Kinetic model for fluorescence microscopy experiments in disordered media with binding sites and obstacles

2018 ◽  
Vol 98 (3) ◽  
Author(s):  
V. P. Shkilev
Keyword(s):  
Kinetic Model ◽  
Fluorescence Microscopy ◽  
Binding Sites ◽  
Disordered Media

scholarly journals A kinetic model for fluorescence microscopy experiments in disordered media that contains binding sites and obstacles

2018 ◽  
Author(s):  
V.P. Shkilev
Keyword(s):  
Fluorescence Microscopy ◽  
Binding Sites ◽  
Living Cells ◽  
Disordered Media ◽  
Mutual Arrangement ◽  
Binding Properties ◽  
Disordered Lattice ◽  
Multiple Trapping ◽  
Multiple Trapping Model ◽  
Trapping Model

AbstractA model is proposed that describes the diffusion of molecules in a disordered medium with binding sites (traps) and obstacles (barriers). The equations of the model are obtained using the subordination method. As the parent process, random walks on a disordered lattice are taken, described by the random barriers model. As the leading process, the renewal process that corresponds to the multiple-trapping model is taken. Theoretical expressions are derived for the curves obtained in experiments using fluorescence microscopy (FRAP, FCS and SPT). Generalizations of the model are proposed, allowing to take into account correlations in the mutual arrangement of traps and barriers. The model can be used to find parameters characterizing the diffusion and binding properties of biomolecules in living cells.

scholarly journals Glycodendrimers: tools to explore multivalent galectin-1 interactions

2015 ◽  
Vol 11 ◽  
pp. 739-747 ◽  
Author(s):  
Jonathan M Cousin ◽  
Mary J Cloninger
Keyword(s):  
Light Scattering ◽  
Dynamic Light Scattering ◽  
Fluorescence Microscopy ◽  
Binding Sites ◽  
Carcinoma Cells ◽  
Large Excess ◽  
Human Prostate Carcinoma ◽  
Typical Function ◽  
Cellular Aggregation ◽  
Du145 Cells

Four generations of lactose-functionalized polyamidoamine (PAMAM) were employed to further the understanding of multivalent galectin-1 mediated interactions. Dynamic light scattering and fluorescence microscopy were used to study the multivalent interaction of galectin-1 with the glycodendrimers in solution, and glycodendrimers were observed to organize galectin-1 into nanoparticles. In the presence of a large excess of galectin-1, glycodendrimers nucleated galectin-1 into nanoparticles that were remarkably homologous in size (400–500 nm). To understand augmentation of oncologic cellular aggregation by galectin-1, glycodendrimers were used in cell-based assays with human prostate carcinoma cells (DU145). The results revealed that glycodendrimers provided competitive binding sites for galectin-1, which diverted galectin-1 from its typical function in cellular aggregation of DU145 cells.

scholarly journals A kinetic model for Ca2+ efflux mediated by the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1987 ◽  
Vol 245 (3) ◽  
pp. 713-721 ◽  
Author(s):  
J M McWhirter ◽  
G W Gould ◽  
J M East ◽  
A G Lee
Keyword(s):  
Experimental Data ◽  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Atpase Activity ◽  
Binding Site ◽  
Binding Sites ◽  
Adenine Nucleotides ◽  
Contraction Coupling ◽  
High Affinity

We present a model for Ca2+ efflux from vesicles of sarcoplasmic reticulum (SR). It is proposed that efflux is mediated by the Ca2+ + Mg2+-activated ATPase that is responsible for Ca2+ uptake in this system. In the normal ATPase cycle of the ATPase, phosphorylation of the ATPase is followed by a conformational change in which the Ca2+-binding sites change from being outward-facing and of high affinity to being inward-facing and of low affinity. To mediate Ca2+ efflux, it is proposed that the ATPase can adopt a conformation in which the Ca2+-binding sites are of low affinity but still outward-facing. It is shown that experimental data on the rates of Ca2+ efflux can be simulated in terms of this model, with Ca2+-binding-site affinities previously proposed to explain ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227]. Effects of Mg2+ and adenine nucleotides on efflux rates are explained. It is suggested that Ca2+ efflux from SR mediated by the ATPase could be important in excitation-contraction coupling in skeletal muscle.

scholarly journals Effect of pH on the activity of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1990 ◽  
Vol 267 (2) ◽  
pp. 423-429 ◽  
Author(s):  
F Michelangeli ◽  
J Colyer ◽  
J M East ◽  
A G Lee
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Atpase Activity ◽  
Binding Sites ◽  
Ph Dependence ◽  
Effect Of Ph ◽  
High Ph ◽  
Atp Concentration ◽  
Slow Dissociation

A kinetic model for the Ca2(+) + Mg2(+)-activated ATPase of sarcoplasmic reticulum was presented in a previous paper [Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730]. Here, that model is modified to account for the pH-dependence of ATPase activity and for the effects of Mg2+ on activity at high pH. It is shown that effects of Mg2+ on measurements of ATPase activity as a function of ATP concentration at pH 8.0 and pH 8.5 are consistent with binding of Mg2+ to the Ca2(+)-binding sites on the phosphorylated ATPase, such binding inhibiting dephosphorylation of the ATPase. It is also shown that slow dissociation of Ca2+ from the phosphorylated ATPase is consistent with the previously published model.

scholarly journals Quantitation of lectin binding sites in human colon mucins by use of peanut and wheat germ agglutinins.

1988 ◽  
Vol 36 (10) ◽  
pp. 1305-1307 ◽  
Author(s):  
C R Boland ◽  
J A Roberts
Keyword(s):  
Colon Cancer ◽  
Fluorescence Microscopy ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Lectin Binding ◽  
Human Colon ◽  
Peanut Agglutinin ◽  
Normal Colon ◽  
Lectin Binding Sites

We have developed a novel method for quantitation of lectin binding sites in mucins derived from colon tissues. Binding of peanut agglutinin and wheat germ agglutinin was measured in extracts from normal and malignant human colon epithelium. Binding of wheat germ agglutinin was used as an estimate of the total mucin present in the tissue extract. Peanut agglutinin was found to bind to mucin from normal colon, but at levels that may be difficult to appreciate by fluorescence microscopy. The yield of mucin extracted from colon cancer was more variable than that from normal colon, and the binding ratio of peanut agglutinin to wheat germ agglutinin was greater in extracts from tumors than in normal tissues. Our findings confirm the histological observation that peanut agglutinin binds more avidly to mucins from colon cancer than to those from normal colon. The finding of peanut agglutinin binding sites in mucins front normal colon was not expected. The quantitative technique may have detected small numbers of binding sites not readily appreciable by fluorescence microscopy. Alternatively, the chromatographic method for measuring lectin binding may be sufficiently sensitive to detect nonspecific binding of the lectin to terminal galactose residues other than the Thomsen-Friedenreich antigen.

scholarly journals A kinetic model for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1986 ◽  
Vol 237 (1) ◽  
pp. 217-227 ◽  
Author(s):  
G W Gould ◽  
J M East ◽  
R J Froud ◽  
J M McWhirter ◽  
H I Stefanova ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Binding Sites ◽  
Ph Dependence ◽  
High Affinity ◽  
Phosphorylated Form ◽  
Low Concentrations ◽  
Kinetics Of ◽  
Single Binding Site ◽  
Micromolar Range

The Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum exhibits complex kinetics of activation with respect to ATP. ATPase activity is pH-dependent, with similar pH-activity profiles at high and low concentrations of ATP. Low concentrations of Ca2+ in the micromolar range activate the ATPase, whereas activity is inhibited by Ca2+ at millimolar concentrations. The pH-dependence of this Ca2+ inhibition and the effect of the detergent C12E8 (dodecyl octaethylene glycol monoether) on Ca2+ inhibition are similar to those observed on activation by low concentrations of Ca2+. On the basis of these and other studies we present a kinetic model for the ATPase. The ATPase is postulated to exist in one of two conformations: a conformation (E1) of high affinity for Ca2+ and MgATP and a conformation (E2) of low affinity for Ca2+ and MgATP. Ca2+ binding to E2 and to the phosphorylated form E2P are equal. Proton binding at the Ca2+-binding sites in the E1 and E2 conformations explains the pH-dependence of Ca2+ effects. Binding of MgATP to the phosphorylated intermediate E1′PCa2 and to E2 modulate the rates of the transport step E1′PCa-E2′PCa2 and the return of the empty Ca2+ sites to the outside surface of the sarcoplasmic reticulum, as well as the rate of dephosphorylation of E2P. Only a single binding site for MgATP is postulated.

scholarly journals Multiple mechanisms determine the order of APC/C substrate degradation in mitosis

2014 ◽  
Vol 207 (1) ◽  
pp. 23-39 ◽  
Author(s):  
Dan Lu ◽  
Jennifer Y. Hsiao ◽  
Norman E. Davey ◽  
Vanessa A. Van Voorhis ◽  
Scott A. Foster ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Binding Sites ◽  
Spindle Assembly Checkpoint ◽  
Budding Yeast ◽  
Spindle Assembly ◽  
Yeast Cells ◽  
Anaphase Promoting Complex ◽  
Ubiquitin Protein Ligase ◽  
Substrate Degradation ◽  
Quantitative Fluorescence

The ubiquitin protein ligase anaphase-promoting complex or cyclosome (APC/C) controls mitosis by promoting ordered degradation of securin, cyclins, and other proteins. The mechanisms underlying the timing of APC/C substrate degradation are poorly understood. We explored these mechanisms using quantitative fluorescence microscopy of GFP-tagged APC/CCdc20 substrates in living budding yeast cells. Degradation of the S cyclin, Clb5, begins early in mitosis, followed 6 min later by the degradation of securin and Dbf4. Anaphase begins when less than half of securin is degraded. The spindle assembly checkpoint delays the onset of Clb5 degradation but does not influence securin degradation. Early Clb5 degradation depends on its interaction with the Cdk1–Cks1 complex and the presence of a Cdc20-binding “ABBA motif” in its N-terminal region. The degradation of securin and Dbf4 is delayed by Cdk1-dependent phosphorylation near their Cdc20-binding sites. Thus, a remarkably diverse array of mechanisms generates robust ordering of APC/CCdc20 substrate destruction.

scholarly journals Nuclear transport of single molecules

2005 ◽  
Vol 168 (2) ◽  
pp. 233-243 ◽  
Author(s):  
Ulrich Kubitscheck ◽  
David Grünwald ◽  
Andreas Hoekstra ◽  
Daniel Rohleder ◽  
Thorsten Kues ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Nuclear Pore Complex ◽  
Binding Sites ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Far Field ◽  
Permeabilized Cells ◽  
Cytoplasmic Filaments ◽  
Transport Receptor

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 ± 0.2 and 7.1 ± 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs.

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