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Biomedical subjects

K Kuwajima

Publications and source records attributed to K Kuwajima.

At least 91 records · Page 5Linked to original sources
PubMed1987

A Japanese patient with the Dubowitz syndrome.

A 2-year-old Japanese male whose clinical features included intrauterine and postnatal growth retardation, mild mental retardation, microcephaly and characteristic facial appearance including sloping forehead, blepharophimosis, ptosis of unilateral eyelid, broad nasal bridge, dysplastic auricles, and retrognathia, is presented. The clinical findings of this patient are strikingly similar to those of patients with the Dubowitz syndrome. However, all reported cases with the Dubowitz syndrome are Caucasians. This syndrome may be diagnosed even in sporadic cases of any ethnic groups based on the characteristic features.

Child↗
PubMed1986

Evidence for identity between the equilibrium unfolding intermediate and a transient folding intermediate: a comparative study of the folding reactions of alpha-lactalbumin and lysozyme.

The refolding kinetics of alpha-lactalbumin at different concentrations of guanidine hydrochloride have been investigated by means of kinetic circular dichroism and stopped-flow absorption measurements. The refolding reaction consists of at least two stages, the instantaneous accumulation of the transient intermediate that has peptide secondary structure and the subsequent slow process associated with formation of tertiary structure. The transient intermediate is compared with the well-characterized equilibrium intermediate observed during the denaturant-induced unfolding. Stabilities of the secondary structures against the denaturant, affinities for Ca2+, and tryptophan absorption properties of the transient and equilibrium intermediates were investigated. In all of these respects, the transient intermediate is identical with the equilibrium one, demonstrating the validity of the use of the equilibrium intermediate as a model of the folding intermediate. Essentially the same transient intermediate was also detected in the folding of lysozyme, the protein known to be homologous to alpha-lactalbumin but whose equilibrium unfolding is represented as a two-state reaction. The stability and cooperativity of the secondary structure of the intermediate of lysozyme are compared with those of alpha-lactalbumin. The results show that the protein folding occurring via the intermediate is not limited to the proteins that show equilibrium intermediates. Although the unfolding equilibria of most proteins are well approximated as a two-state reaction, the two-state hypothesis may not be applicable to the folding reaction under the native condition. Two models of protein folding, intermediate-controlled folding model and multiple-pathway folding model, which are different in view of the role of the intermediate in determining the pathway of folding, are also discussed.

Guanidine↗
PubMed1986

Innocuous character of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and EDTA as metal-ion buffers in studying Ca2+ binding by alpha-lactalbumin.

The binding of EGTA and EDTA to alpha-lactalbumin, first demonstrated by Kronman and Bratcher (Kronman, M. J., and Bratcher, S. C. (1983) J. Biol. Chem. 258, 5707-5709) and afterwards regarded as a significant source of error in estimating the binding constant of Ca2+ to the protein, has been investigated by comparison of the thermal unfolding curves of the protein in the absence and presence of the chelators and also by measuring the NMR spectra of the protein, the chelators, and the protein-chelator mixtures. The unfolding curve in the presence of a large excess of each chelator has been found to be identical to that in the absence of chelator, indicating that there is essentially no interaction between the chelators and alpha-lactalbumin. The NMR results have also supported this conclusion, and the innocuous character of these chelators as metal-ion buffers in studying the Ca2+-binding properties of alpha-lactalbumin is demonstrated. In order to re-examine the binding constant for Ca2+ of alpha-lactalbumin without the aid of chelating metal-ion buffers, the thermal unfolding curve of the protein in the presence of 0.1 mM excess Ca2+ but without chelators has been compared with the unfolding curve in the absence of Ca2+ at a constant concentration of Na+ (0.010 or 0.10 M) at pH 7.0. The binding constant of alpha-lactalbumin can be calculated from the increment of melting temperature caused by the presence of Ca2+ and from the enthalpy and heat capacity changes in the unfolding. Because Ca2+ binding to the unfolded protein can be neglected under the conditions employed, the binding constant evaluated corresponds to the binding constant to protein that has native structure. The constant obtained is 3-5 X 10(9) M-1 after corrections for binding of Na+ to the protein and for ionic strength, and this shows excellent agreement with the corresponding value previously estimated (2.9 +/- 1.0 X 10(9) M-1), although the latter value was obtained in the presence of EDTA. The apparent Ca2+-binding constant that has been discussed in most previous studies, without taking account of the folding-unfolding equilibrium associated with the binding process, also depends on the concentration of monovalent cations such as Na+, and the present results lead to values of 1.5 X 10(8) and 8.7 X 10(6) M-1 at 0.01 and 0.1 M Na+, respectively.

Buffers↗
PubMed1986

Binding strength of calcium ion to bovine alpha-lactalbumin.

A Ca2+-sensitive electrode was used for determination of the binding strength of Ca2+ to bovine alpha-lactalbumin in 60 mM Tris buffer (pH 7.8-8.5) in the presence of various concentrations of NaCl. The dependence of the apparent binding constant on the concentration of NaCl was consistent with competitive binding of Ca2+ and Na+, and the binding constants of Ca2+ and Na+ were found to be 2.2 (+/- 0.5) X 10(7) M-1 and 99 (+/- 33) M-1, respectively, at 37 degrees C and pH 8.0. The temperature dependence of the binding constant of Ca2+ was examined between 30 and 45 degrees C; extrapolation of the dependence led to a binding constant of approximately 1 X 10(8) M-1 at pH 8.4 and 25 degrees C. The electrostatic contribution and conformational effect of the protein were also taken into consideration, and the intrinsic binding constant of Ca2+ to native alpha-lactalbumin was calculated to be (1.2-1.5) X 10(10) M-1 at 37 degrees C and pH 8.0.

Animals↗
PubMed1986

Ca2+-induced alteration in the unfolding behavior of alpha-lactalbumin.

Comparative studies of the unfolding equilibria of two homologous proteins, bovine alpha-lactalbumin and hen lysozyme, induced by treatment with guanidine hydrochloride have been made by analysis of the peptide and the aromatic circular dichroism spectra. The effect of the specific binding of Ca2+ ion by the former protein was taken into account in interpreting the unfolding equilibria of the protein. Proton nuclear magnetic resonance spectra of alpha-lactalbumin were also measured for the purpose of characterizing an intermediate structural state of the protein. In previous studies, alpha-lactalbumin was shown to be an exceptional protein whose equilibrium unfolding does not obey the two-state model of unfolding, although lysozyme is known to follow the two-state unfolding mechanism. The present results show that the apparent unfolding behavior of alpha-lactalbumin depends on Ca2+ concentration. At a low concentration of Ca2+, alpha-lactalbumin unfolds with a stable intermediate that has unfolded tertiary structure, as evidenced by the featureless nuclear magnetic resonance and aromatic circular dichroism spectra, but has folded secondary structure as evidenced by the peptide circular dichroism spectra. However, in the presence of a sufficiently high concentration of Ca2+, the unfolding transition of alpha-lactalbumin resembles that of lysozyme. The transition occurs between the two states, the native and the fully unfolded states, and the cooperativity of the unfolding is essentially the same as that of lysozyme. Such a change in the apparent unfolding behavior evidently results from an increase in the stability of the native state relative to that of the intermediate induced by the specific Ca2+ binding to native alpha-lactalbumin. The results are useful for understanding the relationship between the protein stability and the apparent unfolding behavior.

Animals↗
PubMed1986

Influence of Ca2+ binding on the structure and stability of bovine alpha-lactalbumin studied by circular dichroism and nuclear magnetic resonance spectra.

Both the Ca2+-bound and Ca2+-free forms of alpha-lactalbumin can assume essentially the same folded conformation as evidenced by similarity in their CD and proton n.m.r. spectra. Thermal unfolding followed by the aromatic CD has shown that the stability of the folded state is markedly enhanced by Ca2+ and that the stabilization is almost entirely entropic; addition of 0.1 mM Ca2+ shifts the transition temperature from 40 degrees to 62 degrees in 0.1M Na+ at pH 7.0. The enthalpy change of the unfolding, coincident between the two forms, is, however, significantly smaller than that known for lysozyme. The n.m.r. spectrum under the condition that both the forms of the protein are in the folded state reflects minor environmental changes of certain protons upon Ca2+ binding, and these changes are shown to afford useful probes for assessment of the location of the binding site. From the pH dependence and temperature dependence of the spectrum and also by using spin decoupling in the aromatic region (6.4-8.7 p.p.m.), it is shown that none of histidyl residues are affected and that at least two tryptophanyl ring protons experience environmental changes upon Ca2+ binding to the folded apo-protein. Effect of free excess Ca2+ on the spectrum has also shown that in native alpha-lactalbumin there is only one Ca2+-binding site that is detectable by the present method.

Animals↗
PubMed1985

Comparison of the transient folding intermediates in lysozyme and alpha-lactalbumin.

Refolding kinetics of two homologous proteins, lysozyme and alpha-lactalbumin, were studied by following the time-dependent changes in the circular dichroism spectra in the aromatic and the peptide regions. The refolding was initiated by 20-fold dilution of the protein solutions originally unfolded at 6 M guanidine hydrochloride, at pH 1.5 for lysozyme and pH 7.0 for alpha-lactalbumin at 4.5 degrees C. In the aromatic region, almost full changes in ellipticity that were expected from the equilibrium differences in the spectra between the native and unfolded proteins were observed kinetically. The major fast phase of lysozyme folding has a decay time of 15 s. The decay time of alpha-lactalbumin depends on the presence or absence of bound Ca2+: 10 s for the holoprotein and 100 s for the apoprotein. In the peptide region, however, most of the ellipticity changes of the two proteins occur within the dead time (less than 3 s) of the present measurements. This demonstrates existence of an early folding intermediate which is still unfolded when measured by the aromatic bands but has folded secondary structure as measured by the peptide bands. Extrapolation of the ellipticity changes to zero time at various wavelengths gives a spectrum of the folding intermediate. Curve fitting of the peptide spectra to estimate the secondary structure fractions has shown that the two proteins assume a similar structure at an early stage of folding and that the intermediate has a structure similar to that of partially unfolded species produced by heat and, for alpha-lactalbumin, also by acid and a moderate concentration of guanidine hydrochloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Experimental studies of folding kinetics and structural dynamics of small proteins.

Main points given in the above reports can be summarized as follows. Multiple unfolded forms exist for lysozyme as well as for RNase. The existence of fast- and slow-folding forms appears to be a general phenomenon; it has been confirmed for a number of globular proteins, which contain proline residues. The major slow refolding reaction of RNase A is a sequential process via structural intermediates. A rapidly formed intermediate has also been detected on the direct UF----N refolding pathway of lysozyme. The activated state for folding of lysozyme shows a conformation similar to the native protein in terms of packing of hydrophobic groups. This suggests that, in terms of compactness, the rate-limiting step occurs at a late stage of the refolding process. A protein homologous to lysozyme, alpha-lactalbumin, shows similar kinetics although alpha-lactalbumin shows an apparent equilibrium unfolding intermediate. The location of the rate-limiting step close to the native state has also been suggested for other proteins. It still remains open whether this is a general property of protein folding reactions. As shown for the unfolding of Mb, the multi-probe kinetic measurements will be a powerful tool for investigating the mechanism of folding, in particular for characterizing structural kinetic intermediates. The dynamics of local fluctuations of a well-defined part of RNase S can be monitored by NMR measurements of NH proton exchange. An increasing number of experimental and theoretical studies are focussing on the problem of protein dynamics. Application of NMR methods to protein folding should give extensive information about the structure of intermediates, which cannot be given by other techniques.

Animals↗
PubMed1983

Nature and locations of the most slowly exchanging peptide NH protons in residues 1 to 19 of ribonuclease S.

The locations have been found of the eight most slowly exchanging peptide protons in residues 1 to 19 of ribonuclease S. The resonance lines of these eight protons are resolved by proton magnetic resonance at 360 MHz when either S-peptide (residues 1 to 19) or peptide 1-15 is bound to S-protein (residues 21 to 124). Other peptide protons have been removed by exchange in the sample preparation [( 1H]S-peptide is added to deuterated S-protein in D2O), and also by exchange-out of the less protected protons in residues 1 to 19. At pH 5.1, 0 degrees C, there is a 100-fold difference in rates of exchange between the eight most protected protons and the less protected protons of S-peptide. The highly protected protons are protected 10(4)-fold compared to free S-peptide. The protected protons have been identified by 1H nuclear magnetic resonance after denaturing ribonuclease S in greater than or equal to 3 M-urea-d4, D2O, pH 2.3, -4 degrees C, followed by comparing the chemical shifts of the remaining eight protons with the known -NH spectrum of the free peptide, which has been assigned from the two-dimensional homonuclear correlated spectrum and by comparison with earlier work. The eight highly protected NH protons are localized in one segment, residues 7 to 14. All eight protons are H-bonded: those of residues 7 to 13 are H-bonded within the 3-13 alpha-helix and that of residue 14 is H-bonded to the beta-sheet. The NH proton of residue 16, which also is H-bonded to the beta-sheet, is not one of the highly protected protons. Both the N atoms of the eight NH groups and also the O atoms of their CO acceptor groups are shielded from solvent in most cases, according to the molecular area calculations of Finney (1978).

Amides↗
PubMed1983

Exchange behavior of the H-bonded amide protons in the 3 to 13 helix of ribonuclease S.

The preceding article shows that there are eight highly protected amide protons in the S-peptide moiety of RNAase S at pH 5, 0 degrees C. The residues with protected NH protons are 7 to 13, whose amide protons are H-bonded in the 3 to 13 alpha-helix, and Asp 14, whose NH proton is H-bonded to the CO group of Val47. We describe here the exchange behavior of these eight protected protons as a function of pH. Exchange rates of the individual NH protons are measured by 1H nuclear magnetic resonance in D2O. A procedure is used for specifically labeling with 1H only these eight NH protons. The resonance assignments of the eight protons are made chiefly by partial exchange, through correlating the resonance intensities in spectra taken when the peptide is bound and when it is dissociated from S-protein in 3.5 M-urea-d4, in D2O, pH 2.3, -4 degrees C. The two remaining assignments are made and some other assignments are checked by measurements of the nuclear Overhauser effect between adjacent NH protons of the alpha-helix. There is a transition in exchange behavior between pH 3, where the helix is weakly protected against exchange, and pH 5 where the helix is much more stable. At pH 3.1, 20 degrees C, exchange rates are uniform within the helix within a factor of two, after correction for different intrinsic exchange rates. The degree of protection within the helix is only 10 to 20-fold at this pH. At pH 5.1, 20 degrees C, the helix is more stable by two orders of magnitude and exchange occurs preferentially from the N-terminal end. At both pH values the NH proton of Asp 14, which is just outside the helix, is less protected by an order of magnitude than the adjacent NH protons inside the helix. Opening of the helix can be observed below pH 3.7 by changes in chemical shifts of the NH protons in the helix. At pH 2.4 the changes are 25% of those expected for complete opening. Helix opening is a fast reaction on the n.m.r. time scale (tau much less than 1 ms) unlike the generalized unfolding of RNAase S which is a slow reaction. Dissociation of S-peptide from S-protein in native RNAase S at pH 3.0 also is a slow reaction. Opening of the helix below pH 3.7 is a two-state reaction, as judged by comparing chemical shifts with exchange rates. The exchange rates at pH 3.1 are predicted correctly from the changes in chemical shift by assuming that helix opening is a two-state reaction. At pH values above 3.7, the nature of the helix opening reaction changes. These results indicate that at least one partially unfolded state of RNAase S is populated in the low pH unfolding transition.

Amides↗
PubMed1981

Kinetic correlations between the disulfide bond reduction and the induced conformational change of proteins.

Kinetic correlations between the disulfide bond reduction in excess dithioerythritol and the induced conformational change were studied on two proteins, bovine alpha-lactalbumin and soybean trypsin inhibitor, at 25 degrees C and pH 8.0-8.5 by measuring the absorbance of oxidized dithioerythritol at 310 nm and the ellipticity at 270 nm, respectively. With alpha-lactalbumin, in the absence of guanidine hydrochloride (Gdn x HCl) or in dilute Gdn x HCl, the kinetics for the bond reduction and the conformational change were both of a biphasic type. The fast phase was complete within a few seconds and was associated with the reduction of some of the disulfide bonds and with almost complete loss of the tertiary structure. The slow phase was associated with the reduction of other disulfide bonds. In concentrated Gdn x HCl, the kinetics of both processes were observed as a single phase, the rate of which was similar to that of the slow phase in the absence of Gdn x HCl or in dilute Gdn x HCl. In all cases studied, the rate of the bond reduction was similar to that of the conformational change induced. By correcting the change in absorbance at 310 nm to a contribution from the protein due to the conformational change, the number of bonds which are reduced in the fast phase in the absence of Gdn x HCl was determined to be 1.0-1.1. It was shown, taking observations of others and theoretical results into account, that the bond reduced in the fast phase might be the one between Cys 6 and Cys 120. On the other hand, on of two bonds of soybean trypsin inhibitor in the native form was reduced in the fast phase without any loss of the tertiary structure, and the other was reduced in the slow phase. Considering the results of other researchers, it was concluded that the bond reduced in the fast phase of the inhibitor is a 136-145 bond.

Animals↗
PubMed1981

Role of the interaction between ionizable groups in the folding of bovine alpha-lactalbumin.

Previous studies of the reversible unfolding of alpha-lactalbumin in the acid to neutral pH region have shown that the unfolding transition with guanidine hydrochloride involves a stable intermediate which is similar to a partially unfolded state produced by acid transition. In order to clarify how the interaction of ionizable groups takes part in stabilization of the native structure during the folding of the protein, the transitions were further investigated in the alkaline region in the presence of the denaturant by means of circular dichroism, difference spectra and pH-jump measurements, and the effects of pH on the equilibrium and kinetics of the unfolding in the whole pH region are discussed. The alkaline state is indistinguishable from the acid state in equilibrium and kinetic properties. Thus, as a first approximation, the total unfolding in the whole pH region can be expressed as a three-state mechanism involving the native (N), the intermediate (A), and the fully unfolded (D) states. The strong pH dependence of the N Equilibrium A transition above pH 10 is almost entirely ascribable to the abnormal tyrosines in the N state previously detected by the pH-jump titration method, while the dependence between pH 7 and 10 also suggests the presence of an abnormal alpha-amino group. The normalization of most of the alkaline and the acidic abnormally ionizable groups in the N state occurs simultaneously in the first step of the unfolding pathway, i.e., the forward activation of the N Equilibrium A transition, and the final step of the folding may be associated with the interaction of the ionizable groups. Among the abnormally ionizable groups detected, the tyrosyl and carboxyl groups are most important in view of the large changes in their pK values, suggesting the presence of some interactions, even if only indirect, between these groups. Alignment data of amino acid residues also suggest that at least one such abnormal tyrosyl residue (Tyr 50) and its neighbors are conserved throughout in the alpha-lactalbumin-lysozyme group of proteins. Possible mechanisms of the interaction between the tyrosyl and carboxyl groups are discussed.

Animals↗
PubMed1980

Intramolecular perturbation of tryptophans induced by the protonation of ionizable groups in goat alpha-lactalbumin.

Goat alpha-lactalbumin shows two septral changes at acid pH. One of these corresponds to the acid denaturation, while the other has been regarded as a result of the intramolecular perturbation of tryptophans induced by the protonation of ionizable groups in the native molecule. In order to distinguish between these spectral changes and to investigate the perturbation caused by the ionizable groups, the absorption changes with a change in pH have been followed by the stopped-flow pH-jump method. Only the acid denaturation can be observed kinetically while the perturbation is too rapid to follow by the kinetic method. Extrapolations of the time-dependent absorption changes to zero time in the unfolding and refolding pH jumps give the apparent titration curves of the ionizable groups that participate in the perturbations in the native and in the acid-denatured molecule. The perturbation of the native protein is induced by the protonation of at least two ionizable groups, while the acid-denatured molecule does not show the perturbation because of the loss of the unique spatial arrangement of the ionizable groups around tryptophans. The results of circular dichroism measurements and the comparison with known results of the charge perturbation of lysozyme are also discussed.

Animals↗
PubMed1979

Application of the pH-jump method to the titration of tyrosine residues in bovine alpha-lactalbumin.

A stopped-flow technique has been developed for the zero-time spectrophotometric titration of tyrosine residues in the purely native or in the purely alkaline denatured state of alpha-lactalbumin that undergoes an alkaline conformational transition in the pH region of tyrosine ionization. The progressive absorption change at 298 nm caused by a pH jump from neutral pH is shown to result from the change in ionization of the tyrosine residues brought about by a first-order process of the conformational transition. Extrapolation to zero time gives the titration curve for purely native alpha-lactalbumin. Similarly, the pH jump from highly alkaline pH gives the titration curve for the purely alkaline denatured protein. The method should be generally applicable to other proteins that contain tyrosines. Analysis of the titration curves suggests that the four tyrosines in native alpha-lactalbumin have pK values of 10.5, 11.8, 11.8, and 12.7, respectively. After the alkaline transconformation, all of them become titrated normally with a pK value of 10.3. A comparison of these results with the ionization behavior of tyrosines in hen egg white and human lysozymes is presented and discussed in terms of differences in the sequences of the proteins.

Animals↗
PubMed1979

Lipoma of the corpus callosum: report of a case and review of the literature.

A case of lipoma of the corpus callosum associated with subcutaneous lipoma, whose symptoms were headache and trembling of the extremities, was reported. Roentgenograms showed characteristic findings and computerized tomography revealed a mass of very low density. The literature was reviewed and 15 cases not included in previous compilations were found. Clinical and radiological findings in 89 cases including the present case were analyzed.

Adolescent↗