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Y Kawata

Publications and source records attributed to Y Kawata.

At least 91 records · Page 5Linked to original sources
PubMed1995

The role of ATP hydrolysis in the function of the chaperonin GroEL: dynamic complex formation with GroES.

In order to understand the role of ATP hydrolysis of the chaperonin GroEL during protein folding, we have studied GroEL-GroES complex formation in the presence of ATP or ADP by using capillary electrophoresis and surface plasmon resonance. Capillary electrophoresis analysis showed that the GroEL 14-mer and GroES 7-mer formed a 1:1 complex in the presence of ATP. In the presence of ADP, both the association and dissociation rates of the complex were slower by about one order of magnitude than the rates in the presence of ATP at 25 degrees C. The implications of such a stable complex on the overall mechanism of chaperonin function are discussed.

Adenosine Diphosphate↗
PubMed1995

Stability of ribonuclease T2 from Aspergillus oryzae.

The stability of ribonuclease T2 (RNase T2) from Aspergillus oryzae against guanidine hydrochloride and heat was studied by using CD and fluorescence. RNase T2 unfolded and refolded reversibly concomitant with activity, but the unfolding and refolding rates were very slow (order of hours). The free energy change for unfolding of RNase T2 in water was estimated to be 5.3 kcal.mol-1 at 25 degrees C by linear extrapolation method. From the thermal unfolding experiment in 20 mM sodium phosphate buffer at pH 7.5, the Tm and the enthalpy change of RNase T2 were found to be 55.3 degrees C and 119.1 kcal.mol-1, respectively. From these equilibrium and kinetic studies, it was found that the stability of RNAse T2 in the native state is predominantly due to the slow rate of unfolding.

Aspergillus oryzae↗
PubMed1995

The folding characteristics of tryptophanase from Escherichia coli.

The unfolding and refolding characteristics of Escherichia coli tryptophanase (tryptophan indole-lyase) [EC 4.1.99.1] in guanidine hydrochloride were studied. Tryptophanase unfolded by first dissociating its coenzyme, pyridoxal 5'-phosphate, from the active site. This dissociation caused a significant destabilization of structure, and global unfolding of the protein followed. During this global unfolding step, an intermediate was formed which had a strong tendency to aggregate irreversibly, as detected by light scattering experiments. Tryptophanase was unable to refold quantitatively after unfolding in 4 M guanidine hydrochloride. The low refolding yield was due to non-specific aggregation which occurs during refolding. Various conditions which limited this aggregation were probed, and it was found that by initiating the refolding reaction at low temperature, the aggregation of tryptophanase folding intermediates during the reaction could be avoided to a certain extent, and the refolding yield improved.

Apoenzymes↗
PubMed1995

Crystal structure of Streptomyces erythraeus trypsin at 1.9 A resolution.

A trypsin-like serine protease from Streptomyces erythraeus (abbreviated as SET) has been crystallized at pH 7, which is within its active pH range. The crystal structure of SET has been solved by molecular replacement using the atomic model of Streptomyces griseus trypsin (SGT), which is 37% homologous with SET, and refined to the crystallographic R factor of 0.199 for 15,878 reflections with Fo/sigma(F) > 3 between 7 and 1.9 A resolution. The final model of SET contains 1,619 protein atoms and 97 water molecules. No Ca2+ ion is present in SET apparently because (i) the two carboxylate groups from two Glu residues, which bind a Ca2+ ion in bovine trypsin (BT) or SGT, have disappeared; and (ii) a guanidino group from an Arg residue is unfavorably present in the potential binding region. There is an unusual type II beta-turn in which the third residue is Asp instead of Gly. This Asp residue is the only non-Gly residue significantly outside the allowed regions in the Ramachandran map. The three-dimensional structure of SET is essentially the same as those of other trypsins of mammalian origin. The 211 C alpha atoms of SET exhibit an r.m.s. deviation of 1.16 A with equivalent atoms of BT, and the 208 equivalent C alpha atoms between SET and SGT exhibit an r.m.s. deviation of 1.09 A. The large deviations in C alpha positions between SET and BT or between SET and SGT are mainly observed in the first domain. The conformations of the side-chains of the catalytic triad are mutually similar to each other in these three proteases. Each of the chi 1 torsion angles of the three residues is distributed within +/- 5 degrees from each corresponding mean value. The hydrogen bond distances related to the side-chains in the triad coincide fairly well, though the relative disposition of the side-chains differs by 0.1-0.6 A among SET, BT, and SGT. The hydrogen bond network concerned with Ser(195), Asp(189), and water molecules in the substrate binding pocket differs from that in BT or SGT.

Amino Acid Sequence↗
PubMed1995

In vivo effect of GroESL on the folding of glutamate racemase of Escherichia coli.

The overexpression of the murI (glr) gene, which encodes the glutamate racemase of Escherichia coli, resulted in the formation of inclusion bodies of the enzyme, and little activity was found in the soluble fraction of the transformant cells. The coexpression of the groESL gene with murI caused an in vivo solubilization of glutamate racemase in an active form. We isolated the active enzyme and purified it effectively.

Amino Acid Isomerases↗
PubMed1995

Porphyromonas gingivalis fimbria-stimulated bone resorption in vitro is inhibited by a tyrosine kinase inhibitor.

Our previous study (Y. Kawata, S. Hanazawa, S. Amano, Y. Murakami, T. Matsumoto, K. Nishida, and S. Kitano, Infect. Immun. 62:3012-3016, 1994) showed that Porphyromonas gingivalis fimbriae stimulate bone resorption in vitro. Since it has recently been demonstrated that tyrosine kinase encoded by the c-src gene plays an important role in osteoclastic bone resorption, in the present study we examined the effect of a tyrosine kinase inhibitor on the fimbria-stimulated bone resorption. Genistein, a potent inhibitor of tyrosine kinase, markedly inhibited bone resorption stimulated by the fimbriae. Genistein also inhibited induction of several tyrosine-phosphorylated proteins in the fimbria-treated calvarial bone cells from mouse embryos.

Animals↗
PubMed1994

The guanidine-induced conformational changes of the chaperonin GroEL from Escherichia coli. Evidence for the existence of an unfolding intermediate state.

Equilibrium unfolding experiments of the E. coli chaperonin GroEL were performed in guanidine hydrochloride. A reversible unfolding intermediate was observed in very low concentrations of denaturant (< 0.5 M guanidine hydrochloride). This intermediate was characterized by a decreased light scattering intensity and an increased binding of the fluorescent probe 1-anilino-8-naphthalene sulfonate. No significant changes in circular dichroism spectra were observed for this unfolding intermediate. A second decrease in fluorescence intensity and light scattering was observed in higher concentrations of guanidine hydrochloride, with a transitional midpoint of 1.15 M. This transition was accompanied by the complete loss of secondary structure, as monitored by circular dichroism spectroscopy. This second transition agreed well with the results previously reported in this journal (Price et al. (1993) Biochim. Biophys. Acta 1161, 52-58).

Chaperonin 60↗
PubMed1994

Chaperonin GroE and ADP facilitate the folding of various proteins and protect against heat inactivation.

In the presence of ADP, the molecular chaperones GroEL and GroES from Escherichia coli not only facilitated the refolding of various proteins, but also prevented their irreversible heat inactivation in vitro. Without nucleotides the refolding reactions were arrested by GroEL. Addition of GroES and ADP to the reaction mixture initiated the refolding reactions and the enzyme activities were regained efficiently. The presence of GroE (GroEL and GroES) and ADP also protected against heat inactivation of native enzymes at various temperatures. These findings suggest that in the presence of GroES, nucleotide binding is an important event in the mechanism of GroEL-facilitated protein folding.

Adenosine Diphosphate↗
PubMed1994

Phorbol myristate acetate stimulates osteoclast formation in 1 alpha,25-dihydroxyvitamin D3-primed mouse embryonic calvarial cells by a prostaglandin-dependent mechanism.

Our previous study provided a novel assay system utilizing devitalized bone slices for study of the differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts among calvarial cells of mouse embryos. Using this assay system, we examined the effect of phorbol myristate acetate (PMA) on osteoclast formation as assessed by the appearance of tartrate-resistant acid phosphatase (TRAP)-positive cells and bone resorption lacunae. PMA alone was directly unable to induce the appearance of TRAP-positive cells and bone resorption lacunae of calvarial bone cells of mouse embryos. However, PMA markedly stimulated increases in the number of TRAP-positive cells and area of the resorption lacunae of the calvarial cells when the bone cells were primed by 1 alpha,25-(OH)2D3. This stimulatory effect of PMA was dose dependent. H-7, having relatively high affinity for protein kinase C, strongly inhibited in a dose-dependent fashion the stimulatory effect of PMA on the bone resorption of the hormone-primed calvarial cells. We also examined the involvement of prostaglandin in this stimulatory effect of PMA. Indomethacin, a cyclooxygenase inhibitor, markedly abolished the stimulatory effect of PMA on the bone resorption of the calvarial cells. PMA stimulated prostaglandin E2 (PGE2) production by the calvarial cells primed with 1 alpha,25-(OH)2D3 in a dose-dependent fashion. However, the PMA stimulation of the PGE2 production was significantly inhibited by H-7 and also by indomethacin. Furthermore, we observed that the addition of PGE2 to the calvarial cells primed with 1 alpha,25-(OH)2D3 for 1 or 3 days resulted in an increased number of TRAP-positive cells and increased bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Porphyromonas gingivalis fimbriae stimulate bone resorption in vitro.

Our previous study demonstrated that Porphyromonas gingivalis fimbriae induce the expression of interleukin-1, a potent bone-resorbing cytokine, in macrophages. This demonstration suggested to use the possibility that the fimbriae may stimulate bone resorption via the generation of an inflammatory cytokine(s). The present study was performed to test this suggestion. The bone-resorbing activity was evaluated by measuring the area of resorption lacunae on bone slices incubated with calvarial bone cells taken from 14-day-old mouse embryos. Fimbriae at 0.5 micrograms of protein per ml stimulated the bone-resorbing activity significantly, and the effect was dose and treatment time dependent. Since it is well known that interleukin-1 and granulocyte macrophage colony-stimulating factor induce differentiation of osteoclast lineage cells, we examined the involvement of these cytokines in fimbria-stimulated bone resorption. Fimbria-stimulated bone resorption was abolished significantly by antisera against both cytokines. We observed by Northern (RNA) blot assay that both cytokine genes were markedly expressed in the fimbria-treated calvarial bone cells. Our present data demonstrate that P. gingivalis fimbriae stimulate bone resorption in vitro.

Animals↗
PubMed1994

[Ossified epidural hematomas: report of two cases].

Two cases of ossified epidural hematomas were reported. Patients were nine and twelve year-old boys. Initially they were both followed up conservatively after injuries. Subsequently they were found on CT scan to have calcifications in the capsules of epidural hematomas which were found four months after injury in one case and twelve days after injury in the other. Both underwent craniotomy and histological ossification was found in the capsules of the hematomas. Epidural hematomas in children are known to ossify, and this condition may prevent natural absorption of epidural hematomas. Therefore, careful follow up of hematomas seems to be mandatory on conservative therapy of epidural hematomas in children.

Child↗
PubMed1993

Use of hybridization for distance measurement by fluorescence energy transfer in oligomeric proteins: distance between two functional sites in aspartase.

Our previous studies suggested that in a tetrameric enzyme aspartase, Cys-140 and Trp-430 are located at or near the catalytic and activator sites, respectively. To estimate the distance between these two sites, fluorescence energy transfer between a single tryptophan (Trp-430) and a fluorescent group specifically attached to Cys-140 has been measured. From the fluorescence spectra of the enzyme, the distance was calculated to be 22.2 A according to the Förster's theory. To estimate the contribution of energy transfer between subunits, we prepared hybrids composed of non-fluorescent and fluorescent subunits and showed that the energy transfer occurred mainly within one subunit in the tetramer. These results indicate that the use of hybridization is very effective as a general method for evaluation of intersubunit energy transfer in oligomeric proteins.

Aspartate Ammonia-Lyase↗
PubMed1993

Refolding of yeast enolase in the presence of the chaperonin GroE. The nucleotide specificity of GroE and the role of GroES.

GroE, a chaperonin protein from Escherichia coli, facilitates the folding of numerous proteins by binding to protein-folding intermediates and suppressing aggregation (Gething, M., and Sambrook, J. (1992) Nature 355, 33-45). The specific mechanism of GroE-facilitated folding involves numerous interactions between GroEL, GroES, the refolding protein, and ATP. In the present study, we have probed the molecular characteristics of the refolding reaction of yeast enolase in the presence of GroE. We have found that (a) GroEL interacts specifically with enolase during the folding reaction, resulting in folding arrest; (b) the release of partially folded molecules of enolase from the GroE complex may be mediated by the addition of nucleotides other than ATP (ADP, CTP, and UTP); and (c) GroES is required for enolase to be released from GroEL in the presence of ADP, CTP, and UTP but not required in the presence of ATP. The nucleotide binding mechanism of GroEL and the specific role of GroES during the refolding reaction are discussed in detail.

Adenosine Triphosphate↗
PubMed1993

Identification of an active dimeric form of aspartase as a denaturation intermediate.

The guanidine-HCl (Gu-HCl)-induced denaturation of a tetrameric enzyme, aspartase from Escherichia coli has been studied by size-exclusion chromatography, and circular dichroism and fluorescence spectroscopies. The size-exclusion analysis showed that in the presence of 0.4 M Gu-HCl, the enzyme has a dimeric structure with 45% of the native activity. The fluorescence and CD studies showed that only a small change occurred in the secondary and tertiary structures in 0.4 M Gu-HCl. In the range of 0.4 to 1 M Gu-HCl, decrease in the activity was observed as the secondary and tertiary structures were disrupted, whereas the dimeric enzyme did not dissociate into inactive monomer until 1 M Gu-HCl. When the enzyme was denatured in less than 1 M Gu-HCl, more than 90% of the original activity was recovered from the renaturation reaction, indicating that the dissociation process from tetramer to dimer is reversible. In contrast, the renaturation yield was 43% when the enzyme was diluted from more than 1 M Gu-HCl, indicating that the process of dissociation into monomer is not reversible. Thus, we identified an active dimeric form as a denaturation intermediate in this study, although the intermediates (including dimer) that were detected in renaturation experiments at low temperature were inactive, as reported previously [Imaishi, H., Yumoto, N., & Tokushige, M. (1989) Physiol. Chem. Phys. Med. NMR 21, 221-228].

Aspartate Ammonia-Lyase↗
PubMed1993

Expression of monocyte chemoattractant protein 1 (MCP-1) in adult periodontal disease: increased monocyte chemotactic activity in crevicular fluids and induction of MCP-1 expression in gingival tissues.

The present study shows that monocyte chemotactic activity in crevicular fluids increases with severity of the disease and that a monocyte chemoattractant, monocyte chemoattractant protein 1 (MCP-1), is expressed as the predominant cytokine of gingival tissues and their fibroblasts treated with Porphyromonas (Bacteroides) gingivalis lipopolysaccharide (P-LPS). High monocyte chemotactic activity in the crevicular fluids was neutralized significantly by antiserum specific for the JE/MCP-1 protein. Marked expression of the MCP-1 gene was observed in the gingival tissues of all adult periodontal patients tested, but not in those of healthy subjects. Monocyte chemotactic activity was observed in culture supernatants of human normal gingival tissues treated with P-LPS, and the chemotactic activity increased in a dose-related manner. Expression of MCP-1 in P-LPS-treated human gingival fibroblasts was further examined. P-LPS induced the MCP-1 gene expression in a dose- and treatment time-dependent manner. The MCP-1 gene product in the culture supernatant was detected as two forms with molecular masses of 11,000 and 15,000 Da by immunoprecipitation with the specific antiserum. The MCP-1 gene expression was induced in the fibroblasts treated with interleukin-1 beta and tumor necrosis factor alpha, but not with interleukin-6. These results suggest that gingival fibroblasts can participate in monocyte recruitment in gingival tissues of adult periodontal patients via the MCP-1 gene product and that MCP-1 plays an important role in the inflammatory reaction in the disease.

Adult↗
PubMed1993

[Markedly dilated cervical carotid arteries in a patient with a ruptured aneurysm of the anterior communicating artery: a case report].

We reported a case of ruptured aneurysm of the anterior communicating artery with marked dilatation of bilateral cervical carotid arteries. A 38 year old female suffered a subarachnoid hemorrhage. Angiography on admission revealed markedly dilated cervical carotid arteries with smooth lumen and a few segmental areas with mild constrictions in their entire course up to the carotid canals (their maximal sagittal diameters exceeded those of a cervical vertebral body). A saccular aneurysm was also seen at the junction of right A1, A2, and Acom. External carotid arteries were normal in size. Vertebral arteries were not examined because of failures of selective cannulation. The patient was operated upon and trapping of Acom was performed. During the operation, no definite arteriosclerotic changes were identified in the intracranial arteries. Histopathological examination of the surgical specimens revealed marked hyperplasia of the smooth muscle of the tunica media with intact internal elastic lamina both in a superficial temporal artery and a middle meningeal artery. During the operation, pneumothorax developed due to the rupture of bullae in the right lung. Past history of this patient disclosed hypertension noted a few years previously, and frequent severe bruises following minor trauma. Repeated angiography performed three months after the operation disclosed unchanged dilatation of the cervical carotid arteries as well as mild intraluminal irregularities in the proximal one third of the left renal artery. This patient died of pneumonia one year after the operation, but autopsy was not permitted. Possible diagnosis of this patient was discussed, with particular emphasis on fibromuscular dysplasia and Ehlers-Danlos type IV (arterial, ecchymotic, or Sack-Barabas type).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗