[Electron transfer and interheme interactions in a tetraheme protein, cytochrome c3].
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Cytochrome c Group↗
Biomedical subjects
H Akutsu
Publications and source records attributed to H Akutsu.
Full assignment of heme redox potentials of cytochrome c3 of D. vulgaris Miyazaki F by 1H-NMR.
Site-specific heme assignment of the 1H-NMR spectrum of cytochrome c3 of D. vulgaris Miyazaki F, a tetraheme protein, was established. The major reduction of the heme turned out to take place in the order of hemes I, III, IV and II (numbering in the crystal structure). The hemes with the smallest and greatest solvent accessibility were reduced at the highest and lowest potentials on average, respectively. A cooperative interheme interaction was attributed to a pair of the closest hemes, namely, hemes III and IV. This assignment can provide the physiochemical basis for the elucidation of electron transfer of this protein.
Cytochrome c Group↗
Conformational analysis of the polar head group in phosphatidylcholine bilayers: a structural change induced by cations.
The conformation of the polar head group of phosphatidylcholine in a bilayer in the liquid-crystalline state was deduced by analyzing the deuterium quadrupole splittings of the choline group and the phosphorus chemical shift anisotropy of the phosphate group in combination with the restriction of the choline conformation determined in laser Raman studies. The latter efficiently reduced the number of candidates for the actual conformation. A family of conformations was obtained for both the dynamic-structure and rigid-structure models, respectively. The polar head group is oriented roughly parallel to the membrane surface in both models. Furthermore, they are close to conformation A of the crystal structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The dynamic-structure model was concluded to be more reasonable in view of the fact that the polar head-group structures in most crystals comprise two conformations, which are nearly mirror images of each other. Conformational analysis was also carried out for the polar head group in the presence of multivalent cations. A possible conformational change of the polar head group induced by cations is discussed in the light of the present results.
Cations↗
1H NMR studies on ferricytochrome c3 from Desulfovibrio vulgaris Miyazaki F and its interaction with ferredoxin I.
The 1H NMR signals of the heme methyl, propionate and related chemical groups of cytochrome c3 from Desulfovibrio vulgaris Miyazaki F (D.v. MF) were site-specifically assigned by means of 1D NOE, 2D DQFCOSY and 2D TOCSY spectra. They were consistent with the site-specific assignments of the hemes with the highest and second-lowest redox potentials reported by Fan et al. (Biochemistry, 29 (1990) 2257-2263). The site-specific heme assignments were also supported by NOE between the methyl groups of these hemes and the side chain of Val18. All the results contradicted the heme assignments for D.v. MF cytochrome c3 made on the basis of electron spin resonance (Gayda et al. (1987) FEBS Lett., 217 57-61). Based on these assignments, the interaction of cytochrome c3 with D.v. MF ferredoxin I was investigated by NMR. The major interaction site of cytochrome c3 was identified as the heme with the highest redox potential, which is surrounded by the highest density of positive charges. The stoichiometry and association constant were two cytochrome c3 molecules per monomer of ferredoxin I and 10(8) M-2 (at 53 mM ionic strength and 25 degrees C), respectively.
Binding Sites↗
Psychophysics of reading--X. Effects of age-related changes in vision.
This study examined the influence of age-related changes in vision on reading performance. Maximum reading speed was measured in groups of young (n = 16, mean age 21.6 years) and old (n = 14, mean age 68.3 years) subjects, all with acuities of 20/32 or better. A psychophysical procedure was used for measuring reading speed that has proven reliable and sensitive to visual factors in previous research. Data were collected for character sizes ranging from .15 degrees to 12 degrees. Research revealed that old subjects who were free of eye disease read as fast as the young subjects for character sizes ranging from .3 degrees to 1.0 degrees. This is the range in which reading speed is maximum for young subjects. Research also revealed that old subjects showed a deficit when reading text composed of very small or very large characters. Their speeds dropped to about 70% of the young adult speeds. These deficits may be due to age-related losses in visual contrast sensitivity.
Adult↗
[The metabolic index of nocturnal hypoxia in patients after lung resection and thoracoplasty].
The authors measured urinary uric acid (UA) and creatinine (CR), serum lactate, and CoQ10 prior to retiring at night and on awakening in the morning in 127 patients (PG) after lung resection and thoracoplasty which were done more than 20 years age for treatment of tuberculosis and in 20 controls (NC). delta UA:CR, delta lactate, and delta CoQ10 were calculated respectively as the overnight changes in urinary UA:CR and in serum lactate and CoQ10. delta UA:CR increased in PG (4.0 +/- 43.6%), whereas it decreased in NC (-22.3 +/- 17.7%) (mean +/- SD) (p less than 0.05). Nocturnal hypoxemia suggested from positive balance of delta UA:CR was seen in 37% of PG, but in only 10% of NC. delta UA:CR showed no relationship with delta lactate and delta CoQ10 and also did not correlate with the nadir of arterial oxygen saturation. PG were divided into PG with a positive balance of delta UA:CR (PG-P) and with a negative balance of delta UA:CR (PG-N). The %VC and PaO2 in the PG-P group were lower and PaCO2 was higher than in PG-N, although the difference did not achieve statistical significance.
Aged↗
[Clinical study of centrifugal pump bypass as an auxiliary technique for surgical treatment of descending thoracic aorta].
Left heart bypass or arterial bypass using a centrifugal pump (Bio-pump bypass) with a H-PSD shunt tube was performed as an auxiliary technique for the treatment of descending thoracic aorta in 13 cases. Hemodynamic differences during aortic clamp were compared between cases using Bio-pump bypass and 4 cases of axillo-femoral temporary bypass which were carried out in the first term. Furthermore, in Bio-pump bypass cases, the bypass route was investigated from the point of view of operative complications in relation to bypass technique. Peripheral blood pressure and urinary output during aortic clamp were significantly increased in the Bio-pump bypass group. Those results indicated that the Bio-pump bypass was useful as an auxiliary technique especially for high risk patients with low renal function. In particular, left heart bypass was a beneficial technique which was not affected by modality or region of disorder in the descending thoracic aorta. However, this technique should be selected carefully for patients with cardiac disease, for instance severe left ventricular hypertrophy, because two cases of operative complications consisting of pericardial effusion related to the bypass technique were experienced in this study.
Aorta, Thoracic↗
Direct observation of the phase behavior of the lipid bilayers of phage PM2 and the intact host cells by 1H-31P cross-polarization NMR.
A method for obtaining the 31P NMR spectrum of a particular supramolecular structure in an intact biological system was developed by applying the 1H-31P cross-polarization technique to a lipid-containing bacteriophage, PM2, and its host bacterium, Alteromonas espejiana. It was shown that 31P NMR spectra of nucleic acids and lipid bilayers can be obtained separately with short and long thermal contact times, respectively. The temperature dependence of the chemical shift anisotropy (delta sigma = sigma parallel - sigma perpendicular) was examined for the separately obtained membrane spectra. Referring to the results of thermal analysis and 31P NMR spectra of bilayers of the extracted phospholipids, the phase transition of the biomembrane was identified for the PM2 phage and the host cell. The dynamic state of the biomembrane of the intact bacterium was directly monitored in detail. The phase behavior of the PM2 lipid bilayer showed good agreement with the earlier report (Akutsu et al., 1980). It turned out that the phase behavior of the intact biomembrane is different from that of the bilayer of the extracted lipids for both PM2 and the host cell. Namely, the terminal temperatures of the phase transition of the host cell and PM2 membranes were lower and higher than those of the extracted phospholipids, respectively.
Bacteriophage lambda↗
Assignment of tyrosine resonances in the 1H-NMR spectrum of tryptophan synthase alpha-subunit. Monitoring conformational changes due to substitutions at position 49.
In order to monitor the conformational changes of tryptophan synthase alpha-subunit from Escherichia coli in solution resulting from amino acid substitutions, we have assigned the Tyr resonances in the aromatic region of the 1H-NMR spectrum to specific residues. In the spectrum of the alpha-subunit deuterated with [2,3,4,5,6-2H5]Phe and [3,5-2H2]Tyr, the C2 and C6 protons of Tyr gave completely isolated signals at acidic p2H. Some of the C3 and C5 proton resonances overlapped with each other at acidic p2H. By using a series of mutant alpha-subunits in which each Tyr was singly substituted with His or Phe, we can now assign each of seven Tyr resonances in the aromatic region to a specific residue. We have previously studied the conformational stability of a series of variant alpha-subunits at position 49 [Yutani et al. (1987) Proc. Natl Acad. Sci. USA 84, 4441-4444]. We now compare the 1H-NMR spectra in the aromatic region of the wild-type alpha-subunit and mutant alpha-subunits substituted with Phe or Gly in place of Glu-49. The results suggest that the major conformational effects of substitutions at position 49 are localized close to the position of substitution.
Binding Sites↗
Estimation of microscopic redox potentials of a tetraheme protein, cytochrome c3 of Desulfovibrio vulgaris, Miyazaki F, and partial assignments of heme groups.
The microscopic formal redox potentials of a tetraheme protein, cytochrome c3 from Desulfovibrio vulgaris, Miyazaki F, were estimated from the chemical shifts of the heme methyl signals in its 1H NMR spectrum. All chemical shifts in the five macroscopic oxidation states were determined for eight of the heme methyl protons by the saturation-transfer method. The electron-distribution probability at each heme in each oxidation state was estimated directly from the chemical shifts. To minimize errors due to interheme pseudocontact contributions, the average electron-distribution probability was used for calculation of the microscopic formal redox potentials. By introducing interacting potentials, 32 parameters were reduced to 10. The 10 parameters were determined analytically from the 9 independent electron-distribution probabilities and 2 macroscopic formal redox potentials. The results showed the presence of a strong positive interaction between a pair of particular hemes. The microscopic formal redox potential changes dramatically with the extent of reduction because of the intramolecular interheme interactions. NMR signals of two hemes were assigned to particular hemes in the crystal structures by nuclear Overhauser effect experiments. The results showed that the hemes with the highest and lowest redox potentials in the one-electron reduction process correspond to hemes I and IV in the crystal structure.
Cytochrome c Group↗
Physiological dynamic structures of nucleic acids in A. espejiana cells detected on 1H-31P cross-polarization NMR.
In order to obtain information of the supramolecular structures of nucleic acids in vivo, the 1H-31P cross-polarization NMR technique was applied to intact marine bacterial cells. An asymmetric powder pattern spectrum of nucleic acids in the cell was observed. The major contributor to the spectrum was ribosomes. Furthermore, the powder pattern changed dramatically with the physiological conditions of the cells. The results showed that this is a promising method for the investigation of the supramolecular structures of nucleic acids in vivo.
Bacteria, Aerobic↗
Redox process of iron-sulfur clusters of the soluble-domain of the membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F studied by resonance Raman spectroscopy.
Resonance Raman spectra of the soluble-domain of a membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F were recorded in different oxidation states. In the oxidized state, the Raman band due to the totally symmetric stretching mode of the iron-sulfur cluster was observed at 341 cm-1, which was attributed to the 3Fe-4S cluster. In the hydrogen-reduced state, only a weak and broad band was observed in its vicinity. During the process of reoxidation, a Raman band assignable to the 4Fe-4S cluster was observed at 333 cm-1 in the first step. Then, the band at 341 cm-1 became stronger and eventually dominated the spectrum. Corresponding changes were observed in the visible absorption spectra of the same sample. It was concluded from these observations that this hydrogenase has both 3Fe-4S and 4Fe-4S clusters and takes on at least three oxidation states, namely, oxidized, intermediate, and hydrogen-reduced ones.
Binding Sites↗
[Hemolysis and red cell deformability during cardiopulmonary bypass--the effect of prostaglandin E1 for prevention of hemolysis].
Prevention of hemolysis which is related to renal failure during cardiopulmonary bypass (CPB) is very important. We discussed the relationship between hemolysis and red cell deformability during CPB, and moreover the effect of PGE1 to reduce hemolysis. PGE1 was given during CPB (10 approximately 20 ng/kg/min). Red cell deformability was measured using 20% Ht red cell suspension and Nucleopore Microfilter (pore size: 5 mu). Red cell filtration rate (RFR: microliter/sec) was calculated as a value of red cell deformability. Red cell deformability (RFR: microliter/sec) was reduced during CPB in almost all patients. Secondly, plasma Hb (mg/dl) was measured in the controls (n = 8) and PGE1 group (n = 12). The mean pre-bypass level were 20.6 in the control group and 23.8 in the PGE1 group. The mean values of plasma Hb at 30, 60, 90 and 120 min of CPB were 37.8, 52.4, 52.3 and 61.2 in the control group and 24.1, 25.0, 26.3 and 29.0 in the PGE1 group. Our study showed conclusively that CPB has a detrimental effect on red cell deformability and that this effect is accentuated by prolongation of CPB time. PGE1 lessened the decrease in red cell deformability during CPB and was shown to be very effective for prevention of hemolysis.
Alprostadil↗
[An aged case of peripheral arterial obstructive disease with progressive limb necrosis].
An aged case of peripheral arterial obstructive disease with progressive limb necrosis is reported. A 70-year old man was admitted to our hospital with the complaint of numbness and rest pain on left lower limb. Thereafter, necrosis of the digital region of the upper limbs and the toe region of the lower limbs became progressively worse 3 weeks after onset. Bilateral lower limb amputations after lumbar sympathectomy and digital amputation of upper limbs after thoracic sympathectomy were performed. The histological findings of artery and vein in amputated limbs showed thromboangiitis obliterans. We conclude that Buerger's disease in the aged is rare, but does really exist.
Aged↗
A 2H-NMR study on the glycerol backbone of phospholipids extracted from Escherichia coli grown under high osmotic pressure: evidence for multiconformations of phosphatidylethanolamine.
A glycerol-requiring auxotroph was isolated from mutagenized Escherichia coli K-12 UFAts cells. This auxotroph was used for the specific deuteration of E. coli phospholipids. The cells were grown under high osmotic pressure (in the presence of 2.0% KCl). The membrane had a highly saturated fatty acid composition (76% phosphatidylethanolamine, 20% cardiolipin and 4% phosphatidylglycerol). The deuterium magnetic resonance spectra of coarse liposomes of the extracted phospholipids with perdeuterated glycerol incorporated into them were measured. To obtain well characterized information, phospholipid mixtures reconstituted from the deuterated and nondeuterated components at the same ratios as in the case of the total extract were used. On the analysis of the spectra, the following conclusions were drawn. (1) The whole polar region of cardiolipin is dynamically symmetric and quite rigid in the presence of phosphatidylethanolamine. (2) Although the quadrupole splittings of the deuterons at the C-2 and C-3 positions of the glycerol backbone were similar to each other, those at the C-1 position for phosphatidylethanolamine and cardiolipin are different, even in the same bilayer. (3) Furthermore, each C-1 deuteron of phosphatidylethanolamine gave rise to a doublet, suggesting the presence of two backbone conformations, between which there is slow exchange. (4) The polar head group of phosphatidylethanolamine interacts with cardiolipin and phosphatidylglycerol in different ways, which could be responsible for the different osmotic properties of the vesicles composed of them.
Cardiolipins↗
A photo-chemically induced dynamic nuclear polarization NMR study on rabbit and bovine cytochrome b5.
Although it has been indicated that proteins with chromophoric groups are not suitable for photo-chemically induced dynamic nuclear polarization (photo-CIDNP) measurements, we have successfully obtained these spectra for a heme protein, cytochrome b5. The characteristics of the spectra differed in some points from those so far reported. The intensities of the signals in the aromatic region were very weak, while those of the beta-methylene protons of one histidine and one tryptophan were extremely strong in comparison with the aromatic protons. It was demonstrated, on the basis of the photo-CIDNP spectrum, that one of seven histidines, all three tyrosines and a single tryptophan of the rabbit soluble cytochrome b5 are exposed on the surface of the protein. The results of comparison of the photo-CIDNP spectra for the rabbit soluble and intact, and bovine intact, cytochrome b5 led us to the conclusion that the conformation of the hydrophilic, catalytic part of cytochrome b5 is quite similar among these three proteins. In the presence of Chaps micelles, bovine intact cytochrome b5 was in monomeric form and the histidine signals disappeared from its photo-CIDNP spectrum. When bovine intact cytochrome b5 was reconstituted into egg yolk phosphatidylcholine liposomes, although separate signals due to the protein part were observed in the normal 1H-NMR spectrum, no photo-CIDNP signal could be detected. The normal spectrum suggests that the conformation of the protein embedded in liposomes is similar to that of the oligomeric form without lipids or a detergent.
Animals↗
Proton nuclear magnetic resonance studies on the wild-type and single amino acid substituted tryptophan synthase alpha-subunits.
In order to elucidate the effect of single amino acid substitutions on the conformation of the tryptophan synthase alpha-subunit from Escherichia coli in solution, 1H NMR spectra of the wild-type and mutant proteins were measured at various pHs. Two of the four His C2-proton resonances of the alpha-subunit were assigned to two His residues at positions 92 and 146 by using a mutant protein with Thr substituted for the His at position 92. The replacement did not affect the conformation of the protein significantly. The proton resonances of all the Tyr residues in the aromatic region could be picked up from other resonance peaks, employing the wild-type alpha-subunit deuterated at all of the Phe residues. On comparison of the spectra of the wild-type protein with those of the mutant protein with Met substituted for the Glu at position 49, it was concluded that the substitution affects only the residues close to the substituted residue at acidic pH but that a larger part of the protein is affected at alkaline pH. NOE experiments showed that the five Tyr residues, four of which are located in the proximity of position 49, are close to one another. The present results are discussed in the light of the conformational stability of the protein.
Amino Acids↗
The presence of the 30 nm filament structure of chromatins in intact chicken erythrocytes observed by 31P NMR.
A 31P NMR spectrum of chicken erythrocyte chromatins in vivo was obtained. The spectrum was characterized by examining the spectra of isolated nuclei and nondigested chromatins under various conditions. The results have shown that most chromatins in chicken erythrocyte nuclei assume the 30 nm filament structure as a second step of condensation of DNA in vivo.
Animals↗