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Biomedical subjects

M Tamura

Publications and source records attributed to M Tamura.

At least 937 records · Page 52Linked to original sources
PubMed1983

Microsomal NADH-cytochrome b5 reductase of bovine brain: purification and properties.

Bovine brain microsomal NADH-cytochrome b5 (cyt. b5) reductase [EC 1.6.2.2] was solubilized by digestion with lysosomes, and purified 8,500-fold with a 20% recovery by procedures including affinity chromatography on 5'-AMP-Sepharose 4B. The purified enzyme showed one band of a molecular weight of 31,000 on polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). Polyacrylamide gel electrophoresis of the purified enzyme without SDS revealed a major band with a faint minor band, both of which exhibited NADH-cyt. b5 reductase activity. The isoelectric points of these components were 6.0 (major) and 6.3 (minor). The apparent Km values of the purified enzyme for NADH and ferricyanide were 1.1 and 4.2 microM, respectively. The apparent Km value for cyt. b5 was 14.3 microM in 10 mM potassium phosphate buffer (pH 7.5). The apparent Vmax value was 1,190 mumol cyt. b5 reduced/min/mg of protein. The NADH-cyt. b5 reductase activity of the purified enzyme was inhibited by sulfhydryl inhibitors and flavin analogues. Inhibition by phosphate buffer or other inorganic salts of the enzyme activity of the purified enzyme was proved to be of the competitive type. These properties were similar to those of NADH-cyt. b5 reductase from bovine liver microsomes or rabbit erythrocytes, although the estimated enzyme content in brain was about one-twentieth of that in liver (per g wet tissue). An immunochemical study using an antibody to purified NADH-cyt. b5 reductase bovine liver microsomes indicated that NADH-cyt. b5 reductase from brain microsomes is immunologically identical to the liver microsomal enzyme.

Ammonium Sulfate↗
PubMed1982

Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model.

The subunit structure of islet-activating protein (IAP), pertussis toxin, has been analyzed to study a possibility that this protein is one of the A-B toxins [Gill, D. M. (1978) in Bacterial Toxins and Cell Membranes (Jeljaszewicz, J., & Wadstrom, T., Eds.) pp 291-332, Academic Press, New York]. Heating IAP with 1% sodium dodecyl sulfate caused its dissociation into five dissimilar subunits named S-1 (with a molecular weight of 28 000), S-2 (23 000), S-3 (22 000), S-4 (11 700), and S-5 (9300), as revealed by polyacrylamide gel electrophoresis; their molar ratio in the native IAP was 1:1:1:2:1. The molecular weight of IAP estimated by equilibrium ultracentrifugation was 117 000 which was not at variance with the value obtained by summing up molecular weights of the constituent subunits. The preparative separation of these IAP subunits was next undertaken; exposure of IAP to 5 M ice-cold urea for 4 days followed by column chromatography with carboxymethyl-Sepharose caused sharp separation of S-1 and S-5, leaving the other subunits as two dimers. These dimers were then dissociated into their constituent subunits, i.e., S-2 and S-4 for one dimer and S-3 and S-4 for the other, after 16-h exposure to 8 M urea; these subunits were obtained individually upon further chromatography on a diethylaminoethyl-Sepharose column. Subunits other than S-1 were adsorbed as a pentamer by a column using haptoglobin as an affinity adsorbent. The same pentamer was obtained by adding S-5 to the mixture of two dimers. Neither this pentamer nor other oligomers (or protomers) exhibited biological activity in vivo. Recombination of S-1 with the pentamer at the 1:1 molar ratio yielded a hexamer which was identical with the native IAP in electrophoretic mobility and biological activity to enhance glucose-induced insulin secretion when injected into rats. In the broken-cell preparation, S-1 was biologically as effective as the native IAP; both catalyzed ADP-ribosylation of a protein in membrane preparations from rat C6 glioma cells. In conclusion, IAP is an oligomeric protein consisting of an A (active) protomer (the biggest subunit) and a B (binding) oligomer which is produced by connecting two dimers by the smallest subunit in a noncovalent manner. Rationale for this terminology is discussed based on the A-B model.

Animals↗
PubMed1982

The reactivity of Mg-substituted horseradish peroxidases.

Mg-substituted horseradish peroxidases were oxidized by K2IrCl6 or K3Fe(CN)6 to their porphyrin radical form and the 1:1 stoichiometric relationship was confirmed by spectrophotometric, fluorophotometric, and ESR titration methods. The values of E'0 for oxidations of Mg peroxidases A and C were both 0.63 V at pH 6 and depended on pH in the same way as postulated for the Compound I/Compound II couples of the corresponding enzymes. Unlike Zn peroxidase C, Mg peroxidase C was not directly oxidized by H2O2. The oxidation was catalyzed by the native peroxidase. Mg peroxidase C was photooxidized to the radical form faster than Zn peroxidase C, but its oxidation was accomplished by irreversible changes in the porphyrin in the early stage of reaction. The rate of reduction of the oxidized Mg peroxidases in the presence of various electron donors was measured at varying pH values and compared with the rate of Compound I reduction. A role of porphyrin as a site of electron transfer in the peroxidase catalysis was suggested.

Electron Spin Resonance Spectroscopy↗
PubMed1982

Electron paramagnetic resonance studies of NO-heme-nitrogen base. An interpretation of electron paramagnetic resonance spectra of NO-hemoproteins.

In order to characterize the structure of the heme environment of hemoproteins, electron paramagnetic resonance (EPR) spectra for the NO complex of the iron(II) porphyrin nitrogen bases (pyridine and imidazole derivatives) were measured. The coupling constants of the nitrogen atom of NO (AN1) and the fifth ligand (AN2) and g values were determined from the 9-lined hyperfine using second-derivative display. These EPR parameters varied with changes in trans ligand (trans effect) and heme substitution at positions 2 and 4 (cis effect) as follows. (1) Both AN1 and AN2 increased as the basicity of the nitrogen atom of the fifth ligand increased, while AN1 increased concomitant with the decrease of AN2 by steric hindrance of the fifth ligand. (2) Both AN1 and AN2 increased as the basicity of the porphyrin nitrogen atom decreased. (3) In both cases, the anisotropy of g values (gx and gy) decreased concomitant with the increase of AN2. From the analysis of the EPR spectra of model systems, the substantial difference in the EPR spectra of NO-hemoproteins is discussed in relation to iron-proximal histidine binding and heme-apoprotein interactions.

Binding Sites↗
PubMed1982

Kinetic analysis of the recombination of NO with ferrihemoproteins by the flash photolysis method.

The kinetic analysis of the recombination of NO with some ferric hemoproteins was performed by the use of flash photolysis and stopped-flow methods. The rate constants for recombination of NO with ferrimyoglobin obtained by the two methods were identical with each other in the whole pH range. The rate constants decreased with an increase in pH, giving a pK value of 8.5 (cf. 5.2 x 10(4) M-1 s-1 at pH 6 and 1.3 x 10(4) M-1 s-1 at pH 10). The kinetic difference spectra of NO-ferrimyoglobin at 1 ms after flash were identical with the difference spectra of NO-ferrimyoglobin minus ferrimyoglobin at corresponding pHs. Unlike NO-ferrimyoglobin, NO-ferrihorseradish peroxidase gave different kinetics of NO binding for the two methods. Between pH 9.4 and 11.8, the velocity of NO recombination with the enzyme measured by flash photolysis remained constant, but that by the flow method decreased with increasing pH. Below pH 9.4, both methods gave an identical value of 1.9 x 10(5) M-1 s-1. The kinetic difference spectra showed that the acid form, but not the alkaline form, appeared first upon photolysis of NO-ferrihorseradish peroxidase even at alkaline pH. The acid form of peroxidase isoenzyme C re-formed the NO complex, while that of peroxidase isoenzyme A produced a mixture of the NO complex and the alkaline form. The data obtained here were compatible with the assumption that the formation of the alkaline form of the enzymes is the coordination of OH- at the sixth position, which is vacant at acidic pHs.

Hemeproteins↗
PubMed1982

Some characteristics of hydrogen- and alkylhydroperoxides metabolizing systems in cardiac tissue.

The effect of hydroperoxides on the cardiac tissue was studied by using hemoglobin-free perfused rat heart. Ethylhydroperoxide was degraded mainly through the glutathione peroxidase system of the heart at a maximal rate of about 1.2 mumol/min per g wet wt. When ethylhydroperoxide infused was not degraded completely, the hydroperoxide concentration in the effluent perfusate paralleled the formation of ferrylmyoglobin in the heart. The infusion of ethylhydroperoxide caused release of oxidized glutathione into the effluent perfusate as a result of the enhancement of the cytosolic glutathione peroxidase reaction. The leakage of oxidized glutathione reached the maximal rate of 3.5 nmol/min per g wet wt with the infusion of 175 microM ethylhydroperoxide. At hydroperoxide concentrations above 150 microM, oxidations of pyridine nucleotides and of cytochrome a + a3 occurred, probably through a stimulation of the mitochondrial glutathione peroxidase reaction, and resulted in sudden failure of the heart function. The infusion of t-butyl- and cumene-hydroperoxides, which are unable to react with myoglobin, also caused the oxidations of pyridine nucleotides and cytochrome a + a3, the inhibition of oxygen consumption and the failure of heart function. The results indicate that the cardiac toxicity of hydroperoxides is due mainly to their effect on mitochondrial metabolism.

Animals↗
PubMed1982

Perifoveal capillary network and visual prognosis in diabetic retinopathy.

Fluorescein angiography was carried out in 93 eyes with diabetic maculopathy and the state of the perifoveal capillary network within the zone of one half the disc diameter was studied. Nonperfusing capillary areas were found and the condition was classified into two grades: grade 1, the nonperfusing area less than 50% of the circumference of the network, and grade 2, more than 50% of the network. The visual prognosis was followed for 2 years. The initial and final visual acuities were significantly lower in the grade 2 than in the grade 1 group. There were more eyes showing visual deterioration during the follow-up period in the grade 2 than in the grade 1 group. It was thought that the condition of perfusion of the perifoveal capillary network is one of the determining factors of the visual prognosis in diabetic maculopathy.

Adult↗
PubMed1982

Mathematical formulation of CO2 dissociation curve and buffer line of human blood at rest.

The CO2 content and pH of tonometered blood were measured in nine healthy subjects. The CO2 content in the whole blood (Cb) was found to be expressed by an exponential function of PCO2 including only one parameter (B) as follows: Cb = 1.15 . B-2.548 . PBCO2. The B value was specific to the sampled blood and ranged from 0.4 to 0.45 in the deoxygenated and from 0.45 to 0.52 in the oxygenated blood. The relationship between pH and log PCO2 was also expressed by using one characteristic parameter (D) as follows: for the deoxygenated blood, log PCO2 = 2.144-D . (pH-7.045), and for the oxygenated blood, log PCO2 = 2.037-D . (pH-7.085). The D values were in a range of 1.38 to 1.58. The linear relation between log [HCO3-] and pH was also expressed by using only one parameter. Next, between Cb and [HCO3-] obtained at the same PCO2 of 40 mmHg, a high correlation was observed: the regression line was given, independently of O2 saturation, by Cb40 = 1.942 . [HCO3-]40-3.193, where [HCO3-] was expressed in mM. Using the above equations, it was possible to evaluate the approximate B and D values from a pair of pH and PCO2 measurements and subsequently to depict the CO2 dissociation curve as well as the buffer line in the true plasma.

Adult↗