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K Gekko

Publications and source records attributed to K Gekko.

At least 37 records · Page 2Linked to original sources
PubMed1994

Point mutations at glycine-121 of Escherichia coli dihydrofolate reductase: important roles of a flexible loop in the stability and function.

To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in a flexible loop (residues 117-131), separated by 19 A from active site Asp27, was substituted by site-directed mutagenesis with eight amino acids (Ala, Val, Leu, Asp, Ser, Cys, Tyr, and His). The free energy change of unfolding decreased in the order of G121A > G121D > G121C > G121S, wild-type > G121H > G121Y > G121L > G121V. The thermal denaturation temperature decreased with all mutations, accompanied by a decrease in the calorimetric enthalpy of denaturation. The steady-state kinetic parameter for the enzyme reaction, Km, was only slightly influenced, but kcat was significantly decreased by the mutations, there being 3- (G121C) to 42-fold (G121L) decreases in kcat/Km compared to that of the wild-type enzyme. The effects of mutations on the stability and enzyme activity were statistically examined as a function of the hydrophobicity and volume of amino acids introduced. The diminished stability and activity with increases in the hydrophobicity and volume of amino acids suggest that the main effect of the mutations would be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of the hydrophobic interaction.

Base Sequence↗
PubMed1993

Effects of point mutation in a flexible loop on the stability and enzymatic function of Escherichia coli dihydrofolate reductase.

To elucidate the role of a flexible loop in the stability and function of Escherichia coli dihydrofolate reductase, glycine-121 in the flexible loop (117-131) was substituted to valine and leucine by site-directed mutagenesis. Despite the increased hydrophobicity of the side chains, the free energy changes of unfolding of the two mutants (G121V and G121L) determined by urea denaturation at 15 degrees C were decreased by 1.22 and 0.38 kcal/mol, respectively, compared with that of the wild-type. Thermal denaturation temperature, as monitored by differential scanning calorimetry, was decreased by 2.4 and 5.2 degrees C for G121V and G121L, respectively, accompanying the decrease in enthalpy change of denaturation. These findings indicate that the structure of DHFR is destabilized by the mutations, predominantly due to the large decrease in enthalpy change of denaturation relative to entropy change of denaturation. The steady-state kinetic parameter in the enzyme reaction, Km, was not influenced but kcat was greatly decreased by these mutations, resulting in 240- and 52-fold decreases in kcat/Km for G121V and G121L, respectively. The main effect of the mutations appeared to be modification of the flexibility of the loop due to overcrowding of the bulky side chains, overcoming the enhancement of hydrophobic interaction.

Base Sequence↗
PubMed1991

Flexibility of globular proteins in water as revealed by compressibility.

In order to elucidate the flexibility-structure-function relationships of proteins, the adiabatic compressibility of about 30 globular proteins, including food proteins, was determined by means of sound velocity and density measurements in aqueous solutions. Most proteins studied showed positive compressibility, indicating the large internal flexibility of the molecules. The volume fluctuation was in the range of 30-200 ml/mol, which corresponded to about 0.3% of the total protein volume. From the statistical analyses of the compressibility data, it was found that the flexibility of proteins is closely related to structural factors such as hydrophobicity, helix element, and amino acid composition, and to functional properties such as digestibility and foaming capacity. These results indicate that the dynamics of protein structure should be taken into account in predicting precisely the functions and properties of a protein from its primary or tertiary structure.

Amino Acids↗
PubMed1991

Effect of pressure on the sol-gel transition of gelatin.

The effects of hydrostatic pressure on the sol-gel transition of gelatins were studied in the concentration range 1.5-12.5% under high pressures up to 300 MPa. The gelatin gels were stabilized by pressure: the pressure-induced elevation of melting temperature, (dT/dP)m, was 3.89 x 10(-2), 3.17 x 10(-2) and 2.92 x 10(-2) K/MPa for gelatins of Bloom No. 60, 225 and 304, respectively. The enthalpy, entropy and volume changes accompanying the gel formation were calculated from the Eldridge-Ferry plots and the Clausius-Clapeyron equation. The volume changes of gelation were estimated to be -25.7, -20.8 and -18.3 ml/mol of cross-links for gelatins of Bloom No. 60, 225 and 304, respectively, which were almost independent of pressure. The kinetic process of gelation was suppressed under high pressure, indicating the positive activation volume of gelation. These volume changes were discussed in terms of the characteristic hydration modes of cross-linking junctions of gelatin gels, comparing them with those of native collagen.

Animals↗
PubMed1991

Electroviscosity of polyelectrolyte solutions.

A theoretical expression for the electroviscous effect in polyelectrolyte solutions, caused by the distortion of counterion-distribution and counterion flow around a polyion under a velocity gradient of solvent flow, was obtained to elucidate the characteristic behaviour of the viscosity of highly charged polyelectrolyte solutions observed at low salt concentration. The derivation of the theory was performed on the basis of the Navier-Stokes-Onsager equation, Poisson equation, and diffusion equations for low molecular ions by the use of a cell model (free-volume model) for a polyion. Energy dissipation was obtained without directly solving these equations. It was found that the derived expression of viscosity explained the experimental results satisfactorily, and that the streaming potential effect caused by the counterion flow played an essential role in the increase in viscosity of polyelectrolyte solutions at finite polymer concentration and low salt concentration ranges.

Journal Article↗
PubMed1990

Competing solvent effects of polyols and guanidine hydrochloride on protein stability.

The denaturation of lysozyme and ribonuclease A by guanidine hydrochloride was followed in the presence and absence of glycerol and sorbitol by means of circular dichroism measurements at 25 degrees C. The protein-solvent interactions in the presence of these polyols were also studied by means of density measurements, for discussion of the mechanism of protein stabilization by polyols in terms of the multicomponent thermodynamic theory. The free energy of denaturation depends linearly on the molarity of guanidine hydrochloride at a given polyol concentration, without modification of the cooperativity of the transition. The free energy of denaturation at an infinite dilution of guanidine hydrochloride increases in proportion to the polyol concentration. These results indicate the competing solvent effects of polyols and guanidine hydrochloride on the structures of proteins. In water-protein-polyol systems, protein is preferentially hydrated to elevate its chemical potential, predominantly due to the unfavorable interaction of polyols with the exposed nonpolar amino acid residues. By linkage with the free energy of denaturation, it was quantitatively determined that the chemical potential of denatured protein is more extensively elevated by addition of polyols than that of native protein. These results demonstrate that polyols stabilize the protein structure through strengthening of the hydrophobic interaction, competing with the effect of guanidine hydrochloride.

Animals↗
PubMed1987

Changes in physicochemical properties of mitochondrial membranes during the formation process of megamitochondria induced by hydrazine.

Changes in some biochemical and physico-chemical properties of rat liver mitochondrial membranes during the formation process of megamitochondria induced by hydrazine were analyzed. Hepatic mitochondria obtained from rats placed on a 1% hydrazine diet for 3 days became slightly enlarged and sometimes elongated, while they became gigantic after 7 days of hydrazine intoxication. Changes were observed in mitochondria from rats treated with hydrazine for 3 days. Total amounts of phospholipids extracted from mitochondria and submitochondrial fractions were increased. Among phospholipid species, relative amounts of acidic phospholipids were increased. Contents of Ca2+ in mitochondria were increased. Differential scanning calorimetric analysis of mitochondria, especially that of the outer membrane fraction, showed that the thermotropic lipid phase transition temperatures were elevated accompanying the broadening of thermograms and the increase in transition enthalpy. Contents of water in mitochondria were increased significantly with the ratio of freezable water to unfreezable water unchanged. Among the changes observed was that the total amount of phospholipids (except for that of the outer membrane fraction) and the contents of water and Ca2+ nearly returned to normal in megamitochondria after 7 days of hydrazine intoxication. Relative amounts of phospholipids and thermotropic lipid phase transition temperatures of megamitochondria did not return to normal levels and yet changes were smaller than those obtained from 3 days of hydrazine intoxication. The fluidity of mitochondrial membranes was not affected by hydrazine treatment. These data would suggest that hydrazine-induced megamitochondrial formation is not due simply to the swelling of mitochondria, but might be due to the fusion of adjacent mitochondria by Ca2+-acidic phospholipid interactions, and once megamitochondria are formed the mitochondrial membranes are stabilized.

Animals↗
PubMed1986

Compressibility-structure relationship of globular proteins.

The adiabatic compressibility, -beta s, of 11 globular proteins in water was determined by means of sound velocity measurements at 25 degrees C. All the proteins studied except for subtilisin showed positive -beta s values, indicating the large internal compressibility of the protein molecules. The intrinsic compressibility of proteins free from the hydration effect appeared to be comparable to that of normal ice. The compressibility data for 25 proteins, including 14 reported previously [Gekko, K., & Noguchi, H. (1979) J. Phys. Chem. 83, 2706-2714], were statistically analyzed to examine the correlation of the compressibility with some structural parameters and the amino acid compositions of proteins. It was found that -beta s increases with increasing partial specific volume and hydrophobicity of proteins. The helix element also seemed to be a dynamic domain to increase -beta s. Four amino acid residues (Leu, Glu, Phe, and His) greatly increased -beta s, and another four (Asn, Gly, Ser, and Thr) decreased it. Some empirical equations were derived for the estimation of the -beta s values of unknown proteins on the basis of their amino acid compositions. The volume fluctuations of proteins revealed by the compressibility data were in the range of 30-200 mL/mol, which corresponded to about 0.3% of the total protein volume. The conformational fluctuation seemed to enhance the thermal stability of proteins.

Amino Acids↗
PubMed1983

Increased thermal stability of collagen in the presence of sugars and polyols.

The effects of sugars and polyols on the thermal denaturation temperature, Tm, of acid-soluble collagen from calf skin were studied at pH 4.0 under atmospheric pressure as well as high pressures up to 4,000 atm. Addition of these compounds invariably raised Tm with increases in their concentration over the whole range of pressure. The extent of stabilization by different sugars and polyols is discussed in terms of their different influences on the structure of water. The hydroxymethyl chain length of polyols and equatorial OH groups of the sugars were found to be decisive factors for their stabilizing effect on collagen structure. The similarity in their stabilizing effects on collagen and globular proteins suggests that our stabilization mechanism proposed for globular proteins can be essentially extended to fibrous proteins: such protein stabilization would be dominantly mediated through a preferential hydration of protein, originating in the water-structure-making character of sugars and polyols.

Alcohols↗
PubMed1982

Calorimetric study on thermal denaturation of lysozyme in polyol-water mixtures.

In order to clarify the mechanism of polyol-induced stabilization of protein, the thermal denaturation of lysozyme was studied at pH 4 in aqueous mixtures of some polyols (ethylene glycol, glycerol, erythritol, xylitol, and sorbitol) by a differential scanning calorimetry (DSC). The denaturation temperature, Td, increased with increasing the polyol concentration and the number of hydroxymethyl groups per polyol molecule. The calorimetric enthalpy or denaturation, delta H cal, increased with the increase in polyol concentration, but it was not significantly affected by the chain length of the polyol: delta H cal was about 30 kcal/mol larger in 30% (w/w) aqueous polyols than in water. The standard thermodynamic parameters for denaturation, delta G degrees, delta S degrees, and delta H degrees, which were calculated for glycerol and sorbitol systems using Td and delta H cal and assuming a constant heat capacity change, were an increasing function of polyol concentration. According to the thermodynamics of three component systems, it appeared that one or two polyol molecules are preferentially excluded from the domain of this protein on thermal denaturation. These thermodynamic data support the hypothesis that the thermal stabilization of lysozyme by polyols is due to a preferential solvent interaction effect which strengthens the hydrophobic interaction of the protein.

Calorimetry↗
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

Preferential hydration of bovine serum albumin in polyhydric alcohol-water mixtures.

The preferential solvent interaction with bovine serum albumin in aqueous solution of polyhydric alcohols (ethylene glycol, glycerol, xylitol, sorbitol, mannitol, and inositol) was investigated by a densimetric method with the application of multicomponent theory. This proteins was preferentially hydrated in all solvent systems examined: the extent depended on the number and the steric configuration of the hydroxyl groups of alcohols. The absolute interactions of these alcohols with the protein were estimated by assuming that the amount of hydration of protein at every solvent composition used is identical with that in pure water. The preferential hydration of the protein in 30% aqueous solutions of glycerol and sorbitol was found to decrease as the temperature was increased, indicating that the increase in chemical potential of protein on transferring it from water to both aqueous solvents is generated by a large positive enthalpy change, sufficient to compensate for the positive entropy change in the transfer process. On the basis of these results, and mechanism of stabilization of protein structure by these alcohols was discussed from the viewpoint of the solvation of protein.

Alcohols↗
PubMed1981

Thermodynamics of polyol-induced thermal stabilization of chymotrypsinogen.

In order to clarify the mechanism of polyol-induced stabilization of protein, the thermodynamic parameters (delta G degree, delta H degree, and delta S degree) of thermal denaturation of chymotrypsinogen have been measured in aqeous solutions of some polyols (ethylene glycol, erythritol, adonitol, sorbitol, mannitol, and inositol) by a differential spectrophotometric method. On increasing the alcohol concentration and the number of hydroxymethyl groups of the alcohols, delta G degree increased as a result of a large decrease in delta S degree compensating for a decrease in delta H degree. This result means that the stabilization of this protein by polyols is due to the entropy effect, and that the free energy change of transfer of the denatured protein from water to aqueous media containing these alcohols must be larger than that of the native protein. This strongly supports the previous proposal that the driving force of protein stabilization induced by polyols is a solvent medium effect or a solvent ordering effect. The decreases in delta H degree and delta S degree with polyols are expected to be more due to the effects of polyols on peptide-water interactions than to exposed nonpolar groups of denatured protein.

Alcohols↗
PubMed1981

Mechanism of polyol-induced protein stabilization: solubility of amino acids and diglycine in aqueous polyol solutions.

The solubilities of several amino acids and diglycine have been measured in water and at several concentrations of methanol and various polyols (glycerol, erythritol, xylitol, sorbitol, and inositol). The solubility data were used to calculate the free energy of transfer of amino acid side chains and peptide group from water to the aqueous alcohol solutions. The results for methanol systems were similar to those reported for ethanol and dioxane systems. The free energy of transfer to aqueous solutions of linear polyols was positive for most nonpolar side chains and peptide group, but high concentrations of the polyols may disrupt the hydrophobic interactions of large nonpolar side chains. Moreover, the linear polyols appeared to stabilize the hydrophobic interaction more effectively and the peptide-peptide hydrogen bond less effectively with increasing hydroxymethyl chain length of polyols. A cyclic polyol, inositol, had a very strong stabilizing ability on hydrophobic interactions of nonpolar side chains, but it may act as a destabilizing reagent for peptide-peptide hydrogen bonds. From these results, it was concluded that the protein stabilization by polyols is a manifestation of polyol-induced strengthening of the hydrophobic interaction of protein molecules.

Amino Acids↗
PubMed1981

Enthalpy and entropy of transfer of amino acids and diglycine from water to aqueous polyol solutions.

The enthalpies of transfer of several amino acids and diglycine from water to aqueous polyols (glycerol, xylitol, sorbitol, and inositol) have been calorimetrically determined in order to clarify the mechanism of polyol-induced stabilization of proteins. The obtained enthalpy data were combined with the data on free energy of transfer previously determined to calculate the corresponding entropy of transfer. The enthalpies of transfer of nonpolar side chains of amino acids and peptide group were positive and negative, respectively, depending on the polyol concentration and hydrophobicity of the side chains. This indicates that the non-spontaneous transfer of nonpolar side chains to aqueous polyols is due to an enthalpy effect while that of peptide group is due to an entropy effect. These thermodynamic data are discussed in terms of changes in water structure or solvent ordering around the solute molecules. It was found that the polyol-induced stabilization of proteins is a result of a complicated enthalpy-entropy compensation phenomenon closely related to the solvent ordering around the solute molecules; consequently, the polyol effects on nonpolar side chains do not necessarily dominate those on peptide groups in an enthalpic sense.

Amino Acids↗