Phalloidin enhances actin assembly by preventing monomer dissociation.

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.

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Polymerization of ADP-actin.

The Journal of Cell Biology ◽

10.1083/jcb.99.3.769 ◽

1984 ◽

Vol 99 (3) ◽

pp. 769-777 ◽

Cited By ~ 165

Author(s):

T D Pollard

Keyword(s):

Steady State ◽

Rate Constant ◽

Rate Constants ◽

Actin Filaments ◽

Dissociation Rate ◽

Elongation Rate ◽

Dissociation Rate Constant ◽

Association Rate Constant ◽

Association Rate ◽

Critical Concentrations

Using hexokinase, glucose, and ATP to vary reversibly the concentrations of ADP and ATP in solution and bound to Acanthamoeba actin, I measured the relative critical concentrations and elongation rate constants for ATP-actin and ADP-actin in 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.1 mM nucleotide, 0.1 mM CaCl2, 10 mM imidazole, pH 7. By both steady-state and elongation rate methods, the critical concentrations are 0.1 microM for ATP-actin and 5 microM for ADP-actin. Consequently, a 5 microM solution of actin can be polymerized, depolymerized, and repolymerized by simply cycling from ATP to ADP and back to ATP. The critical concentrations differ, because the association rate constant is 10 times higher and the dissociation rate constant is five times lower for ATP-actin than ADP-actin. These results show that ATP-actin occupies both ends of actin filaments growing in ATP. The bound ATP must be split on internal subunits and the number of terminal subunits with bound ATP probably depends on the rate of growth.

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The rates of formation and dissociation of actin-myosin complexes. Effects of solvent, temperature, nucleotide binding and head-head interactions

Biochemical Journal ◽

10.1042/bj2030453 ◽

1982 ◽

Vol 203 (2) ◽

pp. 453-460 ◽

Cited By ~ 47

Author(s):

S B Marston

Keyword(s):

Rate Constant ◽

Protein Concentration ◽

Order Rate Constant ◽

Dissociation Rate ◽

Dissociation Rate Constant ◽

Light Scatter ◽

Association Rate Constant ◽

Heavy Meromyosin ◽

Association Rate ◽

Low Protein

The rates of formation and dissociation of actin-subfragment 1 and actin-heavy mero-myosin complexes were measured by using light-scatter and the change in fluorescence of N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine (IAEDANS)-labelled acting as probes. Association rate measurements were made at low protein concentration, where the transients approximated to single exponentials with rate constants proportional to the concentration of reactant in excess. Dissociation rate measurements were made by displacing IAEDANS-actin from myosin with excess native actin and by a salt jump. The second-order rate constant of association for actin-subfragment 1 was 3 × 10(6) M-1 . s-1 in 60 mM-KCl at 13 degree C. It was decreased 10-fold in 500 mM-KCl and in 50% (v/v) glycol. It was decreased 6-fold when MgADP or Mg[beta gamma-imido]ATP bound to myosin. The dissociation rate constant was 0.012 s-1 in 60 mM-KCl at 13 degree C. It was increased 4-fold by 500 mM-KCl, 25-fold by 50% glycol, 8-fold by MgADP binding and 170-fold by Mg[beta gamma-imido]ATP binding. Ea for association was 70 kJ . mol-1 and for dissociation 35 kJ . mol-1. Heavy meromyosin associated at twice the rate observed for subfragment 1 and dissociated at less than one-twentieth of the rate for subfragment 1 (60 mM-KCl, 25 degree C), but when Mg[beta gamma-imido]ATP bound actin-heavy meromyosin dissociated at one-half the rate for subfragment 1. There were significant correlations between increase in the dissociation rate constant, decrease in binding constant and increase in magnitude of conformational change. The association rate constant did not correlate with any property of the actin-myosin complex.

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Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes

Biochemical Journal ◽

10.1042/bj2990219 ◽

1994 ◽

Vol 299 (1) ◽

pp. 219-225 ◽

Cited By ~ 59

Author(s):

I Björk ◽

E Pol ◽

E Raub-Segall ◽

M Abrahamson ◽

A D Rowan ◽

...

Keyword(s):

Rate Constant ◽

Cystatin C ◽

Rate Constants ◽

Dissociation Rate ◽

Dissociation Rate Constant ◽

Association Rate Constant ◽

Association Rate ◽

Human Cystatin C ◽

Human Cystatin ◽

Chicken Cystatin

The importance of the N-terminal region of human cystatin C or chicken cystatin for the kinetics of interactions of the inhibitors with four cysteine proteinases was characterized. The association rate constants for the binding of recombinant human cystatin C to papain, ficin, actinidin and recombinant rat cathepsin B were 1.1 x 10(7), 7.0 x 10(6), 2.4 x 10(6) and 1.4 x 10(6) M-1.s-1, whereas the corresponding dissociation rate constants were 1.3 x 10(-7), 9.2 x 10(-6), 4.6 x 10(-2) and 3.5 x 10(-4) s-1. N-Terminal truncation of the first ten residues of the inhibitor negligibly affected the association rate constant with papain or ficin, but increased the dissociation rate constant approx. 3 x 10(4)- to 2 x 10(6)-fold. In contrast, such truncation decreased the association rate constant with cathepsin B approx. 60-fold, while minimally affecting the dissociation rate constant. With actinidin, the truncated cystatin C had both an approx. 15-fold lower association rate constant and an approx. 15-fold higher dissociation rate constant than the intact inhibitor. Similar results were obtained for intact and N-terminally truncated chicken cystatin. The decreased affinity of human cystatin C or chicken cystatin for cysteine proteinases after removal of the N-terminal region is thus due to either a decreased association rate constant or an increased dissociation rate constant, or both, depending on the enzyme. This behaviour indicates that the contribution of the N-terminal segment of the two inhibitors to the interaction mechanism varies with the target proteinase as a result of structural differences in the active-site region of the enzyme.

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Hemoglobin in Frankia, a Nitrogen-Fixing Actinomycete

Applied and Environmental Microbiology ◽

10.1128/aem.68.5.2629-2631.2002 ◽

2002 ◽

Vol 68 (5) ◽

pp. 2629-2631 ◽

Cited By ~ 19

Author(s):

John D. Tjepkema ◽

Robert E. Cashon ◽

Jason Beckwith ◽

Christa R. Schwintzer

Keyword(s):

Optical Absorption ◽

Rate Constant ◽

Dissociation Rate ◽

Oxygen Binding ◽

Association Rate Constant ◽

Association Rate ◽

Absorption Bands ◽

Oxygen Dissociation ◽

Equilibrium Oxygen ◽

Strain Cci3

ABSTRACT Frankia strain CcI3 grown in culture produced a hemoglobin which had optical absorption bands typical of a hemoglobin and a molecular mass of 14.1 kDa. Its equilibrium oxygen binding constant was 274 nM, the oxygen dissociation rate constant was 56 s−1, and the oxygen association rate constant was 206 μM−1 s−1.

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

The Journal of Cell Biology ◽

10.1083/jcb.102.1.282 ◽

1986 ◽

Vol 102 (1) ◽

pp. 282-288 ◽

Cited By ~ 60

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.

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Measurement of the dissociation rate constant of antigen/antibody complexes in solution by enzyme-linked immunosorbent assay

Journal of Immunological Methods ◽

10.1016/0022-1759(94)90392-1 ◽

1994 ◽

Vol 170 (2) ◽

pp. 167-175 ◽

Cited By ~ 21

Author(s):

Marie-Pierre Larvor ◽

Lisa Djavadi-Ohaniance ◽

Barry Nall ◽

Michel E. Goldberg

Keyword(s):

Rate Constant ◽

Immunosorbent Assay ◽

Dissociation Rate ◽

Dissociation Rate Constant ◽

Enzyme Linked Immunosorbent Assay ◽

Antigen Antibody

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Velocity of O2 uptake by human red blood cells

Journal of Applied Physiology ◽

10.1152/jappl.1961.16.3.511 ◽

1961 ◽

Vol 16 (3) ◽

pp. 511-516 ◽

Cited By ~ 51

Author(s):

N. C. Staub ◽

J. M. Bishop ◽

R. E. Forster

Keyword(s):

Rate Constant ◽

Intermediate Compound ◽

Oxygen Electrode ◽

Theoretical Explanation ◽

Association Rate Constant ◽

Association Rate ◽

Human Red Blood Cells ◽

Hemoglobin Saturation ◽

Uptake Process ◽

Normal Human

We have determined the over-all association rate constant, k'c, for the uptake of oxygen by normal human erythrocytes from 0% to 97% initial hemoglobin saturation at pH 7.4 and 37óC. With a modified Hartridge-Roughton rapid-reaction apparatus, we used a small oxygen electrode to follow the oxygen uptake process rather than using the usual photocolorimetric analytical methods. The value of k'c averages 164 mm-1 sec.-1 at 0% initial saturation, rises slowly to over 300 at 50%, and then climbs rapidly to over 1500 at 95%. The theoretical explanation for the increase in k-c with rising initial oxyhemoglobin saturation is based on the increasing prominence of the fourth chemical association rate constant, k-4, in the intermediate compound theory of Adair. It is known that k-4 is several times larger than any of the other three k–s. Submitted on December 19, 1960

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Protein-ligand dissociation rate constant from all-atom simulation

10.21203/rs.3.rs-816562/v1 ◽

2021 ◽

Author(s):

Ekaterina Maximova ◽

Eugene Postnikov ◽

Anastasia Lavrova ◽

Vladimir Farafonov ◽

Dmitry Nerukh

Keyword(s):

Molecular Dynamics ◽

Rate Constant ◽

Md Simulations ◽

Dissociation Rate ◽

Dissociation Rate Constant ◽

Zero Force ◽

Ligand Dissociation ◽

Random Acceleration

Abstract Dissociation of a ligand isoniazid from a protein catalase was investigated using all-atom Molecular Dynamics (MD) simulations. Random Acceleration MD (τ-RAMD) was used where a random artificial force applied to the ligand facilitates its dissociation. We have suggested an approach to extrapolate such obtained dissociation times to the zero-force limit that was never attempted before, thus allowing direct comparison with experimentally measured values. We have found that our calculated dissociation time was equal to 36.1 seconds with statistically significant values distributed in the interval 0.2-72.0 s, that quantitatively matches the experimental value of 50 ± 8 seconds despite the extrapolation over nine orders of magnitude in time.

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