment to a solid supported model membrane. The minimal actin cortex model is compared to entangled networks of the semi-dilute F-actin filament as well as F-actin membrane composites isolated from the apical cortex of living cells by the sandwich cleavage method.In the second part of this thesis, the focus is set on mechanotransduction and rigidity sensing in epithelial monolayers. Cell mechanics in response to surface elasticity are studied by active atomic force microscopy based microrheology and interpreted in terms of (active) soft glassy rheology. Epithelial cells from the kidney (MDCK II) and mammary gland (MCF-10A) are studied as a function of substrate elasticity (E=1-100 kPa). Cells cultured on soft substrates (1 kPa) exhibit similar frequency dependent viscoelastic properties as cells, which are F-actin depleted by latrunculin A. Both cell lines behave stiffer when cultured on surfaces of higher elasticity. Above a certain threshold of substrate stiffness no further changes can be observed upon increase in surface stiffness. This final cortic...

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A Monoclonal Antibody to Avidin Dissociates Quaternary Structure and Curtails Biotin Binding to Avidin and Streptavidin

Cited by 6 publications

References 39 publications

Identification of Nicotinic Acetylcholine Receptor Recycling and Its Role in Maintaining Receptor Density at the Neuromuscular JunctionIn Vivo

Identification of Nicotinic Acetylcholine Receptor Recycling and Its Role in Maintaining Receptor Density at the Neuromuscular JunctionIn Vivo

Novel Biotinylated Phenylarsonous Acids as Bifunctional Reagents for Spatially Close Thiols: Studies on Reduced Antibodies and the Agonist Binding Site of ReducedTorpedoNicotinic Receptors

Active and Passive Microrheology of F-Actin Membrane Composites

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