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S N Timasheff

Publications and source records attributed to S N Timasheff.

At least 91 records · Page 5Linked to original sources
PubMed1982

The interactions of tropolone with magnesium ions and tubulin.

Tropolone, a single analog of colchicine, interacts with Mg2 with the formation of a 1 : 1 complex and an apparent equilibrium binding constant Kb of 1.4 x 10(4) M(-1) in neutral aqueous solution at 25 degrees C. The tropolone-Mg2 complex, but not tropolone, is fluorescent. Since tubulin binds Mg2 (Frigon, R.P. and Timasheff, S.N. (1975) Biochemistry 14, 4567-4573), previous reports of tropolone interaction with tubulin in Mg2 -containing buffers must be critically re-examined. Fluorescence and difference absorption spectroscopy experiments performed at essentially constant Mg2 activity indicate that tubulin does bind tropolone, but the optical effects are too weak to use in quantitative studies.

Animals↗
PubMed1982

Interaction of tubulin with single ring analogues of colchicine.

Simple analogues of the tropolone and trimethoxyphenyl moieties of colchicine have been used as probes for the colchicine binding site of purified calf brain tubulin. [3H]Tropolone methyl ether was found to bind to one site per tubulin molecule with equilibrium constant of (2.2 +/- 0.2) x 10(3) M-1 at 0 degree C, with the interaction having delta H0app = -8.3 +/- 1.0 kcal mol-1 and delta S0app = -15.2 +/- 3.6 eu. The binding of tropolone methyl ether and colchicine was inhibited by each other. Both tropolone and its methyl ether inhibited tubulin polymerization into microtubules in vitro. N-[3H]Acetylmescaline bound to tubulin with a K congruent to 4 x 10(2) M-1 at 37 degrees C. This interaction was inhibited by colchicine and at lower temperatures was below the sensitivity of the measuring method employed. [14C]Mescaline interacted with higher affinity site(s) not related to the colchicine site. Both mescaline and N-acetylmescaline inhibited partially the microtubule assembly at 10(-3) M concentrations. No linkage was observed between the binding of tropolone methyl ether and N-acetylmescaline. The relatively weak interactions of both the two separate parts of colchicine can account quantitatively for the much tighter binding of the complete drug to tubulin within a proposed model which takes into account the entropic advantage of colchicine as a bifunctional ligand.

Colchicine↗
PubMed1982

Tubulin bound to colchicine forms polymers different from microtubules.

The purified tubulin-colchicine complex undergoes in vitro polymerization under the same conditions that promote the assembly of microtubules from purified tubulin. The need for a critical concentration, the apparent free energy change of the reaction, and the effects of divalent cations and nucleotide binding indicate interactions similar to those involved in microtubule formation. The large polymers formed are not microtubules, suggesting that the mode of action of the antimitotic drug may be the production of an incorrect bonding geometry between tubulin molecules.

Animals↗
PubMed1981

Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures.

A densimetric investigation of the interactions between solvent components in glycerol-water mixtures (between 10 and 40 vol % glycerol) and seven proteins have been carried out in the acid pH region. All the proteins were found to be preferentially hydrated at all conditions used, i.e., addition of the proteins to the mixed solvent results in an increase in the chemical potential of glycerol. It is considered that this thermodynamically unfavorable interaction should tend to minimize the surface of contact between proteins and glycerol and in this way stabilize the native structure of globular proteins.

Densitometry↗
PubMed1981

Thermodynamic and kinetic examination of protein stabilization by glycerol.

The effect of concentrated glycerol on the thermal transitions of chymotrypsinogen and ribonuclease has been examined by differential spectrophotometry at 293 and 287 mm, respectively. It was found that for both proteins addition of glycerol raises the transition temperature, the increase in Tm being greater for ribonuclease than for chymotrypsinogen. This increase in the free energy of denaturation appears to reflect primarily a decrease in the entropy change. Analysis in terms of the Wyman linkage equation shows that, for both proteins, the exclusion of glycerol from the protein domain increases on denaturation i.e., the chemical potential of glycerol becomes even more positive when the protein unfolds relative to the native structure. This provides the thermodynamic stabilization free energy. Results of the kinetic examination of the slow unfolding reaction are consistent with the concept that the preferential exclusion of glycerol is related, at least in part, to enhanced solvent ordering.

Animals↗
PubMed1981

The stabilization of proteins by sucrose.

The interactions between proteins and solvent components have been investigated for the sucrose/water system. Thermodynamic and kinetic measurements of the thermal unfolding of alpha-chymotrypsin, chymotrypsinogen, and ribonuclease were performed as a function of sucrose concentration. The alteration in protein-solvent interactions in the presence of sucrose was also studied by density measurements and analyzed by multicomponent thermodynamic theory. Sucrose does not induce a conformational change in three proteins studied, although it does induce a small change in the circular dichroism spectrum of ribonuclease. The enthalpy of thermal unfolding shows little dependence on the concentration of sucrose, while the apparent activation energy of the unfolding process is increased by the addition of sucrose. The results from the protein-solvent interaction study indicate that sucrose is preferentially excluded from the protein domain, increasing the free energy of the system. Thermodynamically this leads to protein stabilization since the unfolded state of the protein becomes thermodynamically even less favorable in the presence of sucrose. The exclusion of sucrose from the protein domain seems to be related to the higher cohesive force of the sucrose water solvent system since all the experimental observations can be correlated with the effect of sucrose on the surface tension of water.

Animals↗
PubMed1980

Stoichiometry of the vinblastine-induced self-association of calf brain tubulin.

The self-association of calf brain tubulin in PG buffer (10(-2) M NaPi and 10(-4) M GTP, pH 7.0) induced by the antimitotic drug vinblastine has been investigated by velocity sedimentation. Schlieren sedimentation patterns were examined at low vinblastine concentrations where the boundary resolves into a bimodal one and at high vinblastine concentration where a single forward-skewed peak prevails. Weight-average sedimentation coefficients of tubulin were determined as a function of protein concentration, the results fitting well a self-association model of an idefinite isodesmic mechanism. This was confirmed by computer simulation of the sedimentation boundary profiles.

Animals↗
PubMed1980

Thermodynamic linkage between tubulin self-association and the binding of vinblastine.

A sedimentation velocity study has been carried out of the vinblastine-induced self-association of calf brain tubulin in PG (0.01 M NaPi and 10(-4) M GTP, pH 7.0) buffer as a function of vinblastine concentration and temperature. The dependence of the weight-average sedimentation coefficients (S20,W) on total protein concentration can be fitted best by an isodesmic, indefinite self-association mechanism. Apparent association constants, derived by computer fittings of the S20,W data, were analyzed in terms of the Wyman linkage equations. Fitting to a variety of reaction models suggested that the self-association is one ligand molecule mediated; i.e., the binding of one vinblastine molecule is coupled to the formation of each intertubulin bond. The intrinsic association equilibrium constant for dimerization of the vinblastine-liganded tubulin was found to be 1.8 x 10(5) M-1. The self-asociation is characterized by an apparent van't Hoff enthalpy change of +8.0 kcal/mol at 5 x 10(-5) M vinblastine and is driven by a positive entropy change. Apparent binding isotherms of vinblastine to tubulin were calculated based on the association mechanism and parameters derived from the linkage analysis and were found to be consistent with the vinblastine binding results previously reported in our laboratory under identical conditions [Lee, J. C., Harrison, D., & Timasheff, S. N. (1975) J. Biol. Chem. 250, 9276---9282]. Comparison of apparent binding curves calculated with different values of the self-association constants suggested that cooperativity between ligand binding and self-association may account for the disparity of the vinblastine-tubulin binding constants reported in the literature.

Animals↗
PubMed1979

Heat capacity microcalorimetry of the in vitro reconstitution of calf brain microtubules.

The self-assembly of calf brain tubulin, purified by the modified Weisenberg procedure, was examined in an adiabatic differential heat capacity microcalorimeter. Tubulin solutions at concentrations between 6 and 17 mg/mL were heated from 8 to 40 degrees C at heating rates between 0.1 and 1.0 deg/min in a pH 7.0 phosphate buffer containing 1 X 10(-3) M GTP, 1.6 X 10(-2) M MgCl2, and 3.4 M glycerol. The heat capacity change, deltaCp of the microtubule growth reaction was found to be -1600 +/- 500 cal/(deg mol) per 110 000 molecular weight tubulin dimer incorporated into microtubules, in agreement with the reported van't Hoff deltaCp value of -1500 cal/(deg mol) [Lee, J.C., & Timasheff, S.N. (1977) Biochemistry 16, 1754-1765]. The assembly reaction is characterized by a complex heat uptake pattern comprising both endothermic and exothermic processes.

Animals↗