scholarly journals Cytochalasin B slows but does not prevent monomer addition at the barbed end of the actin filament.

1986 ◽  
Vol 102 (1) ◽  
pp. 282-288 ◽  
Author(s):  
E M Bonder ◽  
M S Mooseker
Keyword(s):  
Actin Filament ◽  
Rate Constants ◽  
Actin Filaments ◽  
Time Course ◽  
Cytochalasin B ◽  
Critical Concentration ◽  
Dissociation Rate ◽  
Experimental Conditions ◽  
Dissociation Rate Constants ◽  
Pointed End

We used Limulus sperm acrosomal actin bundles to examine the effect of 2 microM cytochalasin B (CB) on elongation from both the barbed and pointed ends of the actin filament. In this paper we report that 2 microM CB does not prevent monomer addition onto the barbed ends of the acrosomal actin filaments. Barbed end assembly occurred over a range of actin monomer concentrations (0.2-6 microM) in solutions containing 75 mM KCl, 5 mM MgCl2, 10 mM Imidazole, pH 7.2, and 2 microM CB. However, the elongation rates were reduced such that the rates at the barbed end were approximately the same as those at the pointed end. The association and dissociation rate constants were 8- to 10-fold smaller at the barbed end in the presence of CB along with an accompanying twofold increase in critical concentration at that end. Over the time course of experimentation there was little evidence for potentiation by CB of the nucleation step of assembly. CB did not sever actin filaments; instead its presence increased the susceptibility of actin filaments to breakage from the gentle shear forces incurred during sample preparation. Under these experimental conditions, the assembly rate constants and critical concentration at the pointed end were the same in both the presence and the absence of CB.

scholarly journals Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores.

1981 ◽  
Vol 88 (3) ◽  
pp. 654-659 ◽  
Author(s):  
T D Pollard ◽  
M S Mooseker
Keyword(s):  
Electron Microscopy ◽  
Growth Rates ◽  
Rate Constants ◽  
Actin Polymerization ◽  
Cytochalasin B ◽  
Monomer Concentration ◽  
Dissociation Rate ◽  
Filament Bundles ◽  
Dissociation Rate Constants ◽  
Pointed End

We used actin filament bundles isolated from intestinal brush-border microvilli to nucleate the polymerization of pure muscle actin monomers into filaments. Growth rates were determined by electron microscopy by measuring the change in the length of the filaments as a function of time. The linear dependence of the growth rates on the actin monomer concentration provided the rate constants for monomer association and dissociation at the two ends of the growing filament. The rapidly growing ("barbed") end has higher association and dissociation rate constants than the slowly growing ("pointed") end. The values of these rate constants differ in 20 mM KCl compared with 75 mM KCl, 5 mM MgSO4. 2 microM cytochalasin B blocks growth entirely at the barbed end, apparently by reducing both association and dissociation rate constants to near zero, but inhibits growth at the pointed end to only a small extent.

scholarly journals Phalloidin enhances actin assembly by preventing monomer dissociation.

1984 ◽  
Vol 99 (2) ◽  
pp. 529-535 ◽  
Author(s):  
L M Coluccio ◽  
L G Tilney
Keyword(s):  
Rate Constant ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Dissociation Rate ◽  
Dissociation Rate Constant ◽  
Muscle Actin ◽  
Actin Assembly ◽  
Association Rate Constant ◽  
Association Rate ◽  
Dissociation Rate Constants

Incubation of the isolated acrosomal bundles of Limulus sperm with skeletal muscle actin results in assembly of actin onto both ends of the bundles. These cross-linked bundles of actin filaments taper, thus allowing one to distinguish directly the preferred end for actin assembly from the nonpreferred end; the preferred end is thinner. Incubation with actin in the presence of equimolar phalloidin in 100 mM KCl, 1 mM MgCl2 and 0.5 mM ATP at pH 7.5 resulted in a slightly smaller association rate constant at the preferred end than in the absence of the drug (3.36 +/- 0.14 X 10(6) M-1 s-1 vs. 2.63 +/- 0.22 X 10(6) M-1 s-1, control vs. experimental). In the presence of phalloidin, the dissociation rate constant at the preferred end was reduced from 0.317 +/- 0.097 s-1 to essentially zero. Consequently, the critical concentration at the preferred end dropped from 0.10 microM to zero in the presence of the drug. There was no detectable change in the rate constant of association at the nonpreferred end in the presence of phalloidin (0.256 +/- 0.015 X 10(6) M-1 s-1 vs. 0.256 +/- 0.043 X 10(6) M-1 s-1, control vs. experimental); however, the dissociation rate constant was reduced from 0.269 +/- 0.043 s-1 to essentially zero. Thus, the critical concentration at the nonpreferred end changed from 1.02 microM to zero in the presence of phalloidin. Dilution-induced depolymerization at both the preferred and nonpreferred ends was prevented in the presence of phalloidin. Thus, phalloidin enhances actin assembly by lowering the critical concentration at both ends of actin filaments, a consequence of reducing the dissociation rate constants at each end.

scholarly journals Kinetics and thermodynamics of the binding of forskolin to the galactose-H+ transport protein, GalP, of Escherichia coli

1995 ◽  
Vol 308 (1) ◽  
pp. 261-268 ◽  
Author(s):  
G E M Martin ◽  
N G Rutherford ◽  
P J F Henderson ◽  
A R Walmsley
Keyword(s):  
Escherichia Coli ◽  
Rate Constants ◽  
Binding Sites ◽  
Cytochalasin B ◽  
Equilibrium Dialysis ◽  
Dissociation Rate ◽  
Protein Fluorescence ◽  
Time Resolved ◽  
Fluorescence Measurements ◽  
Dissociation Rate Constants

The binding of the transport inhibitor, forskolin, to the galactose-H+ symporter, GalP, of Escherichia coli was evaluated by equilibrium and time-resolved fluorescence measurements. A quench in protein fluorescence of 8-12% was observed upon the binding of forskolin. The overall dissociation constant (Kd) for forskolin determined by fluorescence titration ranged between 1.2 and 2.2 microM, which is similar to that reported from equilibrium dialysis measurements of the binding of [3H]forskolin (Kd = 0.9-1.4 microM). The kinetics of forskolin binding were measured by stopped-flow fluorescence methods. The protein fluorescence was quenched in a biphasic manner; the faster of these two rates was dependent on the concentration of forskolin and was interpreted as the initial binding step from which both the association (kon) and dissociation (koff) rate constants were determined. The association and dissociation rate constants were 5.4-6.2 microM-1.s-1 and 5.1-11.5 s-1 respectively, and the Kd was calculated to be 1.5 microM. The binding of forskolin was inhibited by D-galactose, but not by L-galactose, and displacement by sugar provided an additional method to calculate the dissociation rate constant for forskolin (koff = 12.4-13.0 s-1). The rate of the slow change in protein fluorescence (3-5 s-1) was independent of the forskolin concentration, indicating an isomerization of the transporter between different conformations, possibly outward- and inward-facing forms. These kinetic parameters were determined at a series of temperatures, so that the thermodynamics of forskolin binding and transporter re-orientation could be analysed. The binding process was entropically driven (delta S = 83.7 J.K-1.mol-1; delta H = 8.25 kJ.mol-1), similar to that for cytochalasin B, which is also an inhibitor of GalP. Measurements of the binding of [3H]forskolin by equilibrium dialysis revealed competitive displacement of bound forskolin by cytochalasin B, possibly suggesting that the sugar, forskolin and cytochalasin B binding sites are overlapping; the Kds for forskolin and cytochalasin B were calculated to be 0.85 microM and 4.77 microM respectively, and the concentration of binding sites was 10.2 nmol.mg-1.

scholarly journals Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments.

1986 ◽  
Vol 103 (6) ◽  
pp. 2747-2754 ◽  
Author(s):  
T D Pollard
Keyword(s):  
Growth Rates ◽  
Rate Constants ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Detectable Effect ◽  
Type Model ◽  
Phase Dependence ◽  
Model Account ◽  
Wide Range ◽  
Pointed End

I measured the rate of elongation at the barbed and pointed ends of actin filaments by electron microscopy with Limulus sperm acrosomal processes as nuclei. With improvements in the mechanics of the assay, it was possible to measure growth rates from 0.05 to 280 s-1. At 22 degrees C in 1 mM MgCl2, 10 mM imidazole (pH 7), 0.2 mM ATP with 1 mM EGTA or 50 microM CaCl2 or with EGTA and 50 mM KCl, the elongation rates at both ends have a linear dependence on the ATP-actin concentration from the critical concentration to 20 microM. Consequently, over a wide range of subunit addition rates, the rate constants for association and dissociation of ATP-actin are constant. This shows that the nucleotide composition at or near the end of the growing filament is either the same over this range of growth rates or has no detectable effect on the rate constants. Under conditions where polymerization is fastest (MgCl2 + KCl + EGTA) the rate constants have these values: (table; see text) Compared with ATP-actin, ADP-actin associates slower at both ends, dissociates faster from the barbed end, but dissociates slower from the pointed end. Taking into account the events at both ends, these constants and a simple Oosawa-type model account for the complex three-phase dependence of the rate of polymerization in bulk samples on the concentration of ATP-actin monomers observed by Carlier, M.-F., D. Pantaloni, and E. D. Korn (1985, J. Biol. Chem., 260:6565-6571). These constants can also be used to predict the reactions at steady state in ATP. There will be slow subunit flux from the barbed end to the pointed end. There will also be minor fluctuations in length at the barbed end due to occasional rapid dissociation of strings of ADP subunits but the pointed end will be relatively stable.

scholarly journals Dissociation Rate Constants of Human Fibronectin Binding to Fibronectin-binding Proteins on LivingStaphylococcus aureusIsolated from Clinical Patients

2012 ◽  
Vol 287 (9) ◽  
pp. 6693-6701 ◽  
Author(s):  
Nadia N. Casillas-Ituarte ◽  
Brian H. Lower ◽  
Supaporn Lamlertthon ◽  
Vance G. Fowler ◽  
Steven K. Lower
Keyword(s):  
Rate Constants ◽  
Binding Proteins ◽  
Dissociation Rate ◽  
Human Fibronectin ◽  
Fibronectin Binding ◽  
Dissociation Rate Constants

scholarly journals Stimulus-response uncoupling in the neutrophil. Adenosine A2-receptor occupancy inhibits the sustained, but not the early, events of stimulus transduction in human neutrophils by a mechanism independent of actin-filament formation

1992 ◽  
Vol 281 (3) ◽  
pp. 631-635 ◽  
Author(s):  
B N Cronstein ◽  
K A Haines
Keyword(s):  
Actin Filament ◽  
Adenosine Receptor ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
Receptor Occupancy ◽  
Neutrophil Activation ◽  
Filament Formation ◽  
Stimulus Response ◽  
Adenosine A2 Receptor ◽  
A2 Receptors

Generation of superoxide anion (O2-) in response to occupancy of neutrophil chemoattractant receptors requires both early events (‘triggering’) and sustained signals (‘activation’). We have previously demonstrated that occupancy of adenosine A2 receptors inhibits O2- generation by neutrophils. In parallel, adenosine-receptor occupancy promotes association of bound N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors with the cytoskeleton, a process associated with termination of neutrophil activation (stimulus-response uncoupling). We undertook this study to determine whether inhibition of neutrophil function by adenosine-receptor occupancy requires intact actin filaments and to examine the effect of adenosine-receptor occupancy on the stimulated generation of intracellular signals involved in neutrophil triggering and activation. Occupancy of adenosine A2 receptors by 5′-N-ethylcarboxamidoadenosine (NECA, 1 microM) significantly increased (130 +/- 1% of control, P less than 0.001, n = 3) association of [3H]fMLP with cytoskeletal preparations. Cytochalasin B (5 micrograms/ml), an agent which disrupts actin filaments, completely blocked association of [3H]fMLP with cytoskeletal preparations, as previously reported. However, NECA markedly increased association of [3H]fMLP with the cytoskeleton even in the presence of cytochalasin B (P less than 0.0002). Moreover, NECA did not significantly affect either the early (30s) or the late (5 min) formation of actin filaments after stimulation by chemoattractant (fMLP, 0.1-100 nM). Cytochalasin B markedly inhibited actin-filament formation by stimulated neutrophils, and NECA did not reverse the effect of cytochalasin B on actin-filament formation. Adenosine-receptor occupancy did not affect the rapid peak in diacylglycerol generation (less than or equal to 15 s) from either [3H]arachidonate- or [14C]glycerol-labelled phospholipid pools. However, as would be predicted if occupancy of the adenosine receptor was a signal for early termination of cell activation, NECA (1 microM) markedly diminished the slow sustained generation of diacylglycerol. These results suggest that adenosine-A2-receptor occupancy does not affect triggering of the neutrophil, but that occupancy of adenosine receptors is an early signal for the termination of neutrophil activation, i.e. the ‘premature’ finish of signal transduction. Moreover, these data indicate that at least two pathways are available for increasing the association of ligated chemoattractant receptors with the cytoskeleton of neutrophils: F-actin-dependent and -independent.

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