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Biomedical subjects

Y Kyogoku

Publications and source records attributed to Y Kyogoku.

At least 145 records · Page 8Linked to original sources
PubMed1985

Photo CIDNP study on the complex formation of lambda cro protein with OR3.

A photo CIDNP spectrum of lambda cro repressor protein showed that one of the three tyrosines and His 35 are quite accessible to the photosensitive dye. For the remaining two tyrosine residues one is slightly accessible, but the other is inaccessible. In comparison with the result of differential nitration at tyrosine side chains followed by the peptide analysis, it can be concluded that Tyr 26 is mostly exposed and Tyr 51 is slightly exposed on the surface of the cro dimer. On the addition of OR3 17mer, His 35 and Tyr 26 are no longer accessible to the dye, which indicates that they are involved in interaction. However, a similar phenomenon was observed by adding CAP binding site 22mer. The interaction mechanism will be discussed.

Bacteriophage lambda↗
PubMed1985

Phosphorus-31 nuclear magnetic resonance of highly oriented DNA fibers. 1. Static geometry of DNA double helices.

The static geometry of the phosphodiesters in oriented fibers of DNA and a variety of polynucleotides was investigated by solid-state 31P nuclear magnetic resonance (NMR) spectroscopy. The structural parameters of the phosphodiester backbone expressed by two Euler angles beta and gamma were estimated on the basis of the NMR spectra of natural DNA, poly(dA).poly(dT), poly(rA).poly(dT), and poly-(rA).poly(rU). The Euler angles were calculated by using the known single crystal structures of a decamer, r(GCG)d(TATACGC), and a dodecamer, d(CGCGAATTCGCG). The distribution pattern of the Euler angles was quite different between these two oligonucleotides due to the different types of conformation, and it was fully consistent with the 31P NMR results, showing that the conformation of the B form DNA is very heterogeneous while that of the A or A' form is much more invariable with regard to the base composition. The structural parameters were also calculated by using various structures determined by the X-ray fiber diffraction studies, and they were evaluated on the basis of the 31P NMR data. Notably, poly(dA).poly(dT) fibers exhibited abnormal 31P NMR spectra which were very broad in line width and were not appreciably perturbed by hydration; a coiled double-helical structure is proposed as the most plausible model for this polymer.

Animals↗
PubMed1985

Hybrid oligomer of cyclonucleotides and deoxynucleotides. A high anti left-handed double helical DNA structure.

It has been shown by us that oligonucleotides containing cyclonucleosides with a high anti glycosidic conformation take left-handed, single and double helical structures (S. Uesugi, J. Yano, E. Yano and M. Ikehara, J. Am. Chem. Soc. 99,2313 (1977) and references therein). In order to see whether DNA can adopt the high anti left-handed double helical structure or not, a self-complementary hexanucleotide containing 6,2'-O-cyclocytidine (C 0). 8,2'-O-cycloguanosine (G 0), deoxycytidine and deoxyguanosine, C 0 G 0 dCdGC 0 G 0, was synthesized. Corresponding hexanucleotide containing only cyclonucleosides, C 0 G 0 C 0 G 0 C 0 G 0, was also synthesized. Their conformation was examined by UV, CD and 1H NMR spectroscopy. C 0 G 0 C 0 G 0 C 0 G 0 forms an unusually stable, left-handed duplex. Imino proton NMR spectra and the results of nuclear Overhauser effect experiments strongly suggest that C 0 G 0 dCdGC0 G 0 take a left-handed double helical structure where the deoxynucleoside residues are involved in hydrogen bonding and take a high anti glycosidic conformation. Thus it is revealed that DNA could form a high anti, left-handed double helix which is different from that of Z-DNA under some constrained conditions.

DNA↗
PubMed1985

Alcohol induced B-A transition of DNAs with different base compositions studied by circular dichroism.

The circular dichroic (CD) spectra of natural DNAs (from Cl. perfringens, T2 phage, calf thymus, E. coli, and M. lysodeikticus) as well as duplexes of synthetic DNAs (poly(dA) X poly(dT), poly(dA-dT), and poly(dG-dC] were measured in water-ethanol mixtures with 0.3 mM NaCl. A conformational change from the B to the A form was observed for the natural DNAs on adding ethanol. The ethanol concentration that induces the transition and the extent of the change in the CD spectrum are different for the five natural DNAs depending on their GC contents. The higher the GC content is, the more easily the transition to the A form takes place. The results indicate that the GC content of a DNA is an important factor for induction of the B-A transition. The results for the synthetic DNAs show that their properties cannot be inferred by simple extrapolation of those of natural DNAs. Coexisting ions and the molecular weight of a DNA were also found to affect the induction of the B-A transition.

Animals↗
PubMed1985

Exposed tyrosine residues of lambda cro repressor protein evidenced by nitration and photo CIDNP experiments.

The tyrosine residues of lambda cro repressor were partially nitrated with tetranitromethane under mild conditions. After digestion by Achromobacter protease I, the extent of nitration was determined by HPLC and amino acid analysis. Tyr 26 was most easily nitrated and Tyr 51 followed it. Tyr 10 was resistant to nitration. By comparison of the proton magnetic resonance spectrum of the partially nitrated cro protein with the above result, the aromatic proton resonances of the tyrosine side chains could be assigned to individual tyrosine residues. The extent of nitration is parallel to the accessibility to a flavin dye as measured by photo CIDNP (chemically induced dynamic nuclear polarization).

Amino Acids↗
PubMed1985

Dynamic behavior of the imino protons of the gamma OR3 17mer in H2O solution studied by high-resolution NMR.

The imino proton resonances of gamma OR3 17mer in water were observed at 500 MHz with the time-shared Redfield pulse train. All of the 17 imino proton resonances could be assigned specifically to individual base pairs by utilizing the trace of NOE connectivities between the imino and adenine C2H protons and between imino protons themselves. AT1 and 17 showed abnormally high chemical shifts in comparison with the other AT pairs. On raising the temperature, broadening of the signal occurred in a sequential manner from the terminals except for AT10 and AT11, which were broadened at a lower temperature than GC12. The relaxation rates of the imino protons were measured by the inversion recovery method. The rates at higher temperatures represent the exchange rates of the imino protons. From the temperature dependences, activation energies of about 15 kcal/mol for the AT imino protons and 23-26 kcal/mol for the GC imino protons were obtained.

Bacteriophage lambda↗
PubMed1985

Specificity of the interaction between lambda cro repressor protein and operator DNA fragments.

Cro repressor protein is known to interact with specific sites in the operator DNA. The cro protein of lambda phage was isolated and the mode of its interaction with three different DNA fragment, lambda-OR3 17mer, phi 80-OR2 19mer and CAP binding site 22-mer, were examined by the use of proton NMR. Some of the imino proton resonances of lambda-OR3 shifted and were broadened remarkably on addition of lambda-cro protein, which indicated the induction of conformational change with complexation. In the spectrum of phi 80-OR2 which has a six base pair sequence common to lambda-OR3 the signals of the common base pairs revealed slight shifts on addition of lambda-cro protein. The imino proton signals of the CAP site DNA, however, did not show any change at all on mixing with lambda-cro. Combining the data of photo CIDNP of lambda-cro, we could postulate the mode of interaction between lambda-cro repressor and operator DNA.

Bacteriophage lambda↗
PubMed1984

Synthesis and conformational studies of ribooligonucleotides which contain an alternating C-G sequence and show unusual circular dichroism spectra.

The poly [r(C-G)] duplex shows an unusually large negative CD band in the long wavelength region. In order to elucidate this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized by a phosphotriester method and their properties were examined by UV, CD, 1H and 31P NMR spectroscopy. These ribooligomers form self-duplexes at low temperature, the CD spectra of which show negative bands at around 290 nm and positive bands at around 265 nm. The results of 1H nuclear Overhauser effect experiments, 1H chemical shift-temperature profiles of base protons, and the sharp singlet observed for all H1' protons are consistent with a normal A-RNA structure but not with a Z-DNA like structure. The CD-temperature profiles and 31P NMR spectra support this conclusion. These results indicate that RNA duplexes with an alternating C-G sequence can give an unusually large negative CD band in the long wavelength region despite their right-handed helical structure.

Base Sequence↗
PubMed1984

Photochemically induced dynamic nuclear polarization study on microsomal NADPH-cytochrome P-450 reductase.

Sedimentation equilibrium experiments with NADPH-cytochrome P-450 reductase showed that increasing 1-O-n-octyl-beta-D-glucopyranoside levels promoted disaggregation of the flavoprotein. The reductase was monomeric at a molar ratio of detergent to protein above 10(3). Addition of N3-carboxymethyllumiflavin to the flavoprotein in the presence of 1-O-n-octyl-beta-D-glucopyranoside results in photochemically induced dynamic nuclear polarization (CIDNP) signals in the aromatic region. The CIDNP spectrum of the holoprotein shows sharp resonances due to histidine residues. On removal of FMN from the protein, CIDNP signals originating from a tyrosine residue appeared, suggesting that the tyrosine residue is exposed to solvent after the depletion of FMN. However, this tyrosine residue appears to become inaccessible to the external dye after full incubation of FMN-depleted reductase with FMN. This suggests that the tyrosine residue could be located in the vicinity of the FMN-binding domain which constitutes the active center of the reductase.

Animals↗
PubMed1984

Identification of oligonucleotide fragments produced in a strand scission reaction of the d(C-G-C-G-C-G) duplex by bleomycin.

To elucidate the mechanism of DNA strand scission by bleomycin, a d(C-G-C-G-C-G) duplex was treated with the bleomycin-iron ion complex in the presence of H2O2 and degradation products (1, 2, cytosine and deoxyguanosine 5'-phosphate) were identified. 1 and 2 contain a carboxymethyl group attached to the 3'-terminal phosphoryl group of d(C-Gp) and d(C-G-C-Gp), respectively. These compounds were identified by UV, 1H and 31P NMR spectroscopy and paper electrophoresis. 1 was synthesized from the protected dinucleotide and glycolic acid and the proton NMR spectrum was identical to that of 1 obtained as a degradation product. Thus the oligonucleotide fragments produced by the action of bleomycin on DNA were directly identified and cleavage of the C3'-C4' bond of the sugar residues was proved.

Bleomycin↗
PubMed1984

Conformation of ribooligonucleotide duplexes containing an alternating C-G sequence which show an unusual circular dichroism spectrum.

The poly[r(G-C)] duplex shows an unusually large negative band in the long wavelength region of the CD spectrum. In order to elucidate this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized chemically and their properties were examined by UV and CD, and 1H and 31P NMR spectroscopy. These ribooligomers form a self-complementary duplex at low temperature, the CD spectrum of which shows a negative band at around 290 nm and a positive band at around 265 nm with almost equal magnitudes. The proton resonances in the 1H NMR spectra of the oligo[r(C-G)] duplexes were assigned by nuclear Overhauser effect experiments. The chemical shift-temperature profiles of the base proton signals and the sharp singlets observed for all H1' protons are consistent with a normal A-RNA structure but not with a Z-DNA like structure. Moreover, a 500-MHz two-dimensional nuclear Overhauser effect experiment recorded for r(C-G-C-G-C-G) shows that all guanine bases adopt the normal anti-conformation. CD-temperature profiles and 31P NMR spectra of oligo[r(C-G)]s support this conclusion. These results indicate that duplexes of oligo- and polyribonucleotides containing alternating C-G sequences can give an unusually large negative CD band in the long wavelength region despite their right-handed helical structure.

Base Sequence↗
PubMed1984

The effect of protamine on serum complement activity in aminonucleoside nephropathy.

Combined administration of protamine sulfate (Ps) and aminonucleoside (AN) to rats causes severe nephrotic syndrome and, histologically, focal glomerular sclerosis. These changes are more distinct than those produced by AN alone. While the exact mechanism of Ps acting on AN nephropathy is not known, marked hypocomplementemia was observed regularly after the Ps + AN injection, suggesting that complement is activated and consumed in the kidney. It is suggested that complement may play an important role in AN + Ps nephropathy.

Animals↗
PubMed1984

High resolution NMR study of CAP binding site 22mer in H2O solution.

High resolution proton NMR were measured for the deoxyoligonucleotide 22mer duplex corresponding to the CAP (catabolite gene activator protein) binding site of lac promotor. The spectra in the lower field region than the water resonance were taken with the time-shared Redfield pulse method by using a JEOL 500 MHz NMR spectrometer. In the imino proton region 18 peaks were separately observed, but the area intensity at 10 degrees C corresponds to 20 protons. By selective irradiation at each peak position NOEs (nuclear Overhauser effects) were observed between the imino and adenine C2H protons and between imino proton themselves. By tracing sequential NOE train carefully, 17 imino proton signals could be unambiguously assigned to each base pair except five AT base pairs at terminals. With the elevation of temperature the peaks showed gradual broadening and disappeared, which indicates the stepwise base pair opening of the duplex. Referring to the above peak assignments it can be concluded that GC20 and AT4 pairs close to terminals relax first and the base pair opening proceeds toward central GC13 and 14.

Base Sequence↗
PubMed1983

Determination of protonation sites in thermospermine and in some other polyamines by 15N and 13C nuclear magnetic resonance spectroscopy.

The pK values of thermospermine, a novel asymmetric tetraamine (4,8-diazadodecane-1,12-diamine), have been determined for the first time by the conventional titration method with subsequent data analyses. It was found to be the most basic polyamine among naturally occurring tetraamines. The 15N NMR titration method was applied to determine its protonation site. Natural-abundance 15N magnetic resonance spectra of the polyamine have been recorded as a function of pH from its free to the protonated form. Its apparent pK values, as well as the intrinsic chemical shifts for each stage of protonation, have been calculated from the NMR titration curve by using a nonlinear least-squares method. The four protonation sites of thermospermine have been successfully determined to be N12, N1, N-8, and N-4 in that order from the free to the completely protonated form. In a similar manner the protonation sites of spermidine and two symmetric tetraamines, thermine and spermine, have been established. 13C NMR experiments for the four polyamines were almost consistent with those of 15N NMR. Owing to the much higher concentration of the polyamines furnished for NMR titration experiments, the pK values obtained by NMR were larger than those derived by the conventional titration methods.

Binding Sites↗
PubMed1983

Classification of iron-sulfur cores in ferredoxins by 1H nuclear magnetic resonance spectroscopy.

A 1H nuclear magnetic resonance (NMR) study was carried out on various ferredoxins which possess one of three types of iron-sulfur clusters, (2Fe-2S), (3Fe-3S), or (4Fe-4S). In the isolated form, (2Fe-2S) ferredoxins from spinach (Spinacea oleracia), pokeweed (Phytolacca americana), a blue-green alga (Spirulina platensis), and a halobacterium (Halobacterium halobium) exhibited two broad resonances common in chemical shift at the region downfield of 10 ppm. In their reduced forms, seven contact-shifted resonances appeared spread over 30 ppm. Although the positions of the contact-shifted resonances in the reduced state differed among the four, a common trend in the temperature dependence of their resonance positions was recognized. Two (4Fe-4S) ferredoxins from Bacillus stearothermophilus and Bacillus thermoproteolyticus exhibited almost indistinguishable spectral patterns in both the oxidized and reduced forms. The ferricyanide-treated ferredoxins of B. stearothermophilus and B. thermoproteolyticus showed characteristic contact-shifted resonances distinct from the spectra of the original (4Fe-4S) ferredoxins. This corresponds to the recent finding of the interconversion of (4Fe-4S) and (3Fe-3S) clusters with ferricyanide in the ferredoxin. Based on our data together with reported NMR data on other ferredoxins, contact-shift resonances of three types of clusters were tabulated. The reliability of NMR classification increases when we compare the NMR spectra of a ferredoxin with the classification standards at the two redox states. Moreover, not only the absolute values of the chemical shifts of contact-shifted resonances but also their temperature dependence give distinctive information applicable to iron core identification.

Azotobacter↗
PubMed1983

Origin of slow conformer conversion of triostin A and interaction ability with nucleic acid bases.

The n.m.r. pattern of triostin A in weakly polar solvents was explained by the presence of two symmetrical conformers. Its S-benzyl derivative still shows the n.m.r. pattern of two conformers, while des-N-tetramethyltriostin A, which lacks the N-methyl groups, gives one conformer. Therefore the slow interconversion of two conformers arises from the cis-trans isomerization of the N-methyl peptide bonds, but not from the reversal of the chirality around the S-S bond. Only one of the two conformers of triostin A interacts with adenosine and guanosine derivatives. Des-N-tetramethyltriostin A can also interact with the purine nucleosides, but more preferentially with the adenosine derivative.

Adenosine↗