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M Fujioka

Publications and source records attributed to M Fujioka.

At least 253 records · Page 14Linked to original sources
PubMed1984

Purification of messenger RNA coding for glycine methyltransferase from rat liver.

Rat liver messenger RNA coding for glycine methyltransferase was associated preferentially with free polysomes. The mRNA was purified about 1000-fold over the total poly(A)-containing RNA by specific immunoadsorption of polysomes to protein A-Sepharose followed by oligo(dT)-cellulose column chromatography. Sodium dodecyl sulfate-gel electrophoresis of the in vitro translation products in a rabbit reticulocyte lysate system revealed only one major band which migrated to the position of the purified glycine methyltransferase subunit. The result shows that the mRNA isolated is nearly homogeneous and suggests that no precursor form of the enzyme existed. The mRNA sedimented at the position slightly smaller than 18 S rRNA in a sucrose density-gradient centrifugation and was shown to contain about 1,300 nucleotides by the Northern blot hybridization analysis with a cDNA probe.

Animals↗
PubMed1983

Guanidoacetate methyltransferase from rat liver: purification, properties, and evidence for the involvement of sulfhydryl groups for activity.

Guanidoacetate methyltransferase (EC 2.1.1.2) has been purified about 800-fold from rat liver. The purified preparation shows a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme is estimated to be 25,000 and 26,000 by Sephadex gel molecular-exclusion chromatography and by electrophoresis in polyacrylamide gradient gel, respectively. The sodium dodecyl sulfate-denatured enzyme also has a molecular weight of 26,000; thus, the enzyme is a monomeric protein. Guanidoacetate methyltransferase as isolated is catalytically inactive, but is readily reactivated by incubation with a thiol. The reactivated enzyme, which contains 3 mol of sulfhydryl groups/mol of enzyme, is again inactivated by oxidized glutathione. This inactivation is accompanied by the disappearance of two sulfhydryl residues. The relationship between the loss of enzyme activity and the number of residues disappeared indicates that the integrity of these sulfhydryl residues is critical for activity. The oxidized enzyme fails to bind the substrate S-adenosylmethionine as evidenced by the equilibrium dialysis study. Alkylation of the nonoxidizable sulfhydryl by N-ethylmaleimide shows that this residue is also essential for activity. UV absorption, fluorescence, and CD spectra show no difference between the reduced and oxidized enzymes, but the former is more susceptible to proteolytic attack by trypsin. The enzyme has an isoelectric pH of 5.3, and is most active at pH 9.0. From the CD spectrum, an alpha helix content of 15% is calculated. The Km values for guanidoacetate and S-adenosylmethionine are 97.5 and 6.73 microM, respectively, at pH 8.0 and 37 degrees C.

Amino Acids↗
PubMed1983

Chemical modification of arginine residues of rat liver S-adenosylhomocysteinase.

Rat liver S-adenosylhomocysteinase (EC 3.3.1.1) is inactivated by phenylglyoxal following pseudo-first order kinetics. The dependence of the apparent first order rate constant for inactivation on the phenylglyoxal concentration shows that the inactivation is second order in reagent. This fact together with the reversibility of inactivation upon removal of excess reagent and the lack of reaction at residues other than arginine as revealed by amino acid analysis and incorporation of phenylglyoxal into the protein indicate that the inactivation is due to the modification of arginine residue. The substrate adenosine largely but not completely protects the enzyme against inactivation. Although the modification of two arginine residues/subunit is required for complete inactivation, the relationship between loss of enzyme activity and the number of arginine residues modified, and the comparison of the numbers of phenylglyoxal incorporated into the enzyme in the presence and absence of adenosine indicate that one residue which reacts very rapidly with the reagent compared with the other is critical for activity. Although the phenylglyoxal treatment does not result in alteration of the molecular size of the enzyme or dissociation of the bound NAD+, the intrinsic protein fluorescence is largely lost upon modification. The equilibrium binding study shows that the modified enzyme apparently fails to bind adenosine.

Adenosylhomocysteinase↗
PubMed1983

Evidence for an essential histidine residue in S-adenosylhomocysteinase from rat liver.

Rat liver S-adenosylhomocysteinase (EC 3.3.1.1) is inactivated by diethyl pyrocarbonate. The inactivation is first order in enzyme and in reagent, and a second-order rate constant of 77 M-1 min-1 is obtained at pH 6.9 and 0 degree C. The rate of inactivation is dependent on pH, and the pH-inactivation rate data show the involvement of a group with a pK of 6.8. The difference spectrum of the inactivated and native enzymes shows a single peak at 242 nm, indicating the modification of histidine residues. No trough at around 280 nm due to O-carbethoxytyrosine is observed. The sulfhydryl content of the enzyme is unchanged by the reaction. The inactivation was reversed by hydroxylamine. Although the reaction with [3H]diethyl pyrocarbonate reveals that a residue(s) other than histidine is (are) also modified, the agreement of the number of histidine residues modified and the number of carbethoxy groups removed by hydroxylamine treatment indicates that the inactivation is solely due to the modification of histidine. Statistical analysis of the residual enzyme activity and the extent of modification shows that, among six modifiable residues per subunit, one which reacts more rapidly with the reagent than the rest is critical for activity. The modified enzyme still retains the capacity to bind adenosine and S-adenosylhomocysteine and to oxidize the 3'-hydroxyl of these compounds as evidenced by the reduction of the enzyme-bound NAD+. Slow but significant exchange of the 4' proton with solvent also occurs with the modified enzyme. Thus, it may be concluded that the histidine residue essential for activity is involved in a catalytic reaction other than the abstraction of 3'-hydroxyl and 4' protons of the substrates.

Adenosine↗
PubMed1983

Studies on drug metabolism by use of isotopes XXVII: urinary metabolites of rutin in rats and the role of intestinal microflora in the metabolism of rutin.

Analysis of urinary metabolites of orally administered rutin (I) labeled with deuterium [( 2',5',6'-2H]rutin, rutin-d) was carried out by GLC-MS. In rat urine, 3-hydroxyphenylacetic acid (III), 3-methoxy-4-hydroxyphenylacetic acid (IV), 3,4-dihydroxyphenylacetic acid (V), 3,4-dihydroxytoluene (VI), and 3-(m-hydroxyphenyl)propionic acid (VIII) were identified as rutin metabolites and were differentiated from the corresponding endogeneous compounds. Unchanged I and quercetin (II) were not present in the urine. Rutin-d was injected intraperitoneally in rats, administered orally to neomycin-treated rats, and incubated in vitro with the intestinal contents of rats. The experiments suggested the involvement of intestinal microflora in the metabolism of orally administered I.

Animals↗
PubMed1982

Inactivation of rat liver S-adenosylhomocysteinase by iodoacetamide.

S-Adenosylhomocysteine (EC 3.3.1.1) from rat liver is inactivated by iodoacetamide following pseudo-first-order reaction kinetics. The apparent first-order rate constant for inactivation is proportional to the concentration of the modifier, and a value of 7.55 M-1 min-1 is obtained for the second-order rate constant at pH 9.06 and 25 degrees C. Amino acid analysis of the modifier enzyme shows the formation of S-(carboxymethyl)cysteine. No peaks corresponding to N epsilon-(carboxymethyl)- and N epsilon,N epsilon-bis(carboxymethyl)lysines, N-(carboxymethyl)histidines, S-(carboxymethyl)homocysteine, homoserine, and homoserine lactone are detected. Glycolic acid is also not found in the acid hydrolysate of the modified enzyme, indicating the absence of modification at carboxyl residues. These results and the finding that the number of residues modified as determined by the incorporation of iodo[1-14C]acetamide is equal to the number of cysteine residues lost by modification establish the site of modification as cysteine residues. Kinetics of inactivation and incorporation of the label from iodo[1-14C]acetamide show that two among three modifiable residues per enzyme subunit are essential for activity and the modification of either results in complete inactivation. The inactivation by iodoacetamide does not involve alteration in the molecular size of enzyme nor release of the bound NAD+. The modified enzyme still retains the capacity to bind adenosine and to oxidize it as evidenced by the reduction of enzyme-bound NAD+ but does not catalyze the exchange of the 4' proton with solvent. Thus, it is suggested that the inability of the modified enzyme to catalyze the overall reaction is due to the failure to abstract the 4' proton in the catalytic cycle.

Adenine↗
PubMed1982

Purification and properties of glycine N-methyltransferase from rat liver.

Glycine N-methyltransferase (EC 2.1.1.20) has been purified to homogeneity from rat liver. The enzyme has a molecular weight of 132,000 by sedimentation equilibrium method. This value is in good agreement with a value of 130,000 obtained by Sephadex G-150 chromatography. The molecular weight of the denatured enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is 31,500. The numbers of peptides obtained by tryptic digestion and by cyanogen bromide cleavage are one-fourth of those expected from the contents of lysine plus arginine residues and methionine residues, respectively. By Edman degradation, phenylthiohydantoin-leucine is the only amino acid derivative released from the enzyme. Neither sugar nor phospholipid is detected in the purified preparation. These data indicate that the rat liver glycine N-methyltransferase is a simple protein consisting of 4 identical subunits. The enzyme has an isoelectric pH of 6.4, and is most active at pH 9.0. From the circular dichroism spectrum, an alpha helix content of about 11% is calculated. Whereas the initial velocity as a function of glycine concentration gives a Michaelis-Menten kinetics, the enzyme shows a positive cooperativity with respect to S-adenosylmethionine. The concentrations of glycine and S-adenosylmethionine which give a half-maximum velocity are 0.13 mM and 30 microM, respectively, at pH 7.4 and 25 degrees C.

Amino Acids↗
PubMed1982

Rectal pressure and rectal compliance in ulcerative colitis.

Anorectal manometry, coupled with measurement of rectal compliance, was carried out on 11 healthy subjects and 9 patients with ulcerative colitis. Resting pressure profile of the anorectum but basal rhythmic contraction of the anal canal was normal in most of the patients and rectoanal flex was positive in all. Rectal compliance was, however, significantly decreased in 7 of 9 patients. These results suggest that distensibility of the rectum may be decreased in cases of ulcerative colitis and that measurements of resting pressure profile, rectoanal reflex, and rectal compliance may be helpful in selecting patients suitable for elective surgery to preserve sphincter function.

Adult↗
PubMed1982

Comparative effects of heparin, urokinase, and ancrod on intraglomerular coagulation induced in progressive Masugi nephritis.

Effects of heparin, urokinase, and ancrod were concurrently compared on intraglomerular coagulation using a model of progressive Masugi nephritis. Evaluations were performed with light and immunofluorescent microscopy. Treatment significantly reduced crescent formation, more markedly with ancrod. In contrast, endocapillary proliferation was enhanced in the groups treated with urokinase and, especially, ancrod. Ancrod appeared to be most effective in decreasing the glomerular deposition of fibrinogen-related materials as judged from the immunofluorescence, and a relatively high dose was required for urokinase to be comparable with ancrod. However, in some glomeruli from two of 13 animals given ancrod, mesangiolytic lesion developed. Thus, although promising, ancrod has many problems to be solved before using it to control endocapillary proliferation and mesangiolysis.

Ancrod↗
PubMed1982

[Congenital Minamata disease accompanied by arachnoid cyst (author's transl)].

A male, born on December 8, 1956, during the period when many Minamata diseases broke out in a district. His parents who ate much fish and shell fish taken in Minamata Bay suffered from the light, incomplete Minamata disease showing sensory disturbance, the constriction of the visual field, muscular weakness, etc. He weighed 3,225 gr. upon the normal birth given 10 months after pregnancy. His abnormalities were noted since his head was not stabilized on the neck even six months after the birth. Because of the delay in the development of the motor function, he became barely able to sit, stand up and begin walking at the ages of 3, 5 and 6 respectively. In 1962 (at the age of 6), his congenital Minamata disease was diagnosed in view of his clinical symptoms and epidemiological conditions. The mercury value in the hair and blood upon the birth is not known because a considerable time had elapsed after the birth when his mercury poisoning was discovered. However, the clinical symptoms included intelligence disturbance, character change, dysarthria, primitive reflexes, strabismus, hypersalivation, ataxia and hyperkinesia, indicating a typical congenital Minamata disease. Until he became 13 years old (1969) or so, his mental and motor function developed, both gradually. In the same year, he was admitted to a special class for the handicapped. EEG examination revealed that there was a slow alpha activity in the basic pattern and that 6 Hz positive spike was found in the sleep EEG. The constriction of the visual field was classified through examination.2+

Adult↗
PubMed1981

S-Adenosylhomocysteine hydrolase from rat liver. Purification and some properties.

S-Adenosylhomocysteine hydrolase (EC 3.3.1.1) from rat liver was purified to homogeneity. The enzyme had a molecular weight of 188,000 and was composed of 4 subunits with a molecular weight of 47,000. The isoelectric pH of the enzyme was at 5.7. The Km values for S-adenosyl-L-homocysteine, adenosine, and DL-homocysteine were 15.2 microM, 1.05 microM, and 155 microM, respectively, at pH 7.2 and 25 degrees C. The enzyme showed a fluorescence with an emission maximum at 350 nm when excited at 280 nm. The protein fluorescence was partially quenched on addition of adenosine. The fluorescence quenching as a function of adenosine concentration indicated that the enzyme contained four binding sites for adenosine per molecule. The same data provided a value of 0.63 microM for the dissociation constant of adenosine. The enzyme possessed 4 mol of tightly bound NAD+/mol. The addition of adenosine to the enzyme caused the appearance of a peak at 327 nm in a concentration-dependent manner. The enzyme-bound NAD+ readily formed an adduct with bisulfite with the concomitant loss of enzyme activity. These data suggest that the bound NAD+ is involved in the catalytic mechanism of the enzyme.

Amino Acids↗
PubMed1981

Role of arginine residue in saccharopine dehydrogenase (L-lysine forming) from baker's yeast.

The baker's yeast saccharopine dehydrogenase (EC 1.5.1.7) was inactivated by 2,3-butanedione following pseudo-first-order reaction kinetics. The pseudo-first-order rate constant for inactivation was linearly related to the butanedione concentration, and a value of 7.5 M-1 min-1 was obtained for the second-order rate constant at pH 8.0 and 25 degrees C. Amino acid analysis of the inactivated enzyme revealed that arginine was the only amino acid residue affected. Although as many as eight arginine residues were lost on prolonged incubation with butanedione, only one residue appears to be essential for activity. The modification resulted in the change in Vmax, but not in Km, values for substrates. The inactivation by butanedione was substantially protected by L-leucine, a competitive analogue of substrate lysine, in the presence of reduced nicotinamide adenine dinucleotide (NADH) and alpha-ketoglutarate. Since leucine binds only to the enzyme-NADH-alpha-ketoglutarate complex, the result suggests that an arginine residue located near the binding site for the amino acid substrate is modified. Titration with leucine showed that the reaction of butanedione also took place with the enzyme-NADH-alpha-ketoglutarate-leucine complex more slowly than with the free enzyme. The binding study indicated that the inactivated enzyme still retained the capacity to bind leucine, although the affinity appeared to be somewhat decreased. From these results it is concluded that an arginine residue essential for activity is involved in the catalytic reaction rather than in the binding of the coenzyme and substrates.

Amino Acids↗