Probing nucleation, cutting and capping of actin filaments.
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Actins↗
Biomedical subjects
N Selve
Publications and source records attributed to N Selve.
pH-dependent rate of formation of the gelsolin-actin complex from gelsolin and monomeric actin.
The assembly of gelsolin with actin was followed by the increase of the fluorescence intensity of a fluorescence label bound to actin. The time course of the formation of the gelsolin-actin complex in the presence of micromolar [Ca2+] could be quantitatively interpreted by a model in which one actin molecule binds slowly to gelsolin in a rate-determining step and subsequently a second actin molecule is bound at least 40 times more rapidly. The rate of binding of the first actin molecule to gelsolin was found to be remarkably slow and to depend on the pH. The rate constants of formation of the gelsolin-actin complex range from 1.5 X 10(4) M-1 s-1 at pH 8 to 7 X 10(4) M-1 s-1 at pH 6.
Actins↗
Rate constants and equilibrium constants for binding of the gelsolin-actin complex to the barbed ends of actin filaments in the presence and absence of calcium.
The equilibrium constant for binding of the gelsolin-actin complex to the barbed ends of actin filaments was measured by the depolymerizing effect of the gelsolin-actin complex on actin filaments. When the gelsolin-actin complex blocks monomer consumption at the lengthening barbed ends of treadmilling actin filaments, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. By using this effect the equilibrium constant for binding was determined to be about 1.5 X 10(10) M-1 in excess EGTA over total calcium (experimental conditions: 1 mM MgCl2, 100 mM KCl, pH 7.5, 37 degrees C). In the presence of Ca2+ the equilibrium constant was found to be in the range of or above 10(11) M-1. The rate constant of binding of the gelsolin-actin complex to the barbed ends was measured by inhibition of elongation of actin filaments. Nucleation of new filaments by the gelsolin-actin complex towards the pointed ends was prevented by keeping the monomer concentration below the critical monomer concentration of the pointed ends where the barbed ends of treadmilling actin filaments elongate and the pointed ends shorten. The gelsolin-actin complex was found to bind fourfold faster to the barbed ends in the presence of Ca2+ (10 X 10(6) M-1 s-1) than in excess EGTA (2.5 X 10(6) M-1 s-1). Dissociation of the gelsolin-actin complex from the barbed ends can be calculated to be rather slow. In excess EGTA the rate constant of dissociation is about 1.7 X 10(-4) s-1. In the presence of Ca2+ this dissociation rate constant is in the range of or below 10(-4) s-1.
Actins↗
Rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex.
The rate of capping of actin filaments by the gelsolin-actin complex was measured by inhibition of elongation of the barbed ends of actin filaments. Polymeric actin (0.1-1.0 microM) was added to 0.5 microM monomeric actin and various concentrations of the gelsolin-actin complex (0.08-2.4 nM) to induce nucleated polymerization. As under the experimental conditions (2 mM MgCl2, 100 mM KCl, 37 degrees C, actin monomer concentration less than or equal to 0.5 microM) actin filaments treadmilled, filaments elongated only at the barbed ends and the gelsolin-actin complex did not nucleate actin filaments to polymerize towards the pointed ends. The rate of nucleated actin polymerization in the presence of the gelsolin-actin complex was quantitatively analyzed. The rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex was found to be about 10(7) M-1 s-1.
Actins↗
Rate of treadmilling of actin filaments in vitro.
Actin filaments capped at the barbed ends were formed by polymerizing monomeric actin onto a gelsolin-actin complex. The rate of depolymerization and polymerization of the pointed ends was determined by diluting gelsolin-capped actin filaments into various concentrations of monomeric actin. Under the conditions of the experiments (100 mM-KCl, 2 mM-MgCl2 at 37 degrees C) the rate constant of dissociation of subunits both from a shortening and a lengthening filament was found to be 0.21 s-1. As the rate of dissociation of subunits from the slow pointed end determines the rate of treadmilling, it is concluded that actin filaments treadmill with a rate of about 2 micron/h.
Actin Cytoskeleton↗
Increased total body synthesis of prostacyclin in rats with adjuvant arthritis.
In rats with adjuvant arthritis we measured the urinary excretion of 2,3-dinor-6-oxo-PGF1 alpha, 7 alpha-hydroxy-5,11-dioxo-tetranor-prosta-1,16- dioic acid (PGE-M) and 2,3-dinor-thromboxane-B2, reflecting total body synthesis of prostacyclin, thromboxane and the E-prostaglandins, respectively. The urinary prostanoid metabolites were assessed by gas chromatography/tandem mass spectrometry using stable isotope internal standards. We found a more than 10-fold increase of urinary 2,3-dinor-6-oxo-PGF1 alpha excretion and a 5-fold higher urinary excretion of PGE-M in adjuvant arthritic rats as compared to non-arthritic control rats (p < 0.001; n = 12, each). There was no significant difference in urinary 2,3-dinor-thromboxane-B2 excretion between arthritic rats and control animals. Our data show a dramatic increase of urinary 2,3-dinor-6-oxo-PGF1 alpha excretion reflecting increased total body prostacyclin synthesis. It can be assumed that prostacyclin plays a role in generalized inflammatory reactions, comparable to that of the E-prostaglandins.
6-Ketoprostaglandin F1 alpha↗