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Biomedical subjects

H Tojo

Publications and source records attributed to H Tojo.

At least 163 records · Page 9Linked to original sources
PubMed1984

An improved method for destroying mouse blastomeres electrically inside the zona pellucida and the in vitro development of the surviving blastomeres.

An improved method for isolating mouse blastomeres by electrically destroying the other blastomeres inside the zona pellucida is described. Instruments required are two micromanipulators connected to an inverted microscope and an electronic stimulator equipped with an isolator. The target blastomere in which a glass microelectrode is inserted disintegrates completely within a few seconds after a direct current is applied. Complete destruction of a blastomere requires more than 60 microA delivered at 8 V. Destruction of blastomeres occurred mainly as a result of the medium penetrating the blastomere due to the change of membrane potential in both the zona and the blastomere. Most one-half, two-quarter, and three-quarter embryos obtained in this way developed in vitro into normal blastocysts (76.4--86.2%). By contrast, there was a marked decrease (35.3%) of developmental potential in one-quarter embryos. The results indicate that the present method is an improvement over previous methods of obtaining mammalian blastomeres inside the zona pellucida.

Animals↗
PubMed1984

Purification of intracellular phospholipase A2 from rat spleen supernatant by reverse-phase high-performance liquid chromatography.

Intracellular phospholipase A2 was purified to homogeneity from rat spleen supernatant by reverse-phase high-performance liquid chromatography with a trifluoroacetic acid-acetonitrile solvent system. The method simplified the purification procedure, which includes three consecutive chromatographic steps. The recovery of the enzyme activity was greater than 70% with an about 23,000-fold purification. The solvent system did not affect the catalytic properties of the enzyme. Phospholipases A2 from rat spleen, human pancreatic juice, and porcine pancreas were eluted in that order from a column of octadecasilyl silica gel in a similar concentration range of acetonitrile. This result suggests that the phospholipases A2 examined have similar hydrophobicities. This method may be applicable to the purification of phospholipases A2 from other sources.

Animals↗
PubMed1984

Gel chromatographic evidence for the participation of the higher polymers in the self-association system of a flavoenzyme D-amino acid oxidase.

The self-association of subunits of D-amino acid oxidase holoenzyme was studied by high-speed gel filtration with a short column of TSK-GEL G3000 SW in 0.1 M sodium pyrophosphate (pH 8.3) at 25 degrees C. Over the range of the peak concentrations of 0.009-4.45 mg/ml in the presence of FAD the apparent Stokes radii increased with an increase of the concentrations and did not level off. The largest value obtained in this study was 61.5 A. This would correspond to that calculated for the hexamer with linear subunit arrangement which has the largest Stokes radius among the various arrangements. These results provide the first gel chromatographic evidence that the higher polymers greater than the dimer participate in the self-associating system of the enzyme.

Animals↗
PubMed1984

Chemical modification of proteins: plotting of the remaining activity against the number of residues modified.

In the study of chemical modification of proteins, it has been a common practice to plot the fractional remaining activity against the number of residues modified per protein molecule. Extrapolation of the initial, nearly linear portion of the curve to the axis giving numbers of residues has often been presumed to specify the actual number of critical groups modified, i.e., the stoichiometry of the modification and the inactivation reactions. However, this extrapolation method is not generally applicable (Horiike, K. & McCormick, D.B. (1979) J. Theor. Biol. 79, 403-414). This paper describes further examination of the underlying theoretical framework of the extrapolation method. The properties and features of the extrapolated values are considered and presented with numerical and graphical examples. And the theoretical conditions with which the extrapolated value gives the number of essential residues are derived.

Amino Acids↗
PubMed1984

A 13C-NMR study on the interaction of riboflavin with egg white riboflavin binding protein.

The interaction between riboflavin and riboflavin binding protein (RBP) was studied by 13C-NMR spectroscopy. The 13C-NMR spectra of riboflavin selectively enriched at the 2-, 4-, 4a-, and 10a-positions and of (3-[13C]methyl)riboflavin were measured both in the free and RBP-bound forms. The 13C signals of 13C-enriched riboflavin or 3-methylriboflavin bound to RBP are broader than those of the free form, reflecting the restriction of flavin mobility. The 2-, 4-, and 10a-13C signals of riboflavin show no pH-dependent shift in the neutral to acidic pH region either in the bound or free form but the 4a-13C signal of bound riboflavin shifts to lower field in the acidic pH region while that of the free form remains unshifted. The 2-, 4-, 4a-, and 10a-13C signals of free riboflavin exhibited pH-dependent change in the alkaline pH region with a pK value of about 10, in association with the N(3)-H deprotonation. The pH titration profile of the 2-, 4-, and 4a-13C signals of bound riboflavin indicates that the pK of N(3)-H is shifted substantially to the alkaline side when riboflavin is bound to RBP. The 3-methyl-13C signal of 3-methylriboflavin shows no pH-dependent shift whether the compound is free or bound to RBP. The binding of riboflavin and 3-methylriboflavin was also studied spectrofluorometrically. The analysis of the pH dependence of the association constant revealed that one ionizable group in RBP with pK of about 5 and N(3)-H of riboflavin play important roles in the binding. We conclude that RBP preferentially binds the neutral, i.e., N(3)-protonated, form of riboflavin and that the neutral form in turn is stabilized by the hydrophobic environment of RBP surrounding the N(3) region of the bound riboflavin molecule.

Carbon Isotopes↗
PubMed1984

Rat pancreatic phospholipase A2: purification, characterization, and N-terminal amino acid sequence.

Phospholipase A2 was purified from rat pancreas by heat treatment of the homogenate and the sequential use of DEAE-Sepharose chromatography, CM-Sepharose chromatography, and reverse-phase high-performance liquid chromatography (HPLC). Prophospholipase A2 was not separated from the phospholipase A2 by CM-Sepharose chromatography under the conditions used, but it was well resolved by the reverse-phase HPLC. The enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and on analytical HPLC, and its molecular weight was estimated to be 14,000. The enzyme specifically hydrolyzed an acylester bond at the sn-2-position of the phospholipid examined. The purified enzyme has a pH optimum in the range of pH 9.5 to 10.5 and requires the presence of Ca2+ (3 mM) and sodium deoxycholate (0.1%) for optimum activity. The amino acid sequence of the first 32 residues in the N-terminal region of the enzyme was determined. The sequence revealed a marked homology with those of pancreatic phospholipases A2 of man, pig, ox, and horse, and porcine intestinal phospholipase A2 reported previously.

Amino Acid Sequence↗
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

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

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

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↗
PubMed1982

A resonance Raman study on the reaction intermediates of D-amino acid oxidase.

Resonance Raman (RR) spectra of two reaction intermediates of D-amino acid oxidase with substrate analogs were obtained. The reaction intermediates studied were (1) the one in the aerobic oxidative reaction of the enzyme with beta-cyano-D-alanine and (2) the other in the reverse reductive reaction of the enzyme with chloropyruvate and ammonium. Both intermediates are characterized with the charge transfer absorption bands in the long wavelength region extending beyond 600 nm. The RR spectra of the two intermediates excited at 488.0 or 514.5 nm are those of oxidized flavin, which is consistent with our previous assumption that oxidized flavin is involved in these reaction intermediates. Relatively simple RR spectra were obtained for these intermediates with excitation at 632.8 nm which is within the region of the charge transfer bands. The resonance enhancement for the Raman lines around 1585 and 1350 cm-1 for either of the intermediates with excitation in the region of the charge transfer bands suggests that the charge transfer interaction involves the N(5)-C(4a) region extending to the C(10a)-N(1)-C(2) region of the isoalloxazine nucleus. The Raman line at 1657 cm-1 for the intermediate with chloropyruvate and ammonium was assigned to C = N of an imino acid from the isotopic frequency shift upon 15N-substitution. The assignment substantiates our previous conclusion that the intermediate involves an imino acid, alpha-imino-beta-chloropropionate.

Aerobiosis↗
PubMed1982

Thermodynamic characterization of hog kidney D-amino acid oxidase apoenzyme in concentrated guanidine hydrochloride solution. Preferential interaction with the solvent components and the molecular weight of the monomeric unit.

This paper describes the physical characterization of the monomeric unit of hog kidney D-amino acid oxidase apoenzyme in 6 M guanidine hydrochloride (GuHCl) solution by means of differential refractometry, densimetry, light scattering, equilibrium sedimentation, and high-speed gel filtration chromatography. In 6 M GuHCl solution, the oxidase interacts preferentially with GuHCl: the values of the preferential interaction parameter are 0.11 +/- 0.03 (S.D.) g/g of protein by densimetry and 0.14 +/- 0.04 g/g of protein by refractometry. The volume change, delta V, of the oxidase on transfer from the native to the denatured state is -350 ml/mol. The molecular weight of the monomeric apoenzyme is 39,600 +/- 1,700 by light scattering and 38,000 +/- 1,200 by high-speed equilibrium sedimentation. The values of the molecular weight estimated by the empirical methods, i.e., sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and high-speed gel filtration chromatography in 6 M GuHCl, agree well with those obtained by the thermodynamic methods mentioned above. These results confirm definitely that the complex of the apoenzyme with SDS normally behaves in the same manner as those of standard proteins in SDS-gel electrophoresis. This is also supported in this study by the analysis of the electrophoretic data at several gel concentrations by Ferguson plots. The molecular weight of quasi-D-amino acid oxidase apoenzyme was also examined by the empirical methods.

Animals↗
PubMed1982

Proteolytic enzymes and gonadal hormones of the ovarian follicle wall during ovulation in the domestic fowl (Gallus domesticus).

There were no significant changes in the activities of collagenase, acid and neutral protease up to ovulation. Neutral protease activity increased significantly in the post-ovulatory follicle obtained immediately after ovulation. Acid protease and collagenase showed an increasing activity with time after ovulation. The progesterone concentration of the follicle wall rose 3 h before ovulation, peaked 1 h before ovulation and remained high until ovulation. Plasma progesterone values were high but decreased before those in the follicle. Follicular progesterone concentrations decreased markedly soon after rupture, but oestradiol values then increased.

Animals↗
PubMed1981

Resonance Raman study of D-amino acid oxidase-inhibitor complexes.

The resonance Raman (RR) spectra of the complexes of D-amino acid oxidase (DAO) with benzoate derivatives were measured. The RR spectra of complexes of DAO with benzoate derivatives excited at 514.5 nm are similar to one another and also similar to that of oxidized flavin. In the cases of DAO-o-NH2-benzoate and DAO-o-OH-benzoate complexes, however, the line at 568 or 565 cm-1, derived from the benzoate derivative, was intensified. In the case of DAO-o-NH2-benzoate complex, which has an intense charge-transfer absorption band, the resonance enhancement of the Raman lines at 1583 and 568 cm-1 in the RR spectrum excited at 632.8 nm is striking. The former line is known to involve the vibrational displacements of the N(5) and C(4a) atoms of isoalloxazine and the latter is considered to be derived from a ring deformation mode of o-NH2-benzoate. This suggests that the o-NH2-benzoate molecule lies along the N(5)-C(4a) bond and parallel to the flavin face. A Raman line derived from o-OH-benzoate in the RR spectrum of DAO-o-OH-benzoate complex excited at 514.5 nm was detected. This result supports the view that the complex has a charge-transfer band, as has been pointed out by Massey and Ganther. Also, the spectrum of quasi-DAO-o-OH-benzoate complex is identical with that of the complex of DAO, suggesting that the active sites of these two enzymes have similar structures.

Aminobenzoates↗