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Biomedical subjects

H Mitsuda

Publications and source records attributed to H Mitsuda.

At least 55 records · Page 3Linked to original sources
PubMed1977

Identification and properties of reactive sites in protein capable of binding carbon dioxide in a gas-solid phase system.

In order to identify the functional groups which really contribute to the carbon dioxide gas adsorption by proteins, epsilon-amino groups of lysine residues of egg albumin were chemically modified with trinitrobenzene sulfonic acid to various degrees. About 60% of the total amount of carbon dioxide gas absorbed by solid egg albumin diminished by complete modification. The amount of carbon dioxide gas adsorbed by lysozyme, its hydrolyzates and gelatin hydrolyzates depended upon the lysine content, arginine content and average molecular weight. The good correlation was obtained between the amount of carbon dioxide gas absorbed and the total of lysine and arginine content of them. The ability of carbon dioxide gas adsorption by alpha-amino group of amino acids and oligopeptides was found to be developed by the elongation of the peptide chain of glycine and other amino acid, by the removal of alpha-carboxyl group of histidine and tyrosine to corresponding amines and by the esterification of alpha-carboxyl group of leucine with p-nitrophenol. These results clearly indicate that CO2 binding sites in protein in the gas-solid phase system are epsilon-amino, alpha-amino and guanidinium groups.

Amino Acids↗
PubMed1977

The immediate nucleotide precursor, guanosine triphosphate, in the riboflavin biosynthetic pathway.

In the present paper, the nucleotide precursor of riboflavin was investigated by experiments with labeled purines using non-growing cells of Eremothecium ashybii. The added purines, at 10(-4) M, were effectively incorporated into riboflavin at an early stage of riboflavin biosynthesis under the experimental conditions. In particular, both labeled xanthine and labeled guanine were specifically transported to guanosine nucleotides, GMP, GDP, GDP-Mannose and GTP, in the course of the riboflavin biosynthesis. A comparison of specific activities of labeled guanosine nucleotides and labeled riboflavin indicated that the nucleotide precursor of riboflavin is guanosine triphosphate. From the results obtained, a biosynthetic pathway of riboflavin is proposed under "DISCUSSION."

Ascomycota↗
PubMed1977

Examination of the structure of an unknown green fluorescent compound, compound G2, accumulated in non-growing cells of Eremothecium ashbyii by the addition of dimeric diacetyl.

The addition of dimeric diacetyl to Eremothecium ashbyii caused simultaneous accumulation of two green fluorescent compounds with the inhibition of riboflavin formation in non-growing cells. One compound, referred to as Compound G1, was identified as 6, 7-dimethyl-8-ribityllumazine as reported previously and the other is referred to as Compound G2. The latter compound was considered to be 6-methyl-7-(2-hydroxy-2-methyl-3-oxobutyl)-8-ribityllumazine because a violet fluorescent compound, 6-methyl-7-hydroxy-8-ribityllumazine was derived from Compound G2 in the presence of p-quinone and because the known action mechanism of dimeric diacetyl as a trapping agent of possible intermediates in the biosynthetic pathway. The results indicate that an immediate intermediate to 6, 7-dimethyl-8-ribityllumazine in the biosynthetic pathway of riboflavin is 4-ribitylamino-5-amino-2, 6-dihydroxypyrimidine, whose pyrimidine portion is derived by the elimination of a dimeric diacetyl fragment from the molecule of the isolated lumazine.

Ascomycota↗
PubMed1977

The relation between purine metabolism and flavinogenesis in Eremothecium ashbyii. The identification of S-adenosylmethionine and S-adenosylhomocysteine accumulated in non-growing cells of E. ashbyii.

When adenine was added to the non-growing cell medium of Eremothecium ashbyii, riboflavin production of the mold was increasingly inhibited with increasing concentration of adenine. Under these conditions, a cationic compound was accumulated in the mycelia. The compound was isolated from the mycelia and highly purified. The purified compound was proved to be S-adenosylhomocysteine through IR analysis. In the control experiment, or in the addition of other purines which stimulated riboflavin production of the mold, another cationic compound was accumulated in the non-growing cells. The compound was largely accumulated in the presence of both adenine and methionine, isolated from the mycelia and purified. The purified compound was concluded to be S-adenosylmethionine through IR and NMR analyses. The significance of these compounds on the riboflavin biosynthetic pathway was argued under "Discussion".

Adenine↗
PubMed1976

Formation of guanine ribonucleotidyl-(3'-5')-adenosine in a flavinogenic strain of Eremothecium ashbyii.

The addition of caffeine caused the accumulation of a new nucleotide compound simultaneously with the rigid inhibition of ribofalvin production in non-growing cells of Eremothecium ashbyii. In the present study we tried to identify the structure of the nucleotide compound using non-growing cells of the mold. 1) It became possible to obtain a large amount of mycelia by masscultivation in a reagent tank. 2) A new nucleotide compound, referred to as compound A in the paper, was extracted with perchloric acid solution and purified by the following subsequent procedures: 1) Dowex 1 x 2 (HCOO-) column, 2) charcoal treatment, 3) DEAE-Sephadex A25 (CI-) column, 4) Dowex 1 x 2 (C1-) column, and 5) DEAE-Sephadex A25 (HCO3-) column. 3) The structure of the new nucleotide compound was proved to be guanine ribonucleotidyl-(3'-5')-adenosine (GpA) from the results of the following analyses: 1) alkaline degradation, 2) UV-spectra, IR-spectra and NMR-spectra, and 3) enzymatic treatments with RNase T2 and phosphodiesterase. 4) The roles of caffeine and guanine ribonucleotidyl-(3'-5')-adenosine in connection with flavinogenesis of this mold were discussed.

Adenosine↗
PubMed1976

Isolation of 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine from a high flavinogenic mold Eremothecium ashbyii1.

The addition of glyoxal, a trapping agent, caused simultaneously the accumulation of a green fluorescent compound and the inhibition of riboflavin formation in non-growing cells of Eremothecium ashbyii. The fluorescent compound purified was identified as 8-ribityllumazine from the results of spectrophotometric and fluorometric analyses. Accordingly, the fragment, except for the glyoxal portion on the 8-ribityllumazine molecule, 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine, is believed to be an intermediate in the riboflavin biosynthetic pathway.

Ascomycota↗
PubMed1976

Identification of the second product of the riboflavin synthetase reaction.

This study was concerned with the detailed identification of the second product involved in the riboflavin synthetase reaction with riboflavin synthetase from Eremothecium ashbyii and a trapping agent, glyoxal.Thus, a green fluorescent compound accumulated during the incubation. The compound was purified through various column chromatography steps, and was examined by UV, IR, excitation and emission spectra and paper chromatography to prove that the isolated compound was 8-ribityllumazine. Accordingly it was concluded that a second product in riboflavin synthetase reaction was 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine, the fragment, except for C-6 and C-7 of 8-ribityllumazine, being an incorporated glyoxal portion.

Chromatography, Paper↗
PubMed1975

Purification and properties of thiamine pyrophosphokinase from parsely leaf.

Thiamine pyrophosphokinase was purified about 8,000-fold from extracts of parsely leaves. The enzyme, as prepared, was homogenous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme, estimated by gel filtration with Sephadex G-150, was approximately 30,000. In 0.05 M Tris-HCl, the enzymic activity showed a pH optimum over a range of 8 to 9. A least squares analyses of Lineweaver-Burk and Hofstee plots gave Km values of 0.8mM and 0.15mum for ATP and thiamine, respectively. Thiamine homologues and analogues so far tested, except for cetyl thiamine, were all inactive as substrates. The enzyme was specific for ATP and Mg++, although to a lesser extent a combination with other ribonucleoside triphosphates or divalent cations could replace them. SH reagents, such as PCMB, NEM and iodoacetamide, were potent inhibitors of the enzyme. The inhibition was prevented by the addition of dithiothreitol. Inorganic pyrophosphate exhibited striking inhibition. TMP could not replace thiamine as the substrate, whereas it inhibited the TPP formation from thiamine. These findings are consistent with the views that TMP is not directly converted to TPP but after being dephosphorylated by the action of a monoesterase, thiamine is pyrophosphorylated with ATP by thiamine pyrophosphokinase (EC 2.7.6.2) to form TPP and thus give a clear evidence regarding the mechanism of TPP formation in plant tissues.

Ammonium Sulfate↗
PubMed1975

Semi-automated system for analysis of vitamin B6 complex by ion-exchange column chromatography.

A simple system for semi-automatic analysis of pyridoxal, pyridoxine and pyridoxamine has been developed using diazide of 5-chloroaniline 2,4-disulfonyl chloride as a color-producing reagent in combination with desoxypyridoxine as an internal standard. It employs Aminex A-5 column, 10x0.6ø cm, as an adsorbent. Adsorbates were eluted successively with 3 discrete phosphate buffers (0.4 N Na+). The effluent is mixed continuously with capillary streams of the diazide reagent and of sodium acetate. The mixture, maintained at 65 degrees C in a heating bath, then passes through a spiral of Teflon tubing with a residence time of 2 min. Characteristic orange colored products formed by a diazo coupling reaction are continuously monitored at 440 nm in a flow photometer. The individual peaks on the recorded chromatogram are manually integrated by a conventional HW method. The elution position and recovery of desoxypyridoxine permits correction for sensitivity changes or mechanical losses which might occur during a series of analyses. The analyzer system described allow quantitation from 2 to 25 mug of pyridoxal, pyridoxine and pyridoxamine in a single sample within 2 hr and with a precision of 100 +/- 4%. It is also found suitable as a routine procedure for the analysis of varied biological samples.

Autoanalysis↗
PubMed1975

Carbon dioxide-protein interaction in a gas-solid phase.

In the course of developing the packaging of protein foods under the carbon dioxide atmosphere, various proteins in a solid state were found to adsorb carbon dioxide gas gradually. The results obtained by the Warburg manometry indicated that 100-1000 mul of carbon dioxide gas was adsorbed at 25 degrees C for 24 hr by gram of purified proteins, dried protein foods and other proteinous materials such as the rabbit hair and raw silk when they were placed in the high partial pressure of carbon dioxide gas. Casein, gelatin and raw silk were revealed to be the better adsorbents comparing with egg albumin, hemoglobin, gluten and others tested in this experiment. This adsorption was found to be almost specific to carbon dioxide gas. Amount of carbon dioxide gas adsorbed by casein and gelatin depended on the moisture content of them. The lower the moisture is, the greater the adsorption amount of carbon dioxide gas increase. Peptones and partial hydrolyzates of gelatin also showed the adsorbability. Oligo-peptides, amino acids and amines were examined too. Among these, L-lysine (free base), L-arginine (free base), histamine and tyramine adsorbed a large amount of carbon dioxide gas while others failed to do so. Some differences, however, were observed between temperature dependence and reversibility of the carbon dioxide gas adsorption by proteins and those by amines and amino acids (free bases). The mode of interaction between carbon dioxide and protein in a gas-solid phase was discussed comparing with the results obtained in a gas-liquid phase. Large contribution of physical adsorption and less contribution of chemical reaction or chemisorption were assumed in the mode of the carbon dioxide-protein interaction.

Adsorption↗
PubMed1975

Mechanism and regulation of thiamine pyrophosphokinase from parsely leaf.

Thiamine pyrophosphokinase (EC 2.7.6.2) from parsely leaf showed an absolute requirement for divalent cation such as Mg2+, Mn2+ and Co2+. The activation effect varied with the species and concentrations of such cations. When Mn2+ or Co2+ was used as cofactor, maximal activation was found at a lower level than ATP concentration, whereas the activation by Mg2+ increased hyperbolically with the concentration. Studies of initial velocity and product inhibition led to conclude that the kinase reaction obeys a sequential ordered Bi Bi mechanism; i.e. the enzyme combines in turns with MgATP and thiamine, followed by release of TPP and AMP. The inhibition type revealed for inorganic pyrophosphate was competitive with respect to thiamine with Ki of approximately 2.8 mM. On the other hand, thiamine monophosphate exhibited noncompetitive inhibition with Ki of 0.2 mM. The plots of the reaction rate against MgATP concentrations gave a sigmoidal curve. Addition of either AMP or GMP resulted in restoration of a depressed activity at low concentration of MgATP. The "allosteric" inhibition was also relieved by the addition of an excess amount of magnesium ions. These findings suggest that transphosphorylation is regulated by subcellular concentrations of metal ions relative to ATP or of the products involved in the thiamine biosynthesis.

Phosphotransferases↗
PubMed1975

Enzymic formation of thiamine pyrophosphate in plants.

Evidence was presented by paper chromatographic analysis on the occurrence of an enzyme capable of catalyzing a pyrophosphate transfer from ATP to thiamine in green leaves of various plants. The exclusive localization of the enzyme activity in the 105,000 X g supernatant (in a soluble form) was demonstrated by differential centrifugation of a cell homogentae in 0.25 M sucrose. The enzyme was purified by column chromatography with DEAE-cellulose and by gel filtration with Sephadex G-150. The partially pruified preparation, while contaminated with detectable activity of acid phosphatase, lost the ability of utilizing thiamine monophosphate as the substrate in place of thiamine. These findings lead to the conclusion that thiamine pyrophosphate is formed in green leaves of plants through a direct pyrophosphorylation of thiamine in the presence of ATP and Mg.

Acid Phosphatase↗