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S Kidokoro

Publications and source records attributed to S Kidokoro.

14 recordsLinked to original sources
PubMed1992

Enthalpic destabilization of a mutant human lysozyme lacking a disulfide bridge between cysteine-77 and cysteine-95.

To understand the role of disulfide bridges in protein stability, the thermodynamic changes in the denaturation of two mutant human lysozymes lacking a disulfide bridge between Cys-77 and Cys-95 (C77A and C77/95A) were analyzed using differential scanning calorimetry (DSC). At pH 3.0 and 57 degrees C, the stabilities of both the C77A and C77/95A mutants were decreased about 4.6 kcal.mol-1 in Gibbs free energy change. Under the same conditions, the enthalpy changes (delta H) were 94.8 and 90.8 kcal.mol-1, respectively, which were smaller than that of the wild type (100.8 kcal.mol-1). The destabilization of the mutants was caused by enthalpic factors. Although X-ray crystallography indicated that the mutants preserve the wild-type tertiary structure, removal of the disulfide bridge increased the flexibility of the native state of the mutants. This was indicated both by an increase in the crystallographic thermal factors (B-factors) and by a decrease in the affinity of N-acetylglucosamine trimer [(NAG)3] observed using isothermal titration calorimetry (DTC) due to entropic effects. Thus, the effect of cross-linking on the stability of a protein is not solely explained by the entropy change in denaturation.

Amino Acid Sequence↗
PubMed1992

Thermodynamic characterization of cytochrome c at low pH. Observation of the molten globule state and of the cold denaturation process.

Several reports have pointed out the existence of intermediate states (both kinetic and equilibrium intermediate) between the native and the denatured states. The molten globule state, a compact intermediate state in which the secondary structure is formed but the tertiary structure fluctuates considerably, is currently being studied intensively because of its possible implication in the folding process of several proteins. We have examined the thermal stability of horse cytochrome c at low pH between 2.0 and 3.2 and different potassium chloride concentrations by absorbance of the Soret band, far and near-ultraviolet circular dichroism (u.v. c.d.) and tryptophan fluorescence using a multidimensional spectrophotometer. The concentration of potassium chloride ranged from 0 M to 0.5 M. The experimental thermal denaturation curves show that: (1) the helical content of cytochrome c remains stable at higher temperature when the concentration of salt is increased; whereas (2) the extent of ordering of the tertiary structure is weakly dependent on salt concentration; and (3) for cytochrome c, the stabilization of the molten globule state is induced by the binding of anions. Other salts such as NaCl, LiCl, potassium ferricyanide (K3Fe(CN)6) and Na2SO4 may also be used to stabilize the molten globule state. The thermodynamic analysis of the denaturation curves of c.d. at 222 nm and c.d. at 282 nm shows that, whereas a two-state (native and denatured) transition is observed at low-salt concentration, the far and near-u.v. c.d. melting curves of cytochrome c do not coincide with each other at high-salt concentration, and a minimum of three different thermodynamic states (IIb, intermediate or IIc, and denatured) is necessary to achieve a sufficient analysis. The intermediate state (called IIc) is attributed to the molten globule state because of its high secondary structure content and the absence of tertiary structure. Therefore, at low pH, cytochrome c is present in at least four states (native, IIb, IIc and denatured) depending on the salt concentration and temperature. The thermodynamic parameters, i.e. the Gibbs free energy differences (delta G), the enthalpy differences (delta H), the midpoint temperatures (Tm) of the transition (IIb in equilibrium intermediate (IIc in equilibrium denatured) are determined. We also give estimates of the heat capacity differences (delta Cp) from the temperature dependence of the enthalpy differences. The enthalpy change and the heat capacity difference of the IIc in equilibrium denatured transition are non-zero. The number of charges (protons or chloride anions) released upon transitions are determined by analysing the pH and chloride anion concentration dependence of the Gibbs free energy.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Spectroscopic studies on lambda cro protein-DNA interactions.

Spectroscopic (circular dichroism and fluorescence) and thermodynamic studies were conducted on lambda Cro-DNA interactions. Some base substitutions were introduced to the operator and the effects on the conformation of the complex and thermodynamic parameters for dissociation of the complex were examined. It was found that, (1) in the specific binding of Cro with DNA which has a (pseudo) consensus sequence, DNA is overwound, while in non-specific binding it is unchanged, or rather unwound; (2) substitution of central base-pairs or the introduction of a mismatched base-pair at the center of the operator reduces the extent of DNA conformational change on Cro binding and lessens the stability of the Cro-DNA complex, even though there is apparently no direct interaction between Cro and DNA at these positions; (3) stability of the complex increases with the degree of DNA conformational change of the same type during binding; (4) in some cases of specific binding, there are three states in the dissociation of the complex as observed by salt titration: two conformational states for the complex depending on salt concentration and, in non-specific binding, dissociation is a two-state transition; (5) the number of ions involved in interactions between Cro and 17 base-pair DNA is about 7.7 for NaCl titrations; (6) dissociation free energy prediction of the Cro-DNA complex by simple addition of the dissociation free energy change of a single base-pair substitution agrees with our experimental results when DNA overwinding occurs during binding, i.e. in specific binding.

Base Sequence↗
PubMed1990

Structural organization of flagellin.

The terminal regions of flagellin from Salmonella typhimurium have been reported to be disordered in solution, whereas the central part of the molecule contains protease-resistant, compact structural units. Here, conformational properties of flagellin and its proteolytic fragments were investigated and compared to characterize the domain organization and secondary structure of flagellin. Deconvolution analysis of the calorimetric melting profiles of flagellin and its fragments suggests that flagellin is composed of three co-operative units or domains. The central part of the molecule, residues 179 to 418, consists of two domains (G1 and G2), whereas the third domain (G3) is discontinuous, constructed from segments 67 to 178 and 419 to 448. Secondary structure prediction and analysis of far-ultraviolet circular dichroic spectra have revealed that G1 and G2 consist predominantly of beta-structure with a little alpha-helical content. G3 contains almost equal amounts of alpha and beta-structure, while in the terminal parts of flagellin the ordered secondary structure seems to be entirely alpha-helical.

Bacterial Proteins↗
PubMed1990

Thermal stability of dihydrofolate reductase and its fused proteins with oligopeptides.

Two fused proteins of dihydrofolate reductase (DHFR) with oligopeptides were prepared by a recombinant DNA method. One of these, DHFR-IQI, has three (Ile-Gln-Ile) and the other, DHFR-lek, has eight (Ile-Arg-Met-Tyr-Gly-Gly-Phe-Leu) additional amino acid residues at the C terminals; in both proteins, Cys152 of wild DHFR is replaced by Glu. The thermal transition of the proteins was measured by CD and DSC at pH 7.0 and compared with that of wild DHFR. The results show that the thermal stability of DHFR-IQI is the same as that of the wild DHFR and that of DHFR-lek is less than that of the former two DHFRs. Analysis of the DSC data of DHFR-IQI indicates that the thermal transition is a three-state one. Data from both DSC and CD measurements suggest the association of DHFR-lek molecules.

Calorimetry, Differential Scanning↗
PubMed1989

[The effect of calcium hydroxide eugenol agents on pulp of primary teeth of young dogs. The view of early stages].

Eugenol has such effective anti-corrosion and pain-killing properties that it is widely used for dentistry clinically. We focused our attention on eugenol and made agents for pulpotomy which were mixtures of calcium hydroxide, eugenol and polyethylene glycol 4000 for a base. We attempted to use them for the pulpotomy of primary teeth of young dogs and observed the healing progress of the treated teeth during the early stages. The experiment utilized four young dogs whose primary teeth were pulpotomized by the usual method. Agents of the pulpotomy were mixed calcium hydroxide (1.0 g), eugenol (0.8 ml) and polyethylene glycol 4000 (1.2 ml). After 3 days and 7 days, these teeth were taken out to be made into histopathological specimens. We inspected them microscopically. The results obtained were as follows. 1) After 3 days, the amputated pulp which was in contact with the agents turned into necrotic layers. Degenerative, necrotic, and non-structural states were widely observed. Under the necrotic layers, there were hematoxyline dark stained layers. Also the hematoxyline dark stained layers separated from the proper pulp. In the proper pulp, there were no abnormalities. 2) After 7 days, the necrotic layers were atrophied. In the dark stained layers, the stained areas were darker than that of after 3 days and increased in degeneration. In the proper pulp, round cells appeared and with a chronic inflammatory appearance along with vacuole, atrophy and degeneration. It seemed to be affected by eugenol. Dentin bridges still has not yet formed.

Animals↗
PubMed1975

[Effects of low salt plus upright posture, angiotensin II, ACTH, and potassium upon plasma renin activity, aldosterone, and cortisol (author's transl)].

In order to study the control system of plasma aldosterone in human, we examined the effects of low salt plus upright posture, angiotensin II, ACTH and potassium upon plasma renin activity, aldosterone and cortisol in five subjects who were supposed to be normal. All of the procedures, low salt diet with below 3 g of salt and 2 hr-upright posture, 0.25 mg of Cortrosyn, angiotensin infusion to increase 20mmHG of diastolic pressure for an hour, and 30 mEq of potassium infusion stimulated plasma aldosterone significantly. Furthermore, in each subject the degrees of response to each of these stimulations were almost same. In an old woman aged 68, responses to all of stimulations were significantly lower than those in other subjects. Plasma cortisol was significantly stimulated by ACTH, but slightly reduced by potassium infusion. From these results, it is certain that plasma aldosteron levels are easily affected by a small amount of changes in angiotensin, ACTH, potassium and sodium. However, responses of aldosterone to these changes seem to be decreased in old subjects.

Adrenocorticotropic Hormone↗