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Biomedical subjects

M Fujioka

Publications and source records attributed to M Fujioka.

At least 217 records · Page 12Linked to original sources
PubMed1987

Chemical modification of a functional arginine residue of rat liver glycine methyltransferase.

Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 +/- 0.44 M-1 min-1 is obtained at pH 8.2 and 25 degrees C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constant for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of [14C]phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.

Aldehydes↗
PubMed1987

Rat glycine methyltransferase. Complete amino acid sequence deduced from a cDNA clone and characterization of the genomic DNA.

The amino terminus of glycine methyltransferase from rat liver is blocked. A hexapeptide containing the blocked amino-terminal residue was obtained from a tryptic digest of the purified enzyme and its amino acid sequence was determined to be Ac-Val-Asp-Ser-Val-Tyr-Arg by Edman degradation and fast-atom-bombardment mass spectrometry after fragmentation with Staphylococcus aureus protease V8. A full-length cDNA clone for the enzyme was isolated from a lambda gt11 rat liver cDNA library using the previously obtained pGMT A56 cDNA [Ogawa, H., Gomi, T., Horii, T., Ogawa, H. & Fujioka, M. (1984) Biochem. Biophys. Res. Commun. 124, 44-50] as a probe. The amino acid sequence deduced from the nucleotide sequence contained both amino- and carboxyl-terminal sequences. The predicted amino acid composition and molecular mass were also in agreement with the published data obtained with the purified protein. Five clones for the glycine methyltransferase gene were isolated from a Charon 4A library containing EcoRI digest of rat liver DNA by in situ plaque hybridization. All clones had inserts of 6500 base pairs, consistent with the size of EcoRI genomic DNA fragment determined by Southern blot hybridization. Sequence analysis of a 5400-bp fragment of the insert DNA lacking a 1100-bp 5' region and comparison of the sequence with that of the cDNA showed that the insert DNA entirely encoded glycine methyltransferase and the gene consisted of six exons and five introns. S1 nuclease protection mapping and primer extension analysis allowed us to propose that the A residue located 19 bp upstream from the translation initiation codon is the site of transcription initiation. TATA, CAAT and GC sequences, and the complementary sequence to the enhancer core element, were located upstream of the transcription initiation site.

Amino Acid Sequence↗
PubMed1987

Function and reactivity of sulfhydryl groups of rat liver glycine methyltransferase.

Rat liver glycine methyltransferase is completely inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Treatment of the inactivated enzyme with KCN results in a reactivated enzyme having values of Vmax and S0.5 for S-adenosyl-L-methionine comparable to those of the native enzyme and about a 4-fold greater Km value for glycine. Kinetics of inactivation and reactivation show that one cysteine residue is involved in this process. Reaction of the methyltransferase with iodoacetate leads to partial inactivation of the enzyme; about 22% of the initial activity is retained in the modified enzyme. The relationship between the loss of enzyme activity and the number of iodoacetate molecules incorporated and the sequence analysis of peptides containing the modified residues indicate that carboxymethylation of Cys-282 is responsible for loss of activity. The observations that the activity of the cyanylated glycine methyltransferase shows no decrease upon incubation with iodoacetate and, conversely, the residual activity associated with the iodoacetate-modified enzyme is not abolished by DTNB suggest that Cys-282 is also involved in the inactivation by DTNB. Besides this residue, Cys-185, Cys-246, and Cys-262 are modified upon prolonged incubation with iodoacetate. 5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSBA) inactivates glycine methyltransferase by forming 1 disulfide/subunit [Fujioka, M., & Ishiguro, Y. (1986) J. Biol. Chem. 261, 6346-6351]. Despite this stoichiometry, treatment of the FSBA-inactivated enzyme with unlabeled iodoacetate and then with iodo[14C]acetate after reduction with 2-mercaptoethanol and subsequent peptide analysis show that the incorporated radioactivity is distributed equally among Cys-185, Cys-246, Cys-262, and Cys-282.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1987

Acute suppression of endogenous testosterone levels by exogenous testosterone in normal men.

The effect of exogenous testosterone on endogenous plasma testosterone was studied in normal men. Intramuscularly administered testosterone-19,19,19-d3 rapidly appeared in the systemic circulation in large amounts. Endogenous plasma testosterone was suppressed to near-castrate levels. The suppressed level began to rise between 6 and 10 h, and reached a preinjection level at 24 h after the injection. Plasma LH decreased with a concomitant decrease in endogenous testosterone and began to rise as soon as plasma total testosterone returned to physiological levels.

Adult↗
PubMed1987

Autoregulation of neuromuscular transmission in the guinea-pig saphenous artery.

The participation of presynaptic adrenoceptors and receptors mediating the postsynaptic excitatory junction potential (e.j.p.) responses in feedback regulation of neurotransmission in the guinea-pig saphenous artery was studied by electrophysiological recording of the synaptic potentials. Stimulation of perivascular nerves elicited e.j.p. and slow depolarization in the artery. With repetitive stimulation, the successive e.j.p.s decreased in amplitude. Prazosin abolished the slow depolarization without any effect on the e.j.p. The depression of e.j.p. with repetitive stimulation was converted to facilitation in the presence of yohimbine. Yohimbine inhibited the first e.j.p. in a train but did not have a significant effect on the slow depolarization and contraction. A high concentration of ANAPP3, a specific P2-antagonist, depressed the e.j.p. The slow depolarization and contraction, however, were significantly potentiated by ANAPP3, even at low concentrations. Propranolol had no effect on the neural responses. The results suggest that there are functional presynaptic alpha 2-adrenoceptors and P2-receptors that could play a physiological role in the autoregulation of neuromuscular transmission in the guinea-pig saphenous artery.

Adenosine Triphosphate↗
PubMed1987

Increased CT density of the liver due to cis-diaminedichloro platinum (II).

Unusually increased CT density of the liver parenchyma after administration of cis-diaminedichloro platinum (II) (CDDP) is presented. This patient has neither a history of blood transfusion nor of iron overload. Therefore, CDDP which includes the heavy metal, Platinum, in its molecule was considered to be the only agent responsible for increasing the CT density.

Child, Preschool↗
PubMed1987

Effects of bunazosin on electrical responses of smooth muscle cells of the guinea-pig mesenteric artery and vein to perivascular nerve stimulation and to noradrenaline.

The noradrenaline-induced depolarization of smooth muscle cell membrane was blocked by bunazosin in the mesenteric artery but not in the mesenteric vein. Bunazosin enhanced the excitatory junction potential (e.j.p.) evoked in the mesenteric artery but did not modulate the slow depolarization evoked in the mesenteric vein. Application of noradrenaline decreased the amplitude of e.j.p. enhanced by bunazosin but not by yohimbine. It was concluded that bunazosin is a highly selective alpha 1-adrenoceptor blocker in vascular tissues.

Animals↗
PubMed1987

Amino acid sequence of S-adenosyl-L-homocysteine hydrolase from rat liver as derived from the cDNA sequence.

Rat liver cDNA libraries constructed in lambda gt11 were screened for reactivity with polyclonal antibodies to native S-adenosyl-L-homocysteine (AdoHcy) hydrolase (adenosylhomocysteinase; EC 3.3.1.1). Five clones were isolated and sequenced. The amino acid sequence, deduced from the cDNA sequence, contained the sequence of eight peptides obtained by tryptic and cyanogen bromide fragmentation of rat liver AdoHcy hydrolase. Identification of the amino- and carboxyl-terminal peptides in the amino acid sequence showed that the complete sequence was obtained. A "fingerprint" sequence was found that is characteristic of dinucleotide-binding domains of many proteins. For AdoHcy hydrolase, this region from the lysine at position 213 to the aspartate at position 244, containing the sequence Gly-Xaa-Gly-Xaa-Xaa-Gly at positions 219-224, is presumably the site of binding for NAD+, which is required for the activity of the enzyme.

Adenosylhomocysteinase↗
PubMed1987

Antitumour activity of purified arabinogalactan-peptidoglycan complex of the cell wall skeleton of Rhodococcus lentifragmentus.

Antitumour activity of arabinogalactan peptidoglycan (AP) complex (peptidoglycan and arabinogalactan liberated by an acid or alkaline treatment from Rhodococcus lentifragmentus AN-115 cell wall skeleton) was examined in mice and compared with that of the cell wall skeleton. The growth of syngeneic fibrosarcoma Meth A cells after implantation in BALB/c mice was significantly suppressed by AP complex, and also regressed after intratumoral injection of AP complex on days 1, 4 and 7 after tumour implantation. Although the activity of peptidoglycan was less than that of AP complex, peptidoglycan also showed both tumour-suppressive and regressive activities. Arabinogalactan did not show antitumour activity. It is interesting that peptidoglycan has an important role in the effect against tumours.

Animals↗
PubMed1987

[Reduction of radiation dose to the worker in preparing the radiopharmaceutical solution by a simple shielding equipment].

In order to reduce radiation dose to the hands of examiners who prepare and aspirate radiopharmaceuticals, we made a prototype of simplified manually-operated dispense system, which the syringe and the vial shield with lead were set in the small box made of lead and lead glass. The result showed that our dispense system allowed substantial reduction of radiation dose to the hands and rapid preparation of radiopharmaceuticals compared with the conventional lead shield syringe system, and allowed closer operation, smaller dead volume and lower cost compared with the conventional automatic system.

Gamma Rays↗
PubMed1986

S-adenosylhomocysteinase from rat liver. Amino acid sequences of the peptides containing active site cysteine residues modified by treatment with 5'-p-fluorosulfonylbenzoyladenosine.

5'-p-Fluorosulfonylbenzoyladenosine (FSBA) inactivates rat liver S-adenosylhomocysteinase exhibiting characteristics of an active site-directed reagent. The inactivation is not associated with the covalent binding of the reagent, but is correlated with the loss of 2 sulfhydryl groups/enzyme subunit (Takata, Y., and Fujioka, M. (1984) Biochemistry 23, 4357-4362). Treatment of the FSBA-inactivated enzyme with iodoacetate in the absence of reducing agent and then with [14C] iodoacetate after reduction with 2-mercaptoethanol yielded the enzyme containing approximately 2 mol of radiolabeled S-carboxymethylcysteine/mol of subunit. Analysis of tryptic peptides showed that the radioactivity was associated with 2 carboxymethylcysteine-containing peptides whose amino acid sequences were: Trp-Ser-Ser-Cys(Cm)-Asn-Ile-Phe-Ser-Thr-Gln-Asp-His-Ala-Ala-Ala-Ala-Ile- Ala-Lys and Gly-Glu-Thr-Asp-Glu-Glu-Tyr-Leu-Trp-Cys(Cm)-Ile-Glu-Gln-Thr-Leu-His-Phe- Lys, respectively. These results indicate that the inactivation of S-adenosylhomocysteinase by FSBA is the consequence of formation of a disulfide between two specific cysteine residues on each of the four identical subunits.

Adenosine↗
PubMed1986

Reaction of rat liver glycine methyltransferase with 5'-p-fluorosulfonylbenzoyladenosine.

Rat liver glycine methyltransferase is inactivated by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) in a pseudo-first order fashion at pH 7.5. The addition of dithiothreitol (20 mM) to the reaction mixture results in partial restoration of enzyme activity. A semilog plot of residual activity after dithiothreitol reactivation versus time is also linear, indicating that at least two essential residues are present on the enzyme and the modification of either of which causes total loss of activity. The inactivation is accompanied by incorporation of the radiolabel from adenine-labeled FSBA, but the amount of radioactivity fixed is not altered upon treatment with dithiothreitol. From this fact and the stoichiometry between the loss of dithiothreitol-sensitive activity and the number of sulfhydryl groups disappeared, it is suggested that the dithiothreitol-sensitive inactivation is the consequence of the FSBA-mediated formation of a disulfide between two sulfhydryl groups in close proximity. Although 4 mol of reagent are covalently bound per enzyme subunit, the kinetics of modification and inactivation show that the reaction at 1 residue, which is identified as tyrosine, is responsible for the dithiothreitol-insensitive inactivation. The substrate S-adenosylmethionine provides complete protection against both types of inactivation, but the dithiothreitol-insensitive inactivation is protected much more effectively with a Kd value comparable to the Km value. This suggests that the tyrosine is located at or near the active site of the methyltransferase.

Adenosine↗
PubMed1986

Actions of the novel thromboxane A2 antagonists, ONO-1270 and ONO-3708, on smooth muscle cells of the guinea-pig basilar artery.

The effects of the novel thromboxane A2 (TXA2) antagonists, ONO-1270 and ONO-3708, on the electrical and mechanical responses evoked by various agents, and in particular 9,11-epithio-11,12-methano-thromboxane A2 (STA2), were investigated in the guinea-pig artery. STA2 (up to 0.3 microM), and ONO-1270 and ONO-3708 (up to 1.0 microM) did not modify the membrane potential in smooth muscle cells. Perivascular nerve stimulation induced an excitatory junction potential (e.j.p.), and with frequencies over 0.25 Hz, depression of e.j.ps occurred. STA2 (0.1 microM) and both ONO-1270 and ONO-3708 had no effect on these electrical events. STA2 (over 0.1 microM) produced phasic and tonic contractile responses, in a concentration dependent manner. Both ONO-1270 and ONO-3708 competitively inhibited the phasic contraction induced by STA2 as estimated from parallel shifts in the dose-response curve, and from the Lineweaver-Burk and Schild plots (the PA2 values were 8.22 for ONO-1270 and 8.70 for ONO-3708), but both agents inhibited non-competitively the PGF2 alpha-induced contraction. ONO-1270 and ONO-3708 (up to 0.1 microM) had no effect on contractions induced by K+ and caffeine, but did slightly inhibited contractions induced by 5-hydroxytryptamine (5-HT). Following application of indomethacin, neither agent modified the 5-HT-induced contraction. In Ca2+-free solution, 10 nM STA2 produced a phasic but not a tonic contractile response. ONO-1270 and ONO-3708 (over 1 nM) inhibited this phasic contractile response.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Pharmacokinetic properties of testosterone propionate in normal men.

The pharmacokinetic characteristics of testosterone propionate were studied in normal men after a single im dose of 25 mg testosterone propionate-19,19,19-d3. Plasma levels of testosterone propionate-19,19,19-d3, its active metabolite testosterone-19,19,19-d3, and endogenous testosterone were measured by gas chromatography-mass spectrometry. Testosterone propionate-19,19,19-d3 was gradually transferred from the im injection site to the systemic circulation. The plasma levels of testosterone propionate-19,19,19-d3 were maintained at 2-4 ng/ml between 3 and 36 h after administration. Plasma testosterone-19,19,19-d3 levels were maintained above the physiological testosterone level for 48 h, while plasma levels of endogenous testosterone changed little.

Adult↗