PubMed Health⌕ Search

Biomedical subjects

M Tokushige

Publications and source records attributed to M Tokushige.

At least 37 records · Page 2Linked to original sources
PubMed1990

Overproduction and crystallization of tryptophanase from recombinant cells of Escherichia coli.

We have cloned the tryptophanase structural gene from Escherichia coli B/1t7-A into E. coli K-12 MD55 with a vector plasmid, pBR322. The cloned cells produced a large amount of the enzyme corresponding to more than 30% of the total soluble protein. With the enzyme obtained by this overproduction system, we have prepared three different crystals of tryptophanase, apo-enzyme, holo-enzyme, and a complex of holo-enzyme and L-alanine, by using polyethylene glycol 4000 or potassium phosphate as a precipitant and the hanging drop method. These single crystals appeared to be suitable for X-ray diffraction analysis.

Alanine↗
PubMed1990

Preparation of active hybrid enzymes composed of the native and chemically inactivated aspartase subunits from Escherichia coli.

The hybridization of the native and chemically inactivated aspartase from Escherichia coli was studied. Preparations of the tetrameric enzyme obtained by mixing the native and N-ethylmaleimide (NEM)-inactivated aspartase in 4 M guanidine-HCl followed by 51-fold dilution at room temperature retained catalytic activity. Affinity chromatography on AF-Red TOYO-PEARL separated several active components in the hybridized preparations, and the presence of [14C]NEM-inactivated subunits in the active hybrids was demonstrated. The addition of the native aspartase to Sepharose-bound NEM-inactivated enzyme in 4 M guanidine-HCl resulted in the formation of an immobilized enzyme with enzyme activity. The specific activity of the various hybrids, composed of unmodified and [14C]NEM-inactivated subunits, was roughly proportional to the number of unmodified subunits in each tetramer. Furthermore, when reversible denaturation was conducted on mixtures of the native and NEM-inactivated enzyme at various proportions, the enzyme activity recovered was proportional to the amount of the native enzyme added. These results strongly suggest that each subunit makes an independent contribution to the overall enzyme activity regardless of the presence of other subunits in the same molecule. The theoretical and practical implications of this work are discussed.

Ammonia-Lyases↗
PubMed1989

Role of cysteine residues in tryptophanase for monovalent cation-induced activation.

We cloned and sequenced the tryptophanase structural gene of Escherichia coli B/1t7-A strain. The results indicate that tryptophanase proteins of E. coli B/1t7-A and K-12 are identical. When cysteine residues in tryptophanase were chemically modified with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), the stabilizing effect of the active cations such as K+ and NH4+ was abolished. In consideration of our previous results that Cys-298 was selectively modified by SH reagents [Honda T. et al. (1986) J. Chromatogr. 371, 353-360], Cys-298 seems to have a close relation to the expression of the effect of monovalent cations. Fluorescence decay measurement of the holoenzyme revealed that the fluorescence lifetime derived from the coenzyme, pyridoxal 5'-phosphate (PLP), was dependent on coexisting monovalent cations, whereas that of the tryptophyl residue was not, in either the apo- or the holoenzyme preparation. The results of the synchrotron small-angle X-ray scattering measurements showed that radii of gyration which reflect the size and shape of the enzyme were constant at around 38 A irrespective of the presence or absence of the K+ ion. These results suggest that the monovalent cations interact specifically with the PLP-binding site, and that the conformational change of enzyme protein caused by the monovalent-cation binding is limited to a small range. The above results are compatible with the possibility that Cys-298 is involved in the formation of "monovalent cation binding site" in the holoenzyme.

Amino Acid Sequence↗
PubMed1989

Detection of immunoregulatory factors in retroplacental serum in human pregnancy.

Retroplacental serum (RPS) obtained from pregnant women at term deliveries was studied for regulatory effects on T-lymphocyte proliferation and for pregnancy-associated substances and compared with peripheral serum (PS) of the same donor. Proliferative response to phytohemagglutinin and alloantigens in RPS was lower than that in PS. RPS contained higher levels of human placental lactogen, progesterone, estradiol, and prostaglandin E2 than of PS. However, there were no differences in concentrations of pregnancy-associated alpha 2-glycoprotein, pregnancy-specific beta 1-glycoprotein, prostaglandin F2 alpha, alpha-fetoprotein, human chorionic gonadotropin, cortisol, carcinoembryonic antigen, and immunoglobulins between RPS and PS. The amounts of human placental lactogen, progesterone, or prostaglandin E2 seen in RPS did not inhibit T-cell proliferation. Mixtures of various doses of these three substances were still not inhibitory. Thus, the suppressive activity of RPS could not be explained by these pregnancy-associated substances, but a possible involvement of unknown immunoregulatory factors at fetomaternal interface might be suggested.

Dinoprostone↗
PubMed1989

Characterization of intermediate species during the molecular assembly of aspartase.

Molecular assembly of aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli was studied during the reversible denaturation. Although previous studies [Tokushige, M., Eguchi, G., and Hirata, F. (1977) Biochem. Biophys. Acta 480, 479-488] were unable to identify intermediate species during the course of reversible denaturation of aspartase, temperature-controlled HPLC and cross-linking with dimethyl suberimidate of the renaturation products showed that monomeric, dimeric and trimeric species occupied over 80% of the total oligomeric molecules below 13 degrees C; unlike the tetramer, these intermediates were without the activity. The degree of active tetramer formation was a linear function of the restoration of the activity below 18 degrees C, while above 23 degrees C, the activity regain was less than 70% restoration of tetrameric molecules. Upon examination by fluorescence spectroscopy, structural changes during reconstitution exhibited such complex kinetics that the rapid formation of structured oligomers proceeds first with a half-time of less than 10 sec, followed by slow subunit association. These results strongly suggest that the tetramer formation is an essential prerequisite, though not sufficient for the active enzyme.

Ammonia-Lyases↗
PubMed1989

[Intraperitoneal chemotherapy in gastric cancer with peritoneal metastasis using totally implantable peritoneal access system].

Fifteen patients with peritoneal metastasis of gastric cancer were treated with mainly ip-ETP (Etoposide: i.p., THP-ADM: i.v. and CDDP: i.p.) or other drugs by the use of a totally implantable peritoneal access system. In principle, intraperitoneal drug delivery was carried out every two weeks. CDDP was administered into the intraperitoneal cavity with intravenous sodium thiosulfate delivered simultaneously to protect against cisplatin-induced nephrotoxicity. RI-scintigram showed that the intraperitoneal catheter was fully useful even six months after the operation. As a result, performance status has been improved in 12 out of 15 cases, and ascites disappeared in 3 out of 6 cases with same. Ten cases have been alive for more than 6 months after operation. There have been no severe complications (e.g., nephrotoxicity or myelosuppression) even in the cases treated at frequent intervals for more than 8 months. The findings in this study indicated that ip-ETP using totally implantable peritoneal access system is beneficial for advanced gastric cancer with peritoneal metastasis.

Adult↗
PubMed1989

[Two-route chemotherapy under AT-II induced hypertension using totally implanted injection port system in liver metastases derived from digestive cancers].

Two-route chemotherapy (TRC) with intraarterial infusion of cis-diamminedichloroplatinum and intravenous infusion of sodium thiosulfate was carried out on 8 cases of digestive cancer with liver metastases, using totally implanted injection port system. The metastases occurred from gastric cancer in 3 cases and from colonic cancer in 5 cases. Computed tomography and/or ultra-sonography revealed an overall response rate of 50% (4/8). Complete response (CR) was obtained in two cases. The therapy was repeated 12 times in one case of gastric cancer with multiple liver metastases and 5 times in another rectal cancer with a solid metastatic tumor. In the latter case, a right hepatic lobectomy was performed thereafter. The histology of the hepatic tumor showed mucin lakes and necrotic lesions, and no viable cancer cells were observed. This mode of chemotherapy was therefore considered a useful measure for the treatment of liver metastases derived from digestive cancers. Furthermore, no serious side effects occurred.

Adult↗
PubMed1988

Characterization of multiple fumarase proteins in Escherichia coli.

Two different types of fumarase were found in sonic extracts of Escherichia coli; one required Fe-S for the enzyme activity, and the other did not. When the cells were grown without aeration, the Fe-S-independent enzyme occupied over 80% of the overall fumarase activity. Highly purified Fe-S-independent enzyme was suggested to be composed of four subunits (Mr = 48 kDa) by SDS-polyacrylamide gel electrophoresis and gel filtration. Amino acid and N-terminal sequence analyses supported the possibility that the enzyme is a product of fumC gene (FUMC). In aerobically grown cells, however, the content of FUMC was low and the Fe-S-dependent fumarase occupied over 80% of the overall activity. The Fe-S-dependent enzyme appeared to be labile and the activity was rapidly lost during purification. Although the spontaneous inactivation was previously ascribed to thermal lability (S.A. Woods & J.R. Guest (1987) FEMS Microbiol. Lett. 48, 219), the activity could be restored by anaerobic incubation with ferrous ions and SH-compounds.

Amino Acid Sequence↗
PubMed1988

Effect of ovarian steroids on the secretion of immunosuppressive factor(s) from human endometrium.

To elucidate the effect of ovarian steroids on the secretion of immunosuppressive factor(s) from endometrium, supernatants from human endometrial cultures with or without the addition of progesterone or estrogen were compared for their effects on mixed lymphocyte reaction and phytohemagglutinin-induced lymphocyte reactivity. The suppressive activities of supernatants from the proliferative endometria were significantly increased by the addition of progesterone into their explant cultures. The addition of both progesterone and estrogen was also effective in increasing the inhibitory degree of proliferative endometria but that of estrogen alone showed no influence. Supernatants from the secretory endometria had higher suppressive activities than those from proliferative ones and showed no change in suppressive activity by the addition of ovarian steroids. The concentrations of progesterone in supernatants were too low to inhibit immune reactivity. These findings suggest that exogenous or endogenous progesterone may indirectly play an important role in the release of immunosuppressive factor(s) from human endometrium.

Culture Techniques↗
PubMed1988

Hyporeactivity to mitogens of retroplacental blood lymphocytes in human pregnancy.

Retroplacental blood lymphocytes (RPL) obtained from women without complications at the term delivery were studied for proliferative response to mitogens and surface phenotype and were compared with autologous peripheral blood lymphocytes (PBL). Proliferation of RPL induced by phytohemagglutinin (PHA) was significantly lower than that of PBL in 11 of 13 cases. Also RPL proliferated poorly in response to concanavalin A (Con A) in 8 of 10 cases. On the other hand, flow cytometry analysis of T cell subsets revealed that RPL contain comparable or higher number of CD3+ cells and CD4+ cells and similar numbers of CD8+ cells as compared with PBL in most cases. These results indicate that T cell proliferative response in retroplacental space is impaired, which could be due to a functional defect of RPL.

Antigens, Surface↗
PubMed1988

Depression of proliferative response to fetal lymphocytes of retroplacental blood lymphocytes in human pregnancy.

Retroplacental blood lymphocytes (RPL) in human pregnancy were studied for proliferative response to related fetal lymphocytes and it was compared with that of peripheral blood lymphocytes (PBL) of the same donor. RPL proliferated poorly to related cord blood lymphocytes in 9 of 10 cases when compared with autologous PBL. RPL also showed lower proliferative response to unrelated adult lymphocytes in 7 of 12 cases. Proliferation of RPL was weaker to related cord blood lymphocytes than to allogeneic adult lymphocytes. These results indicate that the response of T cells at feto-maternal interface is impaired, especially to fetal antigens.

Female↗
PubMed1988

Chemical modification of tryptophanase by chloramine T: a possible involvement of the methionine residue in enzyme activity.

Tryptophanase purified from Escherichia coli B/1t7-A was irreversibly inactivated by chloramine T (sodium N-chloro-p-toluenesulfonamide). The mode of inactivation was rather complex and did not follow pseudo-first-order kinetics. The inactivation of the apoenzyme was much faster than that of the holoenzyme. The Km value for the synthetic substrate S-o-nitrophenyl-L-cysteine (SOPC) increased concomitantly with the modification. In contrast, the Km value for the coenzyme, pyridoxal 5'-phosphate (PLP), was not altered. L-Serine, another substrate, and L-alanine, a competitive inhibitor, protected the enzyme from inactivation. Determination of SH groups in the enzyme protein with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) showed that modification of two SH groups per enzyme subunit resulted in a complete inactivation. When the enzyme was subjected to chloramine T-modification following the SH group modification with DTNB, further inactivation was still observed, even after the addition of dithiothreitol. The SH-blocked enzyme preparation thus obtained, however, exhibited less pH dependency of inactivation by chloramine T than that of the native enzyme. The amino acid analysis of the chloramine T-modified enzyme showed that modification of four or five methionine residues among the 16 residues per subunit proceeded concomitantly with the complete inactivation. Modification of the enzyme with chloramine T quenched the absorption peak near 500 nm, characteristic of a quinoidal structure formed by labilization of the alpha-proton. These results suggest the possibility that chloramine T modifies not only the SH groups, but also methionine residues important for the catalytic activity of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1988

Fumaraldehydic acid-induced inactivation of aspartase.

Fumaraldehydic acid (FAA) induced a time-dependent inactivation of aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli at 30 degrees C and pH 7.4 following pseudo-first order kinetics. The rate of inactivation increased in proportion to the FAA concentration. In addition, the rate of inactivation increased, as the pH was increased. Determination of sulfhydryl groups showed that approximately one among 11 sulfhydryl groups was modified by FAA concomitant with the inactivation. L-Aspartate and fumarate protected the enzyme against FAA-inactivation, when Mg2+ ions were present. Unlike E. coli aspartase, P. fluorescens aspartase was not inactivated by FAA.

Ammonia-Lyases↗
PubMed1988

Specific inhibition of aspartase by S-2,3-dicarboxyaziridine.

Aspartase of Escherichia coli was inhibited in a competitive manner by S-2,3-dicarboxyazirdine (DCAZ), an antibacterial substance against Aeromonas salmonesida. The inhibition constant (Ki) was 55 microM, which was as low as less than one tenth that of the Km value for the substrate, L-aspartate. In view of the fact that both aspartase and fumarase (J. Greenhut et al. (1985) J. Biol. Chem. 260, 6684-6686) were inhibited by DCAZ in competitive manners, common features of the reaction mechanism of the two enzymes were discussed.

Ammonia-Lyases↗
PubMed1987

Suppression of lymphocyte reactivity in vitro by supernatants of explants of human endometrium.

To clarify the possible immunologic functions of the endometrium before implantation, supernatants from explant cultures of human endometrial tissues were examined for effects on mixed lymphocyte reaction and phytohemagglutinin M-stimulated cultures. Supernatants obtained from both proliferative and secretory phase endometria showed significant dose-related suppressive effects; the suppressive activity of secretory phase endometria was higher than that of proliferative phase endometria. On the other hand, no inhibitory effect was identified in supernatants from similar cultures of peritoneum and omentum, while supernatants of fallopian tubes showed slight suppressive activity. These results suggest the existence of soluble nonspecific immunosuppressive factor(s) released from human endometrium (especially during the secretory phase), and imply that these factor(s) may exert an important role in protecting the fertilized ovum from maternal rejection.

Adult↗
PubMed1986

Characterization of the reactivity of sulphydryl groups in tryptophanase by a dual-monitoring high-performance liquid chromatographic system with a site-directed fluorescent reagent.

Sulphydryl groups of E. coli tryptophanase (L-tryptophan indole lyase, E.C. 4.1.99.1) were made to react with a fluorescent maleimide derivative, N-(4-anilino-1-naphthyl)maleimide(ANM). By carefully controlling the reaction conditions it was possible to limit the extent of sulphydryl group modification. The modified enzyme was digested with (L-1-tosylamide-2-phenylethyl chloromethyl ketone)-trypsin. The fluorescent peptides obtained were analysed by reversed-phase high-performance liquid chromatography on a C18 column with a dual-monitoring system consisting of a UV and a fluorescence monitor connected in tandem. This was followed by the determination of the amino acid composition of the fluorescent peptides. Comparison of these results with the known, complete primary structure of tryptophanase from the K-12 strain of E. coli allowed the assignment of position 298 to the cysteine residue, which is more selectively modified by ANM under the conditions chosen and is involved in the maintenance of the catalytic activity.

Amino Acids↗