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Biomedical subjects

K Hikichi

Publications and source records attributed to K Hikichi.

At least 55 records · Page 3Linked to original sources
PubMed1987

Communication between two globular domains of calmodulin in the presence of mastoparan or caldesmon fragment. Ca2+ binding and 1H NMR.

Ca2+ binding to calmodulin was measured in the presence of mastoparan or caldesmon fragment. Mastoparan and caldesmon fragment were used as model compounds of enzymes and cytoskeleton proteins, respectively, working as the target of calmodulin. Although the Ca2+ bindings of the two globular domains of calmodulin occur independently in the absence of the target peptide (or proteins), mastoparan and caldesmon fragment increased the affinity of Ca2+ and, at the same time, produced the positive cooperative Ca2+ bindings between the two domains. The result of Ca2+ binding was compared with 1H NMR spectra of calmodulin in the presence of equimolar concentration of mastoparan. It is known that a conformation change of the C-terminal half-region (C-domain) occurs by the Ca2+ binding to C-domain. A further change in conformation of C-domain was demonstrated by the Ca2+ binding to the N-terminal half-region (N-domain) in the presence of mastoparan. It indicates that the two domains of calmodulin get into communication with each other in the associated state with the target, and we concluded that the Ca2+ binding to the N-domain is responsive to the development of calmodulin function.

Animals↗
PubMed1986

X-ray determination of the crystallinity in bone mineral.

The X-ray diffraction method of Ruland (Acta Crystallogr. 14 (1961) 1180-1185) used for the crystallinity determination of synthetic polymers was applied to the mineral present in mature rat cortical bone. The results obtained were compared with those obtained by other X-ray methods of Harper and Posner and Wakelin, Virgin and Crystal. It was concluded that the method of Ruland gives a more reliable determination of the crystallinity of bone mineral than other methods.

Animals↗
PubMed1986

Complete structural analysis of globoseries glycolipids by two-dimensional nuclear magnetic resonance.

Combined two-dimensional proton nuclear magnetic resonance allowed the determination of complete oligosaccharide structures of glycolipids belonging to the globo series, without any other analytical methods. Although a chemical modification by peracetylation was required for the above purpose, the derivatization permitted facile assignment of the pyranose ring proton resonances of the oligosaccharide moiety. Two-dimensional chemical-shift-correlated spectroscopy of the acetylated glycolipid enabled us to elucidate the glycosidic positions from the chemical shifts of the protons at the substituted sites. The monosaccharide species were also identified from the characteristic splitting patterns of the methine protons on individual pyranose rings. The sequence of the monosaccharides was inferred from the interresidue connectivity across glycosidic linkages shown by two-dimensional nuclear Overhauser effect spectroscopy, which also gave intraresidue interaction on the pyranose rings. The linkage sites of long oligosaccharide chains having more than five monosaccharides, such as globopentaosylceramide, were analyzed by two-dimensional J-relayed coherence transfer, which yielded 1,3 interactions along with 1,2 interactions.

Acetylation↗
PubMed1985

Hydrogen bonding in the carboxyl-terminal half-fragment 78-148 of calmodulin as studied by two-dimensional nuclear magnetic resonance.

The C-terminal half-fragment (residues 78-148) of scallop testis calmodulin was investigated by 500-MHz two-dimensional proton NMR in order to clarify the structure and the structural change accompanying Ca2+ binding. The sequential resonance assignment to individual amino acid residues was made in part (27 out of 71 residues) by a combination of correlated spectroscopy and nuclear Overhauser effect spectroscopy of a 90% H2O solution. In the Ca2+-bound state, resonances of backbone amide protons of Gly-98, Gly-134, Ile-100, Asn-137, and Val-136 appear at extremely low fields. These findings suggest that amide protons of these residues are hydrogen bonded. In the Ca2+-free state, the amide resonances of Ile-100 and Gly-134 disappear into the crowded normal shift region. This observation indicates that two hydrogen bonds of Ile-100 and Gly-134 are destroyed (or weakened) as Ca2+ ions are removed from two Ca2+-binding sites. Chemical shifts of amide and alpha-protons of residues located in the Ca2+-binding loop of domain III are similar to those of domain IV. These results suggest that the conformations of the two loops are very similar. The present results can be interpreted in terms of a structure predicted by Kretsinger [Kretsinger, R.H. (1980) Ann. N.Y. Acad. Sci. 356, 14].

Amino Acid Sequence↗
PubMed1985

Calcium binding and conformation of regulatory light chains of smooth muscle myosin of scallop.

Calcium binding was studied with two regulatory light chains (RLC-a and RLC-b) of smooth muscle myosin of scallop. With the equilibrium dialysis method, the binding of 0.98 mol Ca2+ per mol of RLC-b was observed with a dissociation constant of 2.3 X 10(-5) M. Similar values for RLC-b, 1.9 X 10(-5) M, and RLC-a, 1.5 X 10(-5) M, were obtained by measuring the difference absorption spectrum induced by Ca2+. The difference molar absorption coefficient at 288 nm was 159 and 209 M-1 X cm-1 for RLC-a and RLC-b, respectively, while it was -34 M-1 X cm-1 for the regulatory light chain of striated muscle myosin of scallop (RLC-st). Proton NMR spectra of the three light chains were very similar to each other and were broader than those of other Ca2+ binding proteins, parvalbumin and calmodulin. The regulatory light chains may be more rigid than in these Ca2+ binding proteins. CD spectra were measured for the three light chains, and the estimated helix contents were 27, 29, and 24%, respectively, for RLC-a, RLC-b, and RLC-st. All these results in comparison with the primary structures led us to suppose that the polypeptide of regulatory light chains is folded in such a way that domain 4 becomes near to the calcium binding site of domain 1. The decrease in intact light chains on trypsin digestion was determined for the gel electrophoretic patterns. RLC-a was 6 times more susceptible to the tryptic digestion than RLC-b.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

N-terminal region (domain 1) of calmodulin is the low affinity site for Ca2+. A 13C NMR study of S-cyanocalmodulin.

A single cysteine residue (Cys-27) of wheat calmodulin was labeled with 13C by cyanylation. No change in the Ca2+ saturation pattern was observed after the cyanylation. On titration which Ca2+, the chemical shift of the 13C-label showed a downfield shift. The downfield shift was observed at Ca2+/calmodulin molar ratios between 1.8 and 3.5, while a blue shift of the UV absorption of Tyr-139 was observed between 0 and 1.7. The result indicated the domain 1 containing Cys-27 to be the low affinity site for Ca2+.

Amino Acids↗
PubMed1984

Dynamic viscoelasticity of bovine vitreous body.

The dynamic mechanical property of the vitreous body was studied as functions of frequency and temperature. The data at different temperatures were found to be superposable onto a single set of master relaxation curves. It was found that the shape of the composite master relaxation curves of the vitreous body resembles that of the network polymer system except for the very small absolute value of the shear modulus.

Animals↗
PubMed1983

Nuclear magnetic resonance studies on calmodulin: spectral assignments in the calcium-free state.

The 400-MHz proton magnetic resonance spectra of calcium-free scallop testis calmodulin (CaM) and pig brain CaM were observed. Detailed spectral assignments were made by resolution enhancement, spin decoupling, and nuclear Overhauser enhancement (NOE) experiments as well as pH titration. Comparison between spectra of scallop testis CaM and pig brain CaM were also utilized for the assignment. Previous assignments for tyrosine-99, histidine-107, epsilon-trimethyllysine-115, and tyrosine-138 [Seamon, K. B. (1980) Biochemistry 19, 207; Krebs, J., & Carafoli, E. (1982) Eur. J. Biochem. 124, 619] were confirmed. Phenylalanine-99 and threonine-143 of scallop testis CaM were identified. Sixteen methyl resonances from one isoleucine, two valines, nine methionines, and the amino-terminal acetyl group were identified. First-stage assignments were made of resonances arising from seven phenylalanines. The uniquely high field shifted phenylalanine resonance previously reported by Seamon was found to consist of two doublets from the two pairs of delta protons of two phenylalanines. The NOE experiments showed that the two phenylalanines are located closely to each other. The large high-field shifts of these phenylalanines were accounted for the ring-current effects due to their proximity. An isoleucine and a valine of which methyl resonances appear at high fields were found to be situated closely to each other. It was found that two delta protons and two epsilon protons of almost all aromatic residues are magnetically equivalent, suggesting that the local structure of aromatic residues is so flexible as to permit the rapid flipping motion of the ring.

Animals↗
PubMed1983

Nuclear magnetic resonance studies on calmodulin: calcium-induced conformational change.

The 400-MHz 1H nuclear magnetic resonance (NMR) studies were carried out on the Ca2+-induced conformational change of calmodulins (CaM's) isolated from scallop testis and pig brain. The resonances were found to be classified approximately into three groups. The resonances of group I, which are perturbed by the binding of Ca2+ to the high-affinity sites, include those of tyrosine-138, epsilon-trimethyllysine-115, histidine-107, tyrosine-99, etc. The previous assignments for tyrosine- (Tyr) 138 [Seamon, K. B. (1980) Biochemistry 19, 207] were corrected. The resonances of group II, which are affected by the binding of Ca2+ to the low-affinity sites, include those of a phenylalanine (Phe), a high field shifted methyl, and a low field shifted alpha-methine. Group III (related to the binding of Ca2+ to both the high-and low-affinity sites) includes the resonances of a Phe, a high field shifted methyl, and threonine-143. It is concluded that sites III and IV are the high-affinity sites. The off-rate of Ca2+ from the high-affinity sites is slower than 50 s-1 while the off-rate from the low-affinity sites is faster than 600 s-1. In the Ca2+-free state, there exists a hydrophobic region containing three phenylalanine (probably Phe-89, Phe-92, and Phe-141), a valine, and an isoleucine in the vicinity of sites III and IV. Tyr-138 is distant from these amino acids. Upon binding of Ca2+ to the high-affinity sites, one of the Phe residues and the valine approach Tyr-138. Similar structural changes were observed between CaM and troponin C when Ca2+ ions are bound to the high-affinity sites. CaM changes in a somewhat different way from troponin C when Ca2+ ions are bound to the low-affinity sites.

Animals↗
PubMed1983

Dynamic mechanical properties of milk and milk gel.

The dynamic mechanical properties of milk and milk gel were examined as functions of frequency, temperature, and concentration. The real and imaginary parts of the complex rigidity G' and G" measured at various frequencies were found to be superimposable onto a single set of master relaxation curves by performing the horizontal shift along the logarithmic frequency axis except for milk gel which were prepared at different temperatures. The results show that the rigidity of milk gel depends on the concentration of milk C in such a way that G* alpha C2. It was found that G' depends on the temperature as G' alpha T. It was also found that the complex rigidity of skim milk solution depends on the angular frequency omega in such a way that G* alpha omega 1/2. The dependence of G* on frequency seems to indicate the formation of structure by casein micelle in solution.

Animals↗
PubMed1982

Dynamic viscoelastic studies on the mechanism of milk clotting process.

The reaction order of the clotting process of milk-rennet system was determined by an analysis of clotting curves obtained from the measurement of the complex rigidity at various concentrations of milk and rennet under the conditions of fixed weight ratio of rennet to milk. The results obtained show that the clotting process of milk-rennet system after gelation begins is described by a single first order reaction. At constant weight ratio of rennet to skim milk, it was found that G'00 increases almost linearly with increasing temperature. Under the conditions of a constant milk concentration and temperature, the latent period is inversly proportional to the enzyme concentration and the rate constant of gelation is proportional to the enzyme concentration. These results were discussed in terms of Payens' theory of enzymatic clotting processes and Ziff's theory of the kinetics of polymer gelation.

Animals↗
PubMed1982

Application of the theory of gelation to enzymatic clotting process of casein micelle solution.

Dynamic mechanical measurements were carried out to clarify the mechanism of the clotting process of casein micelle solution. It was found that the clotting process of casein micelle solution was formally expressed by a first order reaction. The enzyme concentration dependence of the latent time tL and the rate constant of gelation Kg were found to be tL alpha [E]-1.1, and Kg alpha [E]1.0, respectively. These results were intrepreted on the basis of the theory of gelation. The results obtained here were found to agree with the theoretical conjectures. The casein micelle concentration dependence of the complex rigidity was also studied.

Animals↗