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Biomedical subjects

T Yamano

Publications and source records attributed to T Yamano.

At least 217 records · Page 12Linked to original sources
PubMed1983

One-electron reduction of D-amino acid oxidase. Kinetics of conversion from the red semiquinone to the blue semiquinone.

The reduction of D-amino acid oxidase (DAAO) by hydrated electrons (eaq-) has been studied in the absence and presence of benzoate by pulse radiolysis. The eaq-did not reduce the flavin moiety in DAAO and reacted with the amino acid residues in the protein. In the presence of benzoate, eaq- first reacted with benzoate to yield benzoate anion radical. Subsequently, the benzoate anion radical transferred an electron to the complex of DAAO-benzoate to form the red semiquinone of the enzyme with a second-order rate constant of 1.2 X 10(9) M-1 s-1 at pH 8.3. After the first phase of the reduction, conversion of the red semiquinone to the blue semiquinone was observed in the presence of high concentration of benzoate. This process obeyed first-order kinetics, and the rate increased with an increase of the concentration of benzoate. In addition, the rate was found to be identical with that of the formation of the complex between benzoate and the red semiquinone of DAAO as measured by a stopped-flow method. This suggests that bound benzoate dissociates after the reduction of the benzoate-DAAO complex by benzoate anion radical and that free benzoate subsequently recombines with the red semiquinone of the enzyme to form the blue semiquinone.

Animals↗
PubMed1983

Resonance Raman study on the flavin in the purple intermediates of D-amino acid oxidase.

Resonance Raman (RR) spectra were measured for the purple intermediates of D-amino acid oxidase reconstituted with isotopically labelled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]flavin adenine dinucleotides, and compared with those with the native enzyme. The RR lines around 1605 cm-1 with D-alanine or D-proline as a substrate and at 1548 cm-1 with D-alanine undergo isotopic shifts upon [4a-13C]- and [4,10a-13C2]-labelling. These lines are assigned to the vibrational modes associated with C(10a) = C(4a) - C(4) = O moiety of reduced flavin, providing the first assignment of RR lines of reduced flavin and conclusive evidence that reduced flavin is involved in this intermediate.

Animals↗
PubMed1983

Application of epithelial cells in culture to the biochemical studies of lysosomal hydrolase deficiencies.

Epithelial cells are readily obtained when the primary culture of skin fibroblasts is established. The major lysosomal hydrolase activities in normal epithelial cells are lower than those in normal fibroblasts. The primary deficiency of lysosomal enzymes can be detected both in epithelial cells and in cultured skin fibroblasts. However, epithelial cells of I-cell disease show only beta-galactosidase deficiency. This result indicates that epithelial cells have different biochemical properties from skin fibroblasts and they may be similar to those of visceral organs. Therefore, cultured epithelial cells seem to be useful for the studies on lysosomal hydrolase deficiencies.

Cells, Cultured↗
PubMed1983

8-Fluoro-8-demethylriboflavin as a 19F-probe for flavin-protein interaction. A 19F NMR study with egg white riboflavin binding protein.

Binding of 8-fluoro-8-demethylriboflavin to riboflavin binding protein was studied 19F-NMR spectroscopically. The pH dependence of 19F chemical shift of this fluoro derivative both free and bound to the protein revealed a shift of pKa associated with N(3)-H to alkali when bound to the protein. 19F chemical shift of the fluoro derivative bound to the protein is in a higher magnetic field than that of the free derivative in the neutral to acidic pH region. These results are interpreted in terms of the environmental changes at the 8- and 3-positions of isoalloxazine, indicating the applicability of this derivative for probing flavin-protein interaction at these positions.

Carrier Proteins↗
PubMed1983

A severe infantile sialidosis (beta-galactosidase-alpha-neuraminidase deficiency) mimicking GM1-gangliosidosis type 1.

We observed a 3-month-old Japanese female infant with severe psychomotor retardation, coarse facial appearance, hepatosplenomegaly, and dysostosis multiplex. Only beta-galactosidase was found to be deficient when the routine lysosomal hydrolase assay was performed on the patient's lymphocytes at 6 months of age. At first GM1-gangliosidosis type 1 seemed the most likely diagnosis. Later, however, additional studies (hydrolase assay in cultured skin fibroblasts, urinary oligosaccharide analysis, genetic complementation study, etc.) revealed that biochemical data of this case were in agreement with those of severe infantile sialidosis. The only important exception was that alpha-neuraminidase in the patient's lymphocytes showed normal activity but abnormal pH dependence toward 4-methylumbellyferyl substrate. In addition, a severely damaged kidney suggested that his case may be classified as a unique type of severe infantile sialidosis (possible nephrosialidosis). These observations stress the importance of careful biochemical diagnosis of a case with GM1-gangliosidosis type 1 phenotype.

Diagnosis, Differential↗
PubMed1983

Synaptic changes in Purkinje cell dentritic spine of mouse cerebellum after neonatal administration of cytosine arabinoside.

This study was undertaken to elucidate morphological changes in the synaptic area of the Purkinje cell dendritic spines when granule cells were decreased in number. The mice were injected s.c. with 30 mg/kg b.w. of cytosine arabinoside on days 2, 3, and 4, and on days 7, 8 and 9, and were designated as group I and group II, respectively. The mice injected with saline on days 2, 3, and 4 served as control. The cerebella of the mice in each group were examined by electron microscopy on days 30, 60, and 90. Using photographs thus obtained, the synaptic length and area of Purkinje cell dendritic spines which participated in synapses with axons of granule cells were measured by computer. In the controls, these spines did not increase significantly either in synaptic length or in spine area in the duration from 30 to 90 days after birth. In the 90-day-old mice belonging to group I and group II, however, they increased by about 20% in the synaptic length and by about 35% in the spine area as compared to those in age-matched control. The elongation and enlargement show that the synaptic surface on the spine spreads to compensate for synapses lost by reduction in number of granule cells in experimental groups.

Age Factors↗
PubMed1983

Ultrastructural study on nervous system of fetus with GM1-gangliosidosis type 1.

The nervous system of a 22-year-old fetus with GM1-gangliosidosis type 1 was studied by electron microscopy. The tissues thus examined were the cerebral cortex at the parietal region, the cerebellum, the thoracic spinal cord, the Auerbach's myenteric plexus in the large intestine and the radial nerve fibers. In the cerebral cortex, membrane-bound vacuoles, which occasionally contained stacks of fine fibrils, were observed in the large young neurons in the deeper part of the cortical plate. The neurons in the other part of the cerebral cortex carried no storage materials. In the cerebellum, the membrane-bound vacuoles with stacks of fine fibrils were seen only in the Purkinje cells. The neurons in the spinal cord also contained several zebra-like bodies and the above membrane-bound vacuoles. As for the peripheral nervous system (PNS), neurons in the Auerbach's myenteric plexus carried membranous cytoplasmic bodies and zebra-like bodies. Some of the axons in the radial nerve fibers also contained a lot of pleomorphic electron-dense bodies and a few membranous cytoplasmic ones. These results show that the accumulation of storage materials is started in the large neurons which are produced in the early stage of neurogenesis in the central nervous system (CNS). Additionally, the observed membrane-bound vacuoles are considered to be structures which occur before the membranous cytoplasmic bodies and/or the zebra-like bodies. It is also elucidated that the PNS is affected earlier than the cerebral and cerebellar cortices and thoracic spinal cord.

Cerebellum↗
PubMed1983

Regulation of 18-hydroxycorticosterone formation in bovine adrenocortical mitochondria.

The conversion of deoxycorticosterone to 18-hydroxycorticosterone was examined by using sonicated mitochondrial suspension of bovine adrenocortex. The KM's of deoxycorticosterone for the 11 beta- and 18-hydroxylations were in the same range (1 microM), while the turnover number for the 11 beta-hydroxylation (50 nmol/min/nmol cytochrome P-450) was 6 times as great as that for the 18-hydroxylation (7.3). The KM and turnover number for the 18-hydroxylation of corticosterone were 6 microM and 0.4, respectively. Those for the 11 beta-hydroxylation of 18-hydroxy-11-deoxycorticosterone were 120 microM and 5. When products were analysed in the incubation of deoxycorticosterone with the mitochondrial suspension containing a larger amount of cytochrome P-450, the formation of 18-hydroxycorticosterone was observed in addition to the formation of corticosterone and 18-hydroxy-11-deoxycorticosterone. The kinetic interrelation between the two pathways was further examined together with consideration of the cytochrome P-450-linked hydroxylation system. The result suggests that the pathway via 18-hydroxy-11-deoxycorticosterone substantially participates in the formation of 18-hydroxycorticosterone from deoxycorticosterone. The perturbation of this network by an artificial means, such as the addition of Triton X-100, revealed that the detergent (0.02%) facilitated the production of 18-hydroxycorticosterone from deoxycorticosterone, regardless of its inhibitory effect on the production of corticosterone and 18-hydroxy-11-deoxycorticosterone from deoxycorticosterone. These studies provide an important insight into the regulation mechanism of 18-hydroxycorticosterone formation from the precursors on the mitochondrial level.

11-Hydroxycorticosteroids↗
PubMed1983

Zonal distribution of cytochrome P-450s (P-450(11) beta and P-450scc) and their relation to steroidogenesis in bovine adrenal cortex.

Zona glomerulosa and zonae fasciculata-reticularis of bovine adrenal cortex were separated by our improved method using a remodeled microtome. A considerable amount of fresh mitochondria and microsomes was quickly obtained. Specific content of cytochrome P-450 in the mitochondria was higher in zonae fasciculata-reticularis than in zona glomerulosa. When metyrapone was added to the mitochondria and the drug-induced difference spectra were measured, there appeared two phases in the titration curve; the first phase was saturated at a concentration of 3 microM and the second phase appeared at concentrations higher than 10 microM. The first phase seemed to be due to the substrate-free P-450(11) beta and the second phase to the cholesterol-bound P-450scc. From these spectral studies contents of cytochrome P-450scc and P-450(11) beta were estimated, and those were found to be equal for each zone. From similar studies on substrate-induced difference spectra, there seemed to be no difference between two zones in the affinity of cytochrome P-450(11) beta for deoxycorticosterone and 11-deoxycortisol. This was also the case with 11 beta-hydroxylase activity and substrate specificity for deoxycorticosterone and 11-deoxycortisol.

Adrenal Cortex↗
PubMed1983

pH-Induced conformational change of ATPase inhibitor from yeast mitochondria. A proton magnetic resonance study.

The ATPase inhibitor from yeast, Saccharomyces cerevisiae, was found to strongly inhibit the ATPase activity when it was preincubated in an acidic pH region. This paper describes the conformational changes of the inhibitor in the acidic pH region, as studied by 1H NMR spectroscopy. In the pH range from 7.43 to 4.48, two conformations were detected. With lowering of the pH in this pH region, one conformation increased at the expense of the other. In the two conformations, His 39 had different pKa values which were determined at 23 degrees C to be 6.08 +/- 0.04 and 5.91 +/- 0.03 from the pH-titration curves of His 39. The exchange between the two conformations was reversible with pH and the rate of the conformational change was estimated to be slower than 22 s-1 at 23 degrees C as estimated by the line-widths (7.0 Hz) of the peaks from the C2-proton of His 39 in the two conformations. The equilibrium constant between the two conformations was dependent on the temperature. From the equilibrium constants in the temperature range from 23 to 45 degrees C, the apparent delta H'a of the conformational change was calculated to be 14 kcal/mol at pH 6.95. It was confirmed by 1H NMR spectra that at pH 6.95 the structure of the inhibitor was reversible with temperature at least below 80 degrees C. The reversibility is consistent with the high thermal stability of the protein.

Hydrogen-Ion Concentration↗
PubMed1983

Interpretation of the stokes radius of macromolecules determined by gel filtration chromatography.

From a comparison of the gel chromatographic properties of large randomly-coiled polypeptides in 6 M guanidine hydrochloride and of large globular proteins, we found that the distribution coefficient was more closely correlated with the intrinsic viscosity-based Stokes radius than with the translational frictional coefficient-based Stokes radius. This means that the effect of the hydrodynamic flow of dissolved molecules during gel chromatography should be considered. The ratio of transport of solute by bulk flow as compared with that by net diffusion (i.e., Brownian motion) is large under some conditions. On the other hand, we consider that the distribution coefficient obtained in static equilibrium experiments should be determined by the translational frictional coefficient-based Stokes radius, since the solvent does not flow. On this basis, we discuss the meaning of the Stokes radius and the separation mechanism of macromolecules by gel filtration.

Chemical Phenomena↗
PubMed1983

8-Fluoro-8-demethylriboflavin as a potential active-site-directed reagent for flavoprotein. Reaction with some amino acids.

8-Fluoro-8-demethylriboflavin was shown to be a potential active-site-directed reagent for flavoproteins. It reacted with the nucleophiles of N-acetylcysteine (-SH), N-acetyltyrosine (-OH), alpha-N-acetyllysine, and glycine (epsilon- and alpha-NH2, respectively) under fairly mild conditions, and the reaction products were identified. The reactivity of the fluoroflavin was higher than that of 8-chloro-8-demethylriboflavin, which reacted only with the cysteine derivative among the amino acid derivatives used, and whose pseudo first order rate constant at 23 degrees C was 1/23 of that of the fluoroflavin. The reactivity of the fluoroflavin was also estimated by 13C and 19F NMR spectroscopy. The results showed that this compound is more reactive than the chloroflavin.

Amino Acids↗
PubMed1983

Purification and characterization of renal ferredoxin from bovine renal mitochondria.

A renal ferredoxin was purified from bovine renal mitochondria to electrophoretic purity. The molecular weight of the renal ferredoxin was estimated by gel filtration and SDS-polyacrylamide gel electrophoresis to be 12,500 and 13,000, respectively. The optical absorption spectrum of renal ferredoxin in the oxidized form showed two peaks at 416 and 457 nm in the visible region, and the EPR absorption spectrum showed peaks at gx = gy =1.94 and gz = 2.02 in the reduced form at 13K. These spectra were typical of the 2S-2Fe type ferredoxins. Dissimilarities were recognized in the amino acid composition and isoelectric point between bovine renal ferredoxin and bovine adrenodoxin, but not in the optical, magnetic, and immunochemical properties. The reconstitution of 25-hydroxyvitamin D3-1 alpha-hydroxylase system was performed with the three components of NADPH-adrenodoxin reductase from bovine adrenal mitochondria, renal ferredoxin, and cytochrome P-450(1) alpha from bovine renal mitochondria. The results showed that the renal ferredoxin was essential for the 1 alpha-hydroxylase activity of 25-hydroxyvitamin D3.

Animals↗
PubMed1983

Purification and some properties of rat spleen phospholipase A2.

Intracellular phospholipase A2 was purified to homogeneity from rat spleen. The enzyme was efficiently concentrated by precipitation with trichloroacetic acid and purified by sequential use of column chromatographies on DEAE-Sepharose, octyl-Sepharose and Bio-Gel P-30. The positional specificity of the enzyme for an acylester bond at the sn-2-position of phospholipids was established. The purified enzyme has a pH optimum ranging from 8.0 to 10.5 and the molecular weight of the enzyme was estimated to be 14,700-14,800 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by gel filtration with a TSK-GEL G 2000 SW column. The purified enzyme requires Ca2+ for activity. The activity was not enhanced by the presence of purified calmodulin.

Animals↗
PubMed1983

Purification and characterization of human pancreatic phospholipase A2 and development of a radioimmunoassay.

Human pancreatic phospholipase A2 was purified to homogeneity from pancreatic juice and a reliable radioimmunoassay for the enzyme was developed. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 14,000. Phosphatidylcholine was hydrolyzed well in an alkaline pH range, and the optimum activity was obtained at pH 9. Calcium ion was indispensable for activity. The enzyme was stable to heat treatment at 60 degrees C for 5 min. The radioimmunoassay system was highly sensitive, reproducible and specific. The dilution curves for the sera of patients with acute pancreatitis were parallel to the standard curve. In healthy individuals, serum phospholipase A2 concentrations ranged from 2.0 to 7.9 ng/ml, the average being 5.1 ng/ml (S.D.: 1.7). In patients with acute pancreatitis, significant elevations of serum phospholipase A2 contents were observed, and the highest value found was 4,000 ng/ml.

Antibody Specificity↗
PubMed1983

Spectroscopic studies on the photoreaction of choline oxidase, a flavoprotein, with covalently bound flavin.

Formation of the anionic flavosemiquinone was observed spectrophotometrically during the anaerobic photo-irradiation of Alcaligenes sp. choline oxidase in the presence of EDTA. Further irradiation slowly converted the semiquinone form into the fully reduced state. The presence of a catalytic amount of riboflavin greatly enhances the photoreduction rate not only to the semiquinone state but also to the fully reduced state. This semiquinone species has low reactivity toward the substrate, choline or betaine aldehyde, as well as toward oxygen. This low reactivity toward oxygen is unique to the semiquinone form of a flavoprotein oxidase. The oxidized enzyme forms a complex with betaine, the product of the enzymatic reaction of choline oxidase. The dissociation constant for this complex was found to be 17 mM by spectroscopic titration. Anaerobic photo-irradiation of the enzyme with a saturating amount of betaine in the absence of EDTA produces, with no detectable semiquinone formation, an absorption spectrum which resembles (but significantly differs from) that of the fully reduced form. This species was found to comprise two flavin species. One of them is rapidly oxidized to the oxidized form by oxygen and is thus assigned as the fully reduced state. The other is converted slowly to the oxidized form upon aerobic standing in the dark. We tentatively assigned this latter species as a C(4a)-adduct. Formaldehyde was detected as a product of this photoreaction. The amount of formaldehyde formed coincided with that of the fully reduced enzyme. On the basis of the results obtained we propose a mechanism of the photoreaction of the enzyme in the presence of betaine where a C(4a)-adduct and the fully reduced enzyme via an N(5)-adduct are formed. Betaine also affects the dithionite reduction. In the dithionite reduction of the oxidized enzyme, the semiquinone species is an intermediate in the conversion of the oxidized to the fully reduced form, while the reduction of the oxidized enzyme-betaine complex with dithionite produces the fully reduced form without any significant formation of the semiquinone species.

Alcaligenes↗
PubMed1983

On the structures of flavoprotein D-amino acid oxidase purple intermediates. A resonance Raman study.

Resonance Raman (RR) spectra were obtained in H2O or D2O solution for the purple intermediates of D-amino acid oxidase (DAO) with isotopically labeled substrates, i.e., [1-13C]-, [2-13C]-, [3-13C]-, [15N]-, and [3,3,3-D3]alanine; [carboxyl-13C]- and [15N]proline. RR spectra were also measured for the intermediates of DAO reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]FAD in D2O. The isotopic shift of the 1692 cm-1 band upon [15N]- or [2-13C]-substitution of alanine indicates that the band is due to the C = N stretching mode of an imino acid derived from D-alanine, i.e., alpha-iminopropionate. The 1658 cm-1 band with D-proline was also assigned to the C = N stretching mode of an imino acid derived from D-proline, i.e., delta 1-pyrrolidine-2-carboxylate, since the band shifts to 1633 cm-1 upon [15N]-substitution and its stretching frequency is generally found in this frequency region. Since the band shifts to low frequency in D2O, the imino acid should have a protonated imino group such as the C = N+1H form. The intense band at 1363 cm-1 with D-alanine was assigned to a mixing of the CO2- symmetric stretching and CH3 symmetric deformation modes in alpha-iminopropionate, based on the isotope effects. The 1359 cm-1 band with D-proline has probably contributions of CO2- symmetric stretching and CH2 wagging, considering the isotope effects with [carboxyl-13C]proline. The 1359 cm-1 band with D-proline was split into 1371 cm-1 and 1334 cm-1 bands in D2O. As this splitting of the 1359 cm-1 band with D-proline in D2O can not be interpreted only by the replacement of the C = N+1-H proton by deuterium, the carboxylate of the imino acid probably interacts with the enzyme through some proton(s) exchangeable by deuterium(s) in D2O. The bands around 1605 cm-1 which shift upon [4a-13C]- and [4,10a-13C2]-labeling of FAD are derived from a fully reduced flavin, because the isotopic shifts of the band are very different from those of the bands of oxidized or semiquinoid flavin observed near 1605 cm-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗