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H Tojo

Publications and source records attributed to H Tojo.

At least 145 records · Page 8Linked to original sources
PubMed1988

Interaction of protein with a self-associating ligand. Deviation from a hyperbolic binding curve and the appearance of apparent co-operativity in the Scatchard plot.

We derived a general formula to analyze a binding system in which a ligand self-associates, in terms of experimentally determinable quantities, i.e. r, the average number of bound ligands per protein molecule, and Lft, the total free ligand concentration, which are expressed as a ligand monomer unit. The limiting behaviors of the Scatchard plot (r/Lft vs r plot), that is, the intercepts on the r-axis and the r/Lft-axis, and the limiting slopes, are generally given. Three models that may be encountered are considered in detail. Numerical examples are also presented to illustrate how the self-association of a ligand affects the binding curves. The ligand self-association alone can cause deviation of the profile of the binding curve (r vs Lft plot) from a hyperbola, resulting in a nonlinear Scatchard plot. Therefore, analysis of the binding data without consideration of ligand self-association may lead to erroneous conclusions as to the numbers and classes of binding sites, co-operativity among the sites and binding parameter values.

Allosteric Regulation↗
PubMed1988

Hepatic cysteine dioxygenase activity and sulfur amino acid metabolism in rats: possible indicators in the evaluation of protein quality.

Experiments were carried out to examine the possibility that the sulfur amino acid metabolism of rats may be an indicator of the nutritional value of dietary protein. Rats were fed diets containing 8, 16 or 24% of gluten, soy protein or casein for 3 wk. Hepatic cysteine dioxygenase activity, hepatic concentration of glutathione, cysteine and taurine and urinary taurine were examined. In addition, the sulfur amino acid metabolism of rats fed these diets fortified with the appropriate first limiting amino acid for 7 d was also examined. High urinary taurine excretion was observed in the three gluten groups, whereas very low urinary taurine excretion was observed with up to 24% soy protein or up to 16% casein. The hepatic hepatic cysteine dioxygenase activities of the gluten diet groups were higher than those of corresponding soy protein or casein diet groups, except that of rats fed the 24% casein diet. The hepatic concentrations of both glutathione and cysteine in gluten diet groups were also higher than those of corresponding soy protein or casein diet groups, except 24% soy protein and 16 and 24% casein diet groups. In rats fed the casein or soy protein diets urinary taurine excretion and hepatic cysteine dioxygenase activity increased with increasing methionine supplementation, the first limiting amino acid. Conversely, in rats fed the gluten diet both urinary taurine excretion and hepatic cysteine dioxygenase activity decreased with increasing lysine supplementation, the first limiting amino acid. These findings suggest that urinary taurine excretion and hepatic cysteine dioxygenase activity may be useful as sensitive indicators of the nutritional value of dietary protein.

Amino Acids, Sulfur↗
PubMed1988

Resonance Raman spectra of anionic semiquinoid form of a flavoenzyme, D-amino acid oxidase.

Resonance Raman (RR) spectra of the complex of anionic semiquinoid D-amino acid oxidase (DAO) with picolinate in H2O and D2O were observed in the 300-1,750 cm-1 region. RR spectra were also measured for the complex of the semiquinoid enzyme reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]-FAD. On the basis of the isotope effects, tentative assignments of the observed bands of the anionic semiquinoid flavin were made. The spectra differ from those of oxidized, neutral semiquinoid, and anionic reduced flavins previously reported. The 1,602 cm-1 band was not shifted for any FAD labeled in ring II and/or ring III and was assigned to a ring I mode. The 1,516 cm-1 band underwent an isotopic shift upon [4a-13C]- or [4,10a-13C2]-labeling. The band was assigned to the mode containing C(4a)-C(10a) stretching. The 1,331 and 1,292 cm-1 bands shifted upon [4a-13C]- or [5-15N]-labeling and were assigned to the modes containing C(4a)-N(5) stretching. The 1,217 and 1,188 cm-1 bands were assigned to the skeletal vibrations of ring III coupled with the N(3)-H bending mode. The RR spectrum of the complex of anionic semiquinoid DAO with alpha-iminopropionate or N-methyl-alpha-iminopropionate was essentially identical with that of the complex with picolinate.

Animals↗
PubMed1988

Complex formation between anionic semiquinoid form of a flavoenzyme D-amino acid oxidase and ligands. Stabilizing mechanism of anionic semiquinoid flavoenzyme.

Picolinate binds to the anionic semiquinoid form of D-amino acid oxidase (DAO), and the complex formed has a broad absorption band in the long-wavelength region extending beyond 800 nm, which is reminiscent of a charge transfer interaction. The binding has a stoichiometry of 1:1 with respect to the enzyme. The dissociation constant at 25 degrees C was 30 microM at pH 7.0. The pH dependence (pH 7.0-8.3) of the dissociation constant indicates that one proton is associated with the complex formation, and suggests that picolinate able to bind to the anionic semiquinoid enzyme is in the cationic form protonated at the nitrogen atom. By adding dithionite to the oxidized DAO solution containing pyruvate and various amines, a similar anionic semiquinoid DAO complex having a broad long-wavelength absorption band, appeared. Resonance Raman spectra with excitation at 623.8 nm of the anionic semiquinoid DAO complex formed in the presence of pyruvate and methylamine indicate that the complex consists of the anionic semiquinoid DAO and N-methyl-alpha-iminopropionate produced from pyruvate and methylamine, and that the imino group must be protonated. This supports the proposal that the presence of a positively charged group in the vicinity of flavin is required for the stabilization of the anionic semiquinoid flavin. The results also suggest that the broad absorption band is derived from the charge transfer interaction between the anionic semiquinoid flavin and the imino acid, in which the flavin C(4a)-N(5) locus and the locus containing (Formula: see text) of the amino acid are important for the interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗
PubMed1988

Transgenic mouse carrying a foreign gene on its Y-chromosome.

In an experiment in which a human A gamma/beta-globin construct gene was introduced into the germ line of mice by microinjection, we obtained a transgenic male mouse carrying the construct gene on its Y-chromosome. This mouse carried 13 copies of a 8.9 kb construct gene as a head-to-tail tandem array, and showed a low level of transcription of the human A gamma- and beta-globin gene in the erythroid tissues. This transgenic mouse can be a useful tool in studies of embryology and developmental genetics because it carries a specific DNA marker on only its Y-chromosome.

Animals↗
PubMed1987

Localization of D-amino acid oxidase in Bergmann glial cells and astrocytes of rat cerebellum.

The localization of D-amino acid oxidase in rat cerebellum was systematically studied in serial fixed sections at the levels of both light and electron microscopy using a coupled peroxidation method based on the intensifying effect of nickel ions. Deposits were only seen in astrocytes and Bergmann glial cells, and not in neuronal components, endothelial cells or ependymal cells. In the molecular layer, heavy deposits were present in the profiles of Bergmann glial processes around the complexes of synapses where the parallel fiber varicosities form synapses with the thorns emerging from the spiny branchlets of Purkinje cell dendrites. In the Purkinje cell layer, the oxidase-containing processes of Bergmann glial cells enveloped basket cell axons, their terminals, the terminals of the recurrent collaterals of Purkinje cell axons and Purkinje cell bodies. In the granular layer, the cerebellar glomeruli were enveloped by the heavily stained processes of astrocytes. Based on this characteristic localization of the oxidase, we discussed the physiological role of the oxidase in connection with the function of glial cells.

Animals↗
PubMed1987

Kinetic and equilibrium studies on the interaction of reduced flavoprotein D-amino acid oxidase with pyridine carboxylates.

The equilibrium constants and the rate constants (binding and dissociation constants) between reduced D-amino acid oxidase and pyridine carboxylates were obtained at various pH values (from pH 6.0 to 8.3). The pH dependence of the constants is consistent with the previous conclusion from a resonance Raman study that pyridine carboxylates in the form of a cation protonated at the N atom can bind to the reduced enzyme, but those in the neutral form cannot bind, showing that the positive charge of cationic pyridine carboxylates interacts with the negative charge of the anionic reduced flavin in the reduced enzyme. The binding rate constants of picolinate and nicotinate in the cationic form for the reduced enzyme were quite similar to each other, but the dissociation rate constant of picolinate is several times smaller than that of nicotinate. Thus, it is concluded that the difference in affinity of picolinate and nicotinate for the reduced enzyme is derived from the difference of the dissociation rate constants.

Animals↗
PubMed1987

Experimental production of transgenic mice carrying human beta-globin genes.

To produce transgenic mice carrying human beta-globin genes, we introduced the following two constructs of the genes to male pronuclei of fertilized mouse eggs: 4.4 kb Pst I/Pst I sequences of the human beta-globin gene (experiment 1) and the human beta-globin gene cluster (cosHG 28) containing G gamma, A gamma, delta and beta-globin genes and cosmid vector pJB8 (37.5 kb, experiment 2). In experiment 1, 25 mice were born, and four (one female and three males) carrying the injected gene sequences were identified. One of these mice carried the entire sequence of the human beta-globin gene but three others appeared to carry only a part of the entire sequence. The mouse with the entire sequence showed a slight increase in the minor component of the mouse beta-globin chain in the same position as the human beta-globin chain. In experiment 2, 61 mice were born, and nine (three females and six males) carried the sequences of the injected gene. However, from DNA analysis, no appropriate sequences present within the A gamma- or beta-globin gene were identified in any of the founder mice. In this case, DNA fragments of the gene cluster that were digested in the mouse nucleus after microinjection of the gene might be integrated into host DNA.

Animals↗
PubMed1986

A resonance Raman study on the structures of complexes of flavoprotein D-amino acid oxidase.

Resonance Raman (RR) spectra were obtained for the purple complexes of D-amino acid oxidase (DAO) with D-lysine or N-methylalanine. RR spectra of a complex of oxidized DAO with the oxidation product of D-lysine or D-proline were also measured. The isotope shifts of the observed bands of the purple complex with D-lysine upon 13C- or 15N-substitution of lysine indicate that the ligand is delta 1-piperideine-2-carboxylate. That the band at 1671 cm-1 for the purple intermediate with N-methylalanine shifts to 1666 cm-1 in D2O solution indicates that the imino acid, N-methyl-alpha-iminopropionate, has a protonated imino group. Many bands due to a ligand in the RR spectra of the complex of oxidized DAO with an oxidation product can be observed below 1000 cm-1, but no band for the purple complex is seen in this frequency region. The band associated with the CO2-symmetric stretching mode of the product, such as delta 1-piperideine-2-carboxylate or delta 1-pyrrolidine-2-carboxylate, complexed with the oxidized DAO shifts in D2O solution. This suggests that the product imino acid interacts with the enzyme through some proton(s).

Alanine↗
PubMed1986

Complex formation between reduced D-amino acid oxidase and pyridine carboxylates.

Picolinate binds to a reduced form of D-amino acid oxidase, and the complex formed has a broad absorption band around 600 nm as in the case of the purple intermediate of the enzyme with a substrate. The dissociation constant at 25 degrees C was 35 microns at pH 7.0. The pH dependence (pH 8.3-pH 6.4) of the dissociation constant indicates that one proton is associated with the complex formation, and picolinate protonated at the N atom binds to the reduced enzyme. Resonance Raman spectra of the complex support that picolinate in the complex is a cationic form protonated at the N atom. Nicotinate also binds to the reduced enzyme, but isonicotinate does not.

Animals↗
PubMed1986

Purification and some properties of membrane-associated phospholipase A2 of human spleen.

Membrane-associated phospholipase A2 was purified to homogeneity from human spleen. The enzyme was solubilized from the particulate fraction by the addition of KBr, and purified by reverse-phase high-performance liquid chromatography. The estimated molecular weight of the enzyme was 14,000. The enzyme had a pH optimum around 9.5, required the presence of Ca2+ for its activity, and hydrolyzed phosphatidylethanolamine more efficiently than phosphatidylcholine.

Calcium↗
PubMed1985

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. I. Analysis of apparent weight-average molecular weight data for the apoenzyme in terms of models.

The self-association of D-amino acid oxidase apoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was studied by low-angle laser light scattering. The concentration (c) dependence of the apparent weight-average molecular weight (Mwapp) was determined over a wide concentration range of 0.04 to 6.1 mg/ml. The extrapolated Mwapp value, to zero enzyme concentration, corresponded to the Mr value of the monomer. The self-association mode of the apoenzyme was systematically explored with nonlinear least-squares analysis of the Mwapp versus c data. The simplest model that fitted the data well was a model of isodesmic indefinite self-association of the monomer with the isodesmic association constant of 0.467 +/- 0.034 liter/g. The monomer-dimer model proposed previously, but only in a low enzyme concentration range of less than 0.9 mg/ml at 5-20 degrees C (Henn, S. W., and Ackers, G. K. (1969) Biochemistry 8, 3829-3838), did not fit the Mwapp versus c data either in the limited low concentration range or in the whole concentration range examined at 25 degrees C. To test the validity of the chosen model, the observed sedimentation boundary profiles were compared with the idealized boundary profiles calculated for the better-fit models. The profile calculated with the model of the isodesmic indefinite self-association mechanism was qualitatively consistent with the observed ones. The utility of the nonlinear least-squares procedure for analyzing self-associating systems was demonstrated.

Animals↗
PubMed1985

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. II. Stoichiometry of holoenzyme association and energetics of subunit association.

The self-association pattern of D-amino acid oxidase holoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was examined by the low-angle laser light-scattering method. As to the results of nonlinear least-squares analysis of the apparent weight-average molecular weight (Mwapp) versus protein concentration (c) data, the following three models fitted equally well the data over the concentration range of 0.03-11.4 mg/ml: 1) the model of isodesmic indefinite self-association of the monomer where the dimerization constant differs from the isodesmic association constant, 2) the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, and 3) the model which involves the trimerization of the monomer and isodesmic indefinite self-association of the trimer. In a more limited concentration range (0.3-11.4 mg/ml), a model of isodesmic indefinite self-association of the stable dimer where the dimer does not dissociate into the monomers cannot be excluded from the above three models. Measurements with the concentration range lowered to 0.03 mg/ml enabled us to exclude unequivocally the model involving such a stable dimer and to extrapolate the Mwapp data to the Mr of the monomer at infinite dilution as in the case of the apoenzyme. The observed sedimentation boundary profiles were qualitatively consistent with the idealized boundary profiles calculated with the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, so this model is the most probable of the models examined. These results provide the first evidence that the association mode of the holoenzyme is different from that of the apoenzyme, i.e. isodesmic indefinite self-association of the monomer (Tojo, H., Horiike, K., Shiga, K., Nishina, Y., Watari, H., and Yamano, T. (1985) J. Biol. Chem. 260, 12607-12614). The overall linkage scheme, between binding of coenzyme FAD and subunit association, was considered, and the overall free energy change in each process in the scheme was calculated. The total stabilization energies of the intersubunit interaction in the holoenzyme relative to the apoenzyme were found to be -2.2 kcal/mol at the dimerization step and -0.5 kcal/mol at the step of the addition of the dimer to any 2i-mer (i = 1,2, ...).

Animals↗
PubMed1985

Immunochemical relatedness between secretory phospholipase A2 and intracellular phospholipase A2.

The immunochemical relationship between rat pancreatic phospholipase A2 and rat splenic phospholipase A2 was examined with the use of anti-rat pancreatic phospholipase A2 antibody as a probe. The immunoelectrophoretic patterns showed that the antibody cross-reacted with the splenic enzyme. The immuno-crossreactivity was also shown by counter immunoelectrophoresis. The splenic phospholipase A2, whether it was purified from the cytosolic fraction or the microsomal fraction, formed an immunoprecipitin band with the anti-pancreatic phospholipase A2 antibody. The antibody was shown to inhibit the activity of the pancreatic phospholipase A2 as well as that of the splenic phospholipase A2.

Animals↗
PubMed1985

Kinetic properties of rat kidney D-amino acid oxidase associated with peroxisomes.

In contrast to hog kidney D-amino acid oxidase, the v vs s plots of D-amino acid oxidase in homogenized rat kidney did not have the form of a rectangular hyperbola, and showed an apparent negative cooperativity. After subcellular fractionation of rat kidney, both of the oxidases in the supernatant fraction and the peroxisomal fraction showed Michaelis-Menten type kinetics. The Km values for D-alanine and D-proline of the peroxisomal fraction were significantly lower than those of the supernatant fraction. The partially purified enzyme from the peroxisomal fraction showed the same kinetic properties as the supernatant fraction. These facts suggest that the two types of rat kidney D-amino acid oxidase were originally identical and that some interaction between the enzyme and peroxisomes is physiologically important for the function of the enzyme.

Alanine↗