PubMed Health⌕ Search

Biomedical subjects

T Inubushi

Publications and source records attributed to T Inubushi.

126 records · Page 7Linked to original sources
PubMed1981

Studies on cobalt myoglobins and hemoglobins. The interaction of molecular oxygen with leghemoglobin.

Cobalt-substituted leghemoglobin (CoLb) was prepared from cobaltous protoporphyrin IX and apo-leghemoglobin a, and its functional and spectroscopic properties were examined. The oxygen affinity of CoLb was pH-dependent. The partial oxygen pressure at half-saturation was decreased from 14.3 torr at pH 8 to 3.5 Torr at pH 3.5 with a pK value of 5.8 at 15 degrees C. The oxygen association and dissociation rates were determined by the temperature jump-relaxation technique. The association rate was essentially pH-independent, and the relatively high oxygen affinity of CoLb was found to be due to the fast oxygen association rate in comparison with cobalt myoglobin. The oxygen dissociation rate was pH-dependent with a pK value of 5.7 at 15 degrees C, suggesting that the pH dependence of the oxygen dissociation rate is responsible for the pH-dependent change in the oxygen affinity of CoLb. The EPR spectrum of oxyCoLb exhibited a pH-dependent change with a pK value of 5.7 at 15 degrees C. The EPR spectrum at 77 K of oxyCoLb at neutral pH exhibited a small effect upon the replacement of H2O with D2O, but it was drastically altered in the acidic pH region. The deoxy EPR spectrum was pH-independent between pH 4 and 9. These observations indicate that in oxyCoLb, the bound oxygen is interacting with the distal amino acid group with a pK value of 5.7 and that this interaction is responsible for the increased oxygen affinity in the acidic pH region.

Cobalt↗
PubMed1978

Studies on cobalt myoglobins and hemoglobins. Proton magnetic resonance investigation of the subunit interaction in iron-cobalt hybrid hemoglobins.

The paramagnetically shiftedd proton nuclear magnetic resonance spectra of iron-cobalt hybrid hemoglobins [alpha(Co)2beta(Fe)2 and alpha(Fe)2beta(Co)2], as well as those of deoxy forms of cobalt hemoglobin, iron hemoglobin, and their isolated chains, have been measured at 360 MHz. The proton NMR signals of the deoxy forms of iron and cobalt hemoglobins were individually assigned to each subunit. The NMR spectral characteristics of the alpha subunits in deoxycobalt hemoglobin, as well as those in deoxy-alpha(Co)2beta(Fe)2, were found to be quite different from those of beta(Co)2 subunits or isolated alpha-SH chain. Upon ligation of carbon monoxide to the beta(Fe)2 subunits in alpha(Co)2beta(Fe)2, the spectral properties of deoxy-alpha(Co)2 subunits became similar to those of the deoxy-beta(Co)2 subunits. No significant change in the NMR spectrum of the beta(Co)2 subunits was observed in alpha(Fe)2beta(Co)2 upon ligation of carbon monoxide to the alpha(Fe)2 subunits. These observations show the linkage of the electronic structure of the prosthetic groups with the subunits cooperativity in hemoglobin, as well as the inequivalence of the subunits. This is the first report on the paramagnetically shifte proton NMR spectra of the cobalt-substituted hemoproteins.

Carbon Monoxide↗
PubMed1978

Nuclear magnetic resonance studies of hemoprotein. Proton hyperfine shifts and structural characterization of the different heme environments in methemoglobin and metmyoglobin.

The different features of the nature of heme iron-proximal binding in methemoglobin and metmyoglobin were investigated by high resolution proton nuclear magnetic resonance spectroscopy at 220 MHz. From the comparison of the hyperfine-shifted heme methyl resonances of various methemoglobin and metmyoglobin derivatives it was revealed that the metHb derivatives exhibit smaller hyperfine shifts than the corresponding metMb derivatives. This observation was interpreted in terms of the stronger interaction between proximal histidine and ferric heme iron in methemoglobin than in metmyoglobin. The spectral contributions from the alpha and beta subunits in methemoglobin in high spin state were successfully separated by the analysis of the temperature dependent heme methyl shifts of the methemoglobin azide complex which is in the high and low thermal spin equilibrium. It was shown that the beta heme methyl resonances have smaller hyperfine shifts than the alpha heme ones in ferric high spin state. This tendency in methemoglobin derivatives was also interpreted in terms of the stronger iron-histidine binding in the beta subunits than in the alpha subunits within the tetramer. From the comparisons between methemoglobin and metmyoglobin, and between the alpha and beta subunits in methemoglobin, the order of the strength of the iron-histidine bond interaction was deduced as follows: metmyoglobin less than alpha subunits in methemoglobin less than or equal to beta units in methemoglobin.

Hemeproteins↗
PubMed1978

Nuclear magnetic resonance studies of high-spin ferric hemoproteins.

220 MHz proton Fourier transform (FT) NMR with quadrature phase detection (QPD) technique is applied to observe largely hyperfine-shifted signals of various hemoproteins and hemoenzymes in ferric high-spin state. The binding of F-, OCN-, SCN-, and CH3OH to the ferric heme iron in high-spin state in various hemoproteins has been studied by the use of FT/QPD technique at 220 MHz. The binding of formate ion to metmyoglobin (metMb) has also been studied. The spectrum of the formate complex was compared with that of hemoglobin M Milwaukee where carboxylate groups are bound to the hemes of the beta subunits. The acid-base transition of ferric myoglobin (Mb) was confirmed by monitoring the pH-dependent shift of the heme side methyl signals with the reflection point at pH 9.1. This finding is analyzed on the basis of rapid exchange between alkaline (low spin) and acidic (high spin) forms accompanied by the dissociation and association of one proton in the ferric Mb. The structure of the heme environment of ferric horseradish peroxidase (HRP) was studied. The pH-dependent features of NMR spectra of the ferric enzyme and its complexes with cyanide and azide were discussed in terms of heme environmental structures, comparing with the case of metMb. The results were interpreted as follows: There exists an ionizable amino group near the heme responsible for the ligand binding reactions of the enzyme, which modulates the entry of external azide to the heme iron through protolytic equilibrium of this group. The pK value of this group was determined to be 5.9 by monitoring the pH-dependent shift of the heme peripheral methyl signals of the native enzyme, indicating that the group is probably a histidyl residue. Acid-alkaline transition of metMb was confirmed to associate with the proton dissociation of an iron-bound water molecule, whereas in HRP, pH-dependent spin state change characterized by pK 11 is attributed not to the simple protolytic reaction of the iron-bound water but to the direct coordination of an amino acid residue of the polypeptide chain to the ferric heme iron. Histidyl imidazole is a possible candidate for the new sixth iron ligand in alkaline peroxidase above pH 11. Interaction of HRP with electron donor(indolepropionic acid, IPA) was also studied. The hyperfine-shifted proton signals of the heme peripheral groups of the enzyme showed a small but significant shift with stepwise additions of IPA, indicating that the donor binds at a specific site of HRP. There results are interpreted in terms of the interaction between the enzyme and the donor at the heme edge site.

Animals↗
PubMed1977

Proton magnetic resonance spectra of tRNA-Met-f from Thermus thermophilus.

220 MHz proton magnetic resonance spectra of tRNAs in bulk and tRNA-Met-f from Thermus thermophilus have been measured and compared with those of tRNAs from E. coli. Temperature dependences and chemical shift positions of the bulk tRNAs are well explained by the difference in their GC contents. It is known that the base sequence of the double helical regions in the cloverleaf structure of T. thermophilus tRNA-Met-f is different from that of E. coli tRNA-Met-f only at two positions in TpsiCarm; one more C:G pair is contained instead of a U:G pair of E. coli tRNA-Met-f and a C:G pair of E. coli is replaced by a G:C pair. In spite of the resembrance in the base sequences, nmr patterns around 13 ppm are fairly different from each other. The difference is discussed in relation with their tertiary structures and with the origin of chemical shift displacements.

Base Sequence↗
PubMedDate not supplied

31P NMR and enzymatic analysis of cytosolic phosphocreatine, ATP, Pi and intracellular pH in the isolated working perfused rat heart.

Hearts from fed male Wistar rats (200-350 g) were perfused at low and high workloads with Pi-free Krebs-Henseleit medium containing either 10 mM glucose or 10 mM glucose plus 15 mU/mL insulin. The intracellular pH by 31P NMR ranged between 6.99 and 7.02 and agreed to within 0.1 pH unit of estimates calculated using enzymatically determined total tissue HCO3-/CO2 contents. At high work, where the tissue contents of phosphocreatine (PCr) and ATP were determined on the same heart as NMR areas (n = 16), the proportionality factors, defined as the 31P NMR area units divided by the total enzymatically determined tissue content (area units/mumol/g dry wt), were 112 +/- 8 for PCr, 99 +/- 4 for gamma-ATP, 138 +/- 9 for alpha-ATP and 100 +/- 4 for beta-ATP. These values were normalized by taking beta-ATP as 100 area units/mumol/g dry wt. Since the proportionality factor for PCr and gamma- and beta-ATP were not statistically different (p less than 0.05), it was concluded that each was equally visible by 31P NMR and that no significant breakdown of PCr occurred during freezing or tissue acid extraction procedures. The cytosolic Pi estimated from NMR in glucose plus insulin perfused hearts at low and high work was 4.92 +/- 0.67 and 6.33 +/- 0.42 mumol/g dry wt. Using the near-equilibrium expression of KCK/KG + G and the metabolite levels in heart extracts, the calculated cytosolic Pi was 13.08 +/- 1.83 and 16.17 +/- 3.08 mumol/g dry wt, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMedDate not supplied

Diffusion-weighted MR in experimental sustained seizures elicited with kainic acid.

PURPOSE: To investigate the early changes in diffusion-weighted MR images in the sustained limbic seizures. METHOD: Intraperitoneal injection of kainic acid was used to induce sustained limbic seizures in seven rats. The animals were investigated with serial 2.0-T MR imaging beginning immediately after kainic acid-induced seizures, and at 24 hours, 3 days, and 7 days after the kainic acid injection. Diffusion-weighted spin-echo and T2-weighted images and apparent diffusion coefficients were sequentially assessed and compared with histologic changes. The results were compared with eight control animals given buffered saline intraperitoneally. RESULTS: Diffusion-weighted MR images revealed an increase in signal intensity bilaterally in the amygdala and the piriform cortices immediately after the sustained seizures, whereas T2-weighted images did not show changes in signal intensity at this time. Both diffusion-weighted and T2-weighted images showed marked increase in signal intensities in these same areas 24 hours after kainic acid injection. The apparent diffusion coefficient values were significantly lower in the area of the amygdala and the piriform cortex immediately after and lower again 24 hours after the sustained seizures. The area of hyperintensity in diffusion-weighted images was concordant with the histologic distribution of neuronal pyknosis and neuropile vacuolation. CONCLUSION: Diffusion-weighted MR revealed focal abnormalities in the limbic system after 1 hour of sustained seizures induced with kainic acid, before changes on T2-weighted imaging. Diffusion-weighted MR is a potential method for studying the mechanisms of brain damage caused by sustained seizures.

Amygdala↗
PubMedDate not supplied

Hormonal and dietary regulation of lysosomal cysteine proteinases in liver under gluconeogenesis conditions.

The activities of cathepsin B, L, J and H in rat liver were significantly increased by starvation if compared with normal diet rats. Furthermore, the activity of cathepsin L increased with glucagon treatment, and the activities of cathepsin L and H decreased significantly with insulin treatment. The changes in cathepsin B and J activities showed the same tendencies as those of cathepsin L and H, but the differences were not statistically significant. The changes in the activities of cathepsin B and L on starvation corresponded with the changes of enzyme protein amounts judged from Western blotting analysis. The levels of the lysosomal cysteine proteinases and amino acid deaminases in the liver changed in parallel with the hormonal and dietary conditions. The increases of alanine amino transferase activity (AAT) started from a much earlier stage than those of cathepsins under the starvation condition. Although administration of prednisolone caused marked induction of the deamination enzymes such as AAT, the levels of cathepsins in the liver were not changed.

Animals↗