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M Tokushige

Publications and source records attributed to M Tokushige.

At least 19 recordsLinked to original sources
PubMed1992

Determination of the subunit contact region of aspartase.

The subunits of tetrameric enzyme aspartase from Escherichia coli and Pseudomonas fluorescens were incapable of forming hybrid tetramers, suggesting that the subunit contact regions of these two enzymes are not conserved in spite of significant homology between the total sequences of the enzymes. To locate the subunit contact region, we modified cysteine residues of the intermediate species formed during the assembly of the subunits of aspartase from E. coli. Subunits modified with N-ethylmaleimide were unable to assemble into tetramers. Further experiments showed that Cys-88 was the primary residue that was modified. The sequence flanking Cys-88 is quite different from that in the the enzyme from P. fluorescens, suggesting that this region participates in the mutual recognition of subunits.

Amino Acid Sequence↗
PubMed1992

Cloning and overproduction of biodegradative threonine deaminase from Escherichia coli W strain.

We have cloned the structural gene (tdcB) of biodegradative threonine deaminase from Escherichia coli W strain by utilizing the polymerase chain reaction. The JM109/pUCTDA strain, which was obtained by transforming E. coli JM109 with a vector plasmid (pUCTDA) containing the cloned tdcB gene, produced a large amount of the enzyme corresponding to more than 5% of the total soluble protein. Amino acid sequence analysis of this recombinant enzyme showed that the amino acid sequence is identical to the nucleotide-deduced sequence of biodegradative threonine deaminase from E. coli K-12.

Amino Acid Sequence↗
PubMed1991

Preliminary X-ray crystallographic analysis of tryptophanase from Escherichia coli.

Tryptophanase (L-tryptophan indole-lyase) from Escherichia coli has been crystallized from ammonium sulfate solution using a vapor diffusion method. The crystals are tetragonal and belong to space group P4(1)2(1)2 or its enantiomorph. The cell dimensions of the crystals are a = b = 113.4 A, and c = 232.2 A, with two subunits per asymmetric unit. The crystals diffract to at least 3 A resolution, and are suitable for X-ray structural analysis.

Ammonium Sulfate↗
PubMed1991

Activation of aspartase by site-directed mutagenesis.

To elucidate the role of sulfhydryl groups in the enzymatic reaction of the aspartase from Escherichia coli, we used site-directed mutagenesis which showed that the enzyme was activated by replacement of Cys-430 with a tryptophan. This mutation produced functional alterations without appreciable structural change: The kcat values became 3-fold at pH 6.0; the Hill coefficient values became higher under both pH conditions; the dependence of enzyme activity on divalent metal ions increased; and hydroxylamine, a good substrate for the wild-type enzyme, proved a poor substrate for the mutant.

Amino Acid Sequence↗
PubMed1991

Rapid purification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae.

Homoserine dehydrogenase of Saccharomyces cerevisiae has been rapidly purified to homogeneity by heat and acid treatments, ammonium sulfate fractionation, and chromatography on Matrex Gel Red A and Q-Sepharose columns. The final preparation migrated as a single entity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr of 40,000. The Mr of the native enzyme was 81,000 as determined by gel filtration, suggesting that the enzyme is composed of two identical subunits. This feature was also confirmed by cross-linking analysis using the bifunctional reagent dimethyl suberimidate. Feedback inhibition by L-methionine and L-threonine was observed using the purified enzyme. The enzyme was markedly stabilized against heat treatment at high salt concentrations. Additions of feedback inhibitors or high concentrations of salts failed to cause any dissociation or aggregation of the enzyme subunits unlike enzymes from other sources such as Rhodospirillum rubrum. The enzyme denatured in 3 M guanidine-HCl was refolded by simple dilution with a concomitant restoration of the activity. Cross-linking analysis of the renaturation process suggested that the formation of the dimer is required for activity expression. Amino acid sequence analysis of peptides obtained by digestion of the enzyme protein with Achromobacter lyticus protease I revealed that several amino acid residues are strictly conserved among homoserine dehydrogenases from S. cerevisiae, Escherichia coli, and Bacillus subtilis.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of two types of fumarase from Escherichia coli.

Two distinct types of fumarase were purified to homogeneity from aerobically grown Escherichia coli W cells. The amino acid sequences of their NH2-terminals suggest that the two enzymes are the products of the fumA gene (FUMA) and fumC gene (FUMC), respectively. FUMA was separated from FUMC by chromatography on a Q-Sepharose column, and was further purified to homogeneity on Alkyl-Superose, Mono Q, and Superose 12 columns. FUMA is a dimer composed of identical subunits (Mr = 60,000). Although the activity of FUMA rapidly decreased during storage, reactivation was attained by anaerobic incubation with Fe2+ and thiols. Studies on the inactivation and reactivation of FUMA suggested that oxidation and the concomitant release of iron inactivated the enzyme in a reversible manner. While the inactivated FUMA was EPR-detectable, through a signal with g perpendicular = 2.02 and g = 2.00, the active enzyme was EPR-silent. These results suggested FUMA is a member of the 4Fe-4S hydratases represented by aconitase. After the separation of FUMC from FUMA, purification of the former enzyme was accomplished by chromatography on Phenyl-Superose and Matrex Gel Red A columns. FUMC was stable, Fe-independent and quite similar to mammalian fumarases in enzymatic properties.

Amino Acid Sequence↗
PubMed1991

Identification of lysyl residues at the AMP-binding site of biodegradative threonine deaminase from Escherichia coli.

The biodegradative threonine deaminase from Escherichia coli is activated allosterically by AMP. To identify the residues interacting with the phosphate group of AMP at the binding site, we used the affinity labeling reagent, adenosine diphosphopyridoxal (AP2-PL). In the absence of AMP, the enzyme formed the Schiff base with AP2-PL and Scatchard plot analysis showed a biphasic pattern, the respective Kd values for the high- and low-affinity binding phases being 20 and 110 microM. The former value is comparable to the Kd value of the enzyme for AMP. In the presence of AMP, the Schiff base formation was greatly reduced. Although the maximal activating effect of adenosine diphosphopyridoxine, a non-reactive derivative of AP2-PL, was about 13% of that of AMP, the half-saturation concentration was almost the same. These findings suggest that AP2-PL specifically labeled the lysyl residue(s) at the AMP-binding site of the enzyme. To identify the labeled residue(s), we reduced the modified enzyme with sodium borohydride, then cleaved it with cyanogen bromide and Achromobacter lyticus protease I. Reverse-phase HPLC was used to isolate two labeled peptides from the digest. Their amino acid compositions and sequences showed that Lys-111 and Lys-113 were labeled. We conclude that these two lysyl residues are located around the phosphate group of AMP at the allosteric regulation site of the enzyme.

Adenosine Diphosphate↗
PubMed1990

Role of tryptophan 248 in the active site of tryptophanase from Escherichia coli.

Tryptophan 248, located in the active site of tryptophanase from Escherichia coli, has been replaced with phenylalanine by site-directed mutagenesis. Judging from CD and fluorescence spectra, the global structure of the mutant enzyme was found to be the same as that of the wild-type enzyme. The binding affinity of the mutant enzyme for the coenzyme pyridoxal 5'-phosphate (PLP) was reduced tenfold compared to the wild-type enzyme. Kinetic analyses under PLP-saturated conditions indicated that the Km values of the mutant enzyme for substrates are the same as those of wild-type enzyme but the kcat values are decreased to about 85%, which accounts for the overall activity decrease. These findings suggest that tryptophan 248 interacts closely with PLP and plays an important role in the catalytic reaction.

Animals↗
PubMed1990

Studies on T-lineage cells in human decidua of first trimester pregnancies.

T-lineage cells in human decidua of early pregnancies were tested for surface markers, proliferative response, interleukin-2 (IL-2) production, and natural killer (NK) activity. T-lineage (CD2+) cells that were obtained from decidua by the use of E-rosette formation contained fewer CD3+ mature T cells and CD4+ cells than those from the peripheral blood of the same donors, while no differences were seen in the frequencies of CD8+ cells. P55 molecules of IL-2 receptor (IL-2R/p55, Tac antigen) were hardly detected on fresh decidual T-lineage cells, though approximately 20% were positive for HLA-DR. More than a half of decidual T-lineage cells expressed CD56 molecules on their surface and killed K562 cells, the prototype target of NK cells, while most of them were negative for CD16 and CD57. Upon stimulation with IL-2, decidual T-lineage cells demonstrated dose-dependent proliferative response. In addition, they were induced to produce high amounts of IL-2 by stimulation with mitogens but not with alloantigens. These results suggest that human decidua contains high numbers of CD2+3-CD16 +/- 56+ lymphocytes and that this population responds to IL-2, produces IL-2 and mediates NK activity.

Decidua↗
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↗