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T Iio

Publications and source records attributed to T Iio.

At least 19 recordsLinked to original sources
PubMed2001

Calcium-bindings of wild type and mutant troponin Cs of Caenorhabditis elegans.

Apparent Ca(2+)-binding constant (K(app)) of Caenorhabditis elegans troponin C (CeTnC) was determined by a fluorescence titration method. The K(app) of the N-domain Ca(2+)-binding site of CeTnC was 7.9+/-1.6 x 10(5) M(-1) and that of the C-domain site was 1.2+/-0.6 x 10(6) M(-1), respectively. Mg(2+)-dependence of the K(app) showed that both Ca(2+)-binding sites did not bind competitively Mg(2+). The Ca(2+) dissociation rate constant (k(off)) of CeTnC was determined by the fluorescence stopped-flow method. The k(off) of the N-domain Ca(2+)-binding site of CeTnC was 703+/-208 s(-1) and that of the C-domain site was 286+/-33 s(-1), respectively. From these values we could calculate the Ca(2+)-binding rate constant (k(on)) as to be 5.6+/-2.8 x 10(8) M(-1) s(-1) for the N-domain site and 3.4+/-2.1 x 10(8) M(-1) s(-1) for the C-domain site, respectively. These results mean that all Ca(2+)-binding sites of CeTnC are low affinity, fast dissociating and Ca(2+)-specific sites. Evolutional function of TnC between vertebrate and invertebrate and biological functions of wild type and mutant CeTnCs are discussed.

Aminoquinolines↗
PubMed1999

Kinetic analysis on nitric oxide binding of recombinant Prolixin-S, a nitric oxide transport protein from the bloodsucking bug, Rhodnius prolixus.

Kinetics of the NO binding and removal reaction of recombinant Prolixin-S (rProlixin-S) were analyzed using stopped-flow spectrophotometry. The reaction was observed as a biphasic process. The rate constant of the fast phase increased linearly as NO concentration increased. The rate constant at the slow phase increased as NO concentrations increased at low NO concentration, then reached a plateau at high NO concentration. These NO dependencies of the reaction are characteristic of a bimolecular two-step consecutive reaction. The reaction consisted of the fast NO binding reaction of rProlixin-S and the following slow structural change of NO-protein complex. Kinetic studies revealed that the NO binding rate constant was independent of pH, but the rate constant of the NO removal reaction increased as pH increased. The apparent NO dissociation constant (Kd) of rProlixin-S was also calculated from the values of the kinetic parameters obtained in this work. The Kd value increased as pH and temperature increased. The Kd value of rProlixin-S and NO was 10-300 nM in regular physiological condition, which is 103 higher and 103 lower than those of the other ferric and ferrous hemoproteins and NO, respectively. These results indicate that Prolixin-S is one of NO transport proteins regulating blood pressure.

Animals↗
PubMed1998

Fluorescence energy transfer of complexes of skeletal muscle troponin C and melittin derivatives.

We studied Ca2+-dependent structural change of rabbit skeletal troponin C (TnC)-melittin (ME) complex as a model of TnC-troponin I complex. In previous study, we found that the distance between Met-25 and Cys-98 of TnC in TnC-ME complex increased upon binding of Ca2+ to TnC [H. Sano and T. Iio (1995) J. Biochem. 118, 996-1000]. In this study, we used a fluorescence energy transfer method. As a fluorescent donor, we used the tryptophan residue in four melittin derivatives, in which residue 2, 5, 8, or 13 was replaced with tryptophan. As acceptor, we used dansylaziridine (DANZ) bound to Met-25 of TnC, or N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS) bound to Cys-98 of TnC. For all TnCDANZ-ME complexes, the donor-acceptor distance (11.9-17.7 A) did not remarkably depend on Mg2+ or Ca2+ binding of TnC or on the position of tryptophan in ME derivatives. The same results were obtained for TnCAEDANS-ME complexes in the absence of Ca2+ (distance 15.2-21.7 A). But in the presence of Ca2+, tryptophan residues in the central region of ME were near to Cys-98 of TnC (distance much less than 10.4 A). Based on these results, we conclude that ME is enfolded by the N- and C-lobes of TnC, and the ME rod is almost perpendicular to a line connecting Met-25 and Cys-98 of TnC. The position of the ME rod shifts upon binding of Ca2+ to TnC.

Amino Acid Sequence↗
PubMed1997

Reconstitution of bacteriorhodopsin from a mixture of a proteinase V8 fragment and two synthetic peptides.

The peptide and retinal mixture of bacteriorhodopsin, composed of two synthetic peptides corresponding to helices F (160-197) and G (202-237) and a proteinase V8-derived fragment V1 (1-166), generated the characteristic features of bacteriorhodopsin with absorbance maximum at 550 nm and fluorescence quenching as in two synthetic peptides corresponded to helix A (sequence 7-31) and B (41-65) and a chymotryptic fragment (72-248). The recovery of reconstitution estimated from the absorption and the fluorescence quenching of these mixture was 16-19% and 25-32% of the native purple membrane, respectively, whereas mixtures lacking any one of the peptides exhibited no absorption recovery Circular dichroism of each peptide fragment showed complete formation of alpha-helical structure in a membrane-mimetic medium of sodium dodecyl sulfate. These results indicate that the specific interactions or mutual recognitions between alpha-helices in lipid bilayers are essential for correct bundling of the seven helices and formation of the retinal binding pocket.

Amino Acid Sequence↗
PubMed1997

Cloning and sequence analysis of genes encoding xylanases and acetyl xylan esterase from Streptomyces thermoviolaceus OPC-520.

Three genes encoding two types of xylanases (STX-I and STX-II) and an acetyl xylan esterase (STX-III) from Streptomyces thermoviolaceus OPC-520 were cloned, and their DNA sequences were determined. The nucleotide sequences showed that genes stx-II and stx-III were clustered on the genome. The stx-I, stx-II, and stx-III genes encoded deduced proteins of 51, 35.2, and 34.3 kDa, respectively. STX-I and STX-II bound to both insoluble xylan and crystalline cellulose (Avicel). Alignment of the deduced amino acid sequences encoded by stx-I, stx-II, and stx-III demonstrated that the three enzymes contain two functional domains, a catalytic domain and a substrate-binding domain. The catalytic domains of STX-I and STX-II showed high sequence homology to several xylanases which belong to families F and G, respectively, and that of STX-III showed striking homology with an acetyl xylan esterase from S. lividans, nodulation proteins of Rhizobium sp., and chitin deacetylase of Mucor rouxii. In the C-terminal region of STX-I, there were three reiterated amino acid sequences starting from C-L-D, and the repeats were homologous to those found in xylanase A from S. lividans, coagulation factor G subunit alpha from the horseshoe crab, Rarobacter faecitabidus protease I, beta-1,3-glucanase from Oerskovia xanthineolytica, and the ricin B chain. However, the repeats did not show sequence similarity to any of the nine known families of cellulose-binding domains (CBDs). On the other hand, STX-II and STX-III contained identical family II CBDs in their C-terminal regions.

Acetylesterase↗
PubMed1995

Fluorescence energy transfer study of troponin C-melittin complex.

Fluorescence energy transfer study of rabbit skeletal troponin C (TnC), which binds a donor, dansylaziridine, to Met-25 in helix A and an acceptor, 5-(iodoacetamido)eosin, to Cys-98 in helix E revealed (i) the donor-acceptor distance did not change upon Mg2+ and Ca2+ binding to TnC, (ii) melittin binding to Ca(2+)-free TnC induced a remarkable decrease in the donor-acceptor distance both in the presence and absence of Mg2+, and (iii) Ca2+ binding to TnC in TnC-melittin complex induced a characteristic increase in the donor-acceptor distance. These results indicate that TnC assumes a contracted structure upon binding of the basic amphipathic TnC-binding peptide, melittin, and that it undergoes a characteristic structural change that includes a displacement of helix A away from helix E upon Ca2+ binding to TnC in the TnC-melittin complex.

Calcium-Binding Proteins↗
PubMed1993

Kinetics of structural changes of reconstituted skeletal muscle thin filaments observed by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer was measured between probes attached to Troponin-I and actin in the reconstituted skeletal muscle thin filament. Cys-133 on TnI was labeled with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, and Lys-61 on actin was labeled with fluorescein 5-isothiocyanate. A large difference in the efficiency of resonance energy transfer was observed between the presence (0.55) and absence (0.76) of Ca2+, which corresponds to the change in the distance of 0.53 nm and is in good accordance with a previous report (Miki, M. (1990) Eur. J. Biochem. 187, 155-162). The fluorescence titration curve of this reconstituted thin filament was measured versus free Ca2+ concentration under physiological conditions in the presence of 8 mM MgCl2. The transfer efficiency changed sharply between pCa 6.9 and 5.75, with a midpoint at pCa 6.25, suggesting that a conformational change of thin filaments occurs with a highly cooperative mode. The time course of this conformational change was followed by measuring the fluorescence intensity change (energy transfer efficiency change) of the labeled reconstituted thin filament after rapid decrease or increase of free Ca2+ concentration with a stopped-flow fluorometer. When the free Ca2+ concentration was increased in the presence of 5 mM MgCl2, the fluorescence intensity increased (the transfer efficiency decreased) with a rate constant of 530 +/- 170 s-1 at 20 degrees C. On the other hand, when the free Ca2+ concentration was decreased, the fluorescence intensity decreased with a rate constant of 43 +/- 5 s-1. The results indicate that the conformational change observed by fluorescence resonance energy transfer corresponds well to the regulation mechanism by troponin-tropomyosin on the reconstituted skeletal muscle thin filament.

Actins↗
PubMed1993

Fluorescent molecular rotor binding to actin.

1-(2-Hydroxyethyl)-6-[(2,2-dicyano)vinyl]-2,3,4-trihydroquinoli ne (DCQ) is a fluorescent dye and its fluorescence yield is determined by a freedom of intramolecular rotation. When DCQ was added to an actin solution, its fluorescence intensity increased as the actin molecule transformed from G-actin to F-actin. This fluorescence intensity increase was induced both by the higher binding constant of the dye to F-actin than to G-actin and by the higher fluorescence yield of the dye bound to F-actin than to G-actin. DCQ bound more strongly to Mg(2+)-F-actin than to Ca(2+)-F-actin, resulting in higher fluorescence intensity of DCQ-Mg(2+)-F-actin solution than of DCQ-Ca(2+)-F-actin solution. DCQ did not distinguish Mg(2+)-G-actin and Ca(2+)-G-actin. Thus, DCQ is a good probe for polymerization of actin and polymorphism of F-actin.

Actins↗
PubMed1993

Melittin-binding of troponin C.

Ca(2+)-dependent interaction between skeletal muscle troponin C and a bee venom melittin, which can be regarded as a mimic of the troponin C-binding peptide of troponin I, was investigated. Sephadex gel chromatography revealed that melittin bound to troponin C irrespective of the presence or absence of Ca2+ in 50 mM KCl and 50 mM Tris-HCl, pH 7.5. At high salt concentration, 0.5-1.2 M KCl, melittin was removed from apo troponin C but still bound to Ca(2+)-loaded troponin C. Hydrophobic affinity chromatography revealed that hydrophobic region(s) appeared on the surface of troponin C upon Ca2+ binding but vanished upon Ca2+ release. Therefore, at physiological salt concentration, melittin binds to Ca(2+)-loaded troponin C by both hydrophobic and electrostatic interactions, and it binds to apo troponin C by electrostatic interaction. The midpoint of the fluorescence titration curve of troponin C-melittin complex, observed by Trp-19 fluorescence, scarcely depended on the concentration of MgCl2. This means Trp-19 of melittin detects only the conformational change of troponin C induced by Ca(2+)-binding to the low affinity Ca(2+)-binding sites (sites I and II) of troponin C; that is, the C-terminal of melittin binds to the N-terminal of troponin C. Fluorescence stopped-flow experiments revealed that the time course of Trp-19 fluorescence change induced by the Ca(2+)-release from the low affinity Ca(2+)-binding sites of troponin C in the complex was biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Induction of hepatic microsomal cytochrome P450 and drug-metabolizing enzymes by 4-benzylpyridine and its structurally related compounds in rats. Dose- and sex-related differential induction of cytochrome P450 species.

We examined the abilities of 4-, 3- and 2-benzylpyridine and 4-tert-butylpyridine to induce hepatic microsomal cytochrome P450 and drug-metabolizing enzymes in male and female rats in order to define the effects of pyridine-containing compounds on drug metabolism. 4-Benzylpyridine (0.4 mmol/kg, for 2 consecutive days) induced total cytochrome P450 to about three times that of the controls at 24 hr, and its inducing effect was sustained for 120 hr after the treatment in male and female rats. 4-Benzylpyridine was a more potent inducer of cytochrome P450 than 3- and 2-benzylpyridine, which induced the cytochrome to 71.4 and 43.9%, respectively, of that produced by the 4-substituted isomer. 4-tert-Butylpyridine also induced cytochrome P450. Immunoblot analysis revealed that a single treatment of male rats with 4-benzylpyridine at doses ranging from 0.05 to 0.80 mmol/kg induced cytochrome P450b/e and caused a maximum increase in the level of the isozyme at the 0.2 mmol/kg dose. 4-Benzylpyridine at doses from 0.40 to 0.80 mmol/kg also induced cytochrome P450c/d in male rats. In female rats, 4-benzylpyridine induced cytochrome P450b at doses ranging from 0.1 to 0.80 mmol/kg and produced a maximum increase in the level of this isozyme at 0.40 to 0.60 mmol/kg. Induction of cytochrome P450c/d by 4-benzylpyridine in female rats was observed at a dose of 0.20 mmol/kg, and the magnitude of the induction of the isozyme was increased in a dose-dependent manner. Both 3- and 2-benzylpyridine induced cytochrome P450b/e and/or c/d depending on the increase of total cytochrome P450 without changing the induction patterns of the isozymes. 4-tert-Butylpyridine induced cytochrome P450b at doses ranging from 0.20 to 0.60 mmol/kg and slightly induced P450c/d at doses ranging from 0.10 to 0.40 mmol/kg in male rats. These results and our previous report (Matsuura et al., Biochem Pharmacol 41: 1949-1956, 1991) clearly show that the pyridine compounds having lipophilic groups at the 4- or 3-position of the ring could be inducers of cytochrome P450. The present results also revealed that 4-benzylpyridine shows dose- and sex-related differences in the induction of cytochrome P450b/e and c/d in rats.

Aminopyrine N-Demethylase↗
PubMed1992

Fluorescent rotors and their applications to the study of G-F transformation of actin.

New fluorescent rotor molecules having hydrophilic functional groups, which are derivatives of p-(N,N-dialkylamino)benzylidenemalononitrile, were synthesized. Their properties as fluorescent rotors were confirmed by an observation of solvent viscosity-dependent fluorescence. Incorporation of hydrophilic groups into the molecules increased the solubility of fluorescent rotors in aqueous media; the application of the compounds to biochemical systems became feasible as a consequence. To demonstrate this applicability, we attempted to monitor the G-F transformation of rabbit skeletal muscle actin with these newly synthesized compounds. All the compounds carrying a malononitrile moiety showed greater fluorescence in F-actin. Among them, 1-(2-hydroxyethyl)-6-[(2,2-dicyano)vinyl]-2,3,4-trihydroquinoli ne gave the best result by the criteria of the difference in fluorescence quantum yield for G- and F-actin, solubility, and stability of the compound. The method has the major advantage of not requiring covalent modification of actin.

Actins↗
PubMed1992

Dose-response study on covalent binding to forestomach protein from male F344 rats following oral administration of [14C]3-BHA.

A dose-response study on covalent binding to forestomach protein was performed using male F344 rats following oral administration of [14C]3-tert-butyl-4-hydroxyanisole (3-BHA). The order of tissue distribution of radioactivity 6 h after oral administration of 1% [14C]3-BHA was forestomach greater than glandular stomach greater than liver greater than kidney greater than plasma. The covalent binding levels to forestomach protein were very low until 0.1% 3-BHA, but rapidly increased at concentrations of 1% and 2% 3-BHA. The dose-response relations of 3-BHA levels to the covalent binding to protein coincided well with the incidence of forestomach papilloma reported previously. The binding levels of forestomach and glandular stomach were compared. In case of i.v. administration, both binding levels were almost the same, however, in case of 0.1% p.o. administration, the forestomach level was approximately 8-fold higher than the glandular stomach level. The binding level of forestomach protein by p.o. administration was approximately 54-fold higher than that by i.v. administration. Although the amount of tert-butylhydroquinone (BHQ) was very low compared with the amount of covalent binding, the BHQ levels in forestomach were dependent upon the dose levels of 3-BHA. Our study indicates that the dose-response study on covalent binding to target tissue protein is an efficient method for the quantitative estimation of the active metabolites coming from the chemicals which form the quinone metabolites.

Animals↗
PubMed1992

Static and kinetic studies of calmodulin and melittin complex.

Ca2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca(2+)-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca(2+)-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittin-calmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin.

Calcium↗
PubMed1992

Properties of an albumin inhibiting lysosomal acid cholesteryl ester hydrolase in rat liver.

Lysosomal acid cholesteryl ester hydrolase (acid CEH, EC. 1.1.1.13) activity was inhibited by addition of an increasing amount of d = 1.21 bottom fraction from rat serum (Lipids in press). To clarify the mechanism of this inhibition, rat native and modified albumin were added to the assay mixture and their effects on acid CEH activity were examined. The inhibitory effect on acid CEH activity was dependent on the concentration of rat albumin. Albumin of various mammalian species, including human, bovine and rabbit, also inhibited acid CEH activity. This inhibitory activity was markedly increased by heat treatment, the effect increasing in parallel with the prolongation of the treatment. Moreover, this albumin-dependent inhibition of acid CEH activity was also markedly increased by methylation of albumin. In contrast, the inhibition of acid CEH activity by modified albumins, such as acetyl albumin, succinyl albumin and glycine methyl ester albumin, was much lower than that of albumin, and no stimulatory effect of heat treatment on the albumins was observed. The heat-treated albumin-dependent inhibition of acid CEH activity was not abolished in the presence of sodium deoxycholate. The values of Vmax obtained were similar with or without heat-treated albumin. These results suggest that the inhibitory effects of heat-treated albumin may be due to an intrinsic and characteristic property of the lipid/water interface, and that the stimulatory effects of heat treatment on albumin-dependent inhibition may be due to heat-induced changes in the affinity and conformation of albumin.

Albumins↗
PubMed1991

Structure-activity relationships in the induction of hepatic microsomal cytochrome P450 by clotrimazole and its structurally related compounds in rats.

We investigated the structure-activity relationship in the induction of hepatic microsomal cytochrome P450 by clotrimazole and its structurally related compounds. For this purpose, we synthesized various compounds structurally analogous to clotrimazole and injected them into rats at a fixed dose of 0.2 mmol/kg. We found that the chlorine atom in clotrimazole was not necessary for the induction of cytochrome P450. The imidazole moiety of clotrimazole, however, was a very important structural component for the induction of cytochrome P450; triazole, but not pyrrole, could be substituted for this moiety. 1-Tritylimidazole, 1-diphenylmethylimidazole and 1-benzylimidazole were also found to be inducers of cytochrome P450, but, to a somewhat lesser extent, with a decreasing number of substituted phenyl groups. Thus, 1-benzylimidazole was a minimum structurally active unit for inducing cytochrome P450. In addition, 4-diphenylmethylpyridine and 4-benzylpyridine also induced cytochrome P450 to extents similar to those induced by the corresponding imidazole derivatives, but 4-benzylpiperidine lacked this effect. When the methylene unit of clotrimazole-related compounds was introduced by a hydroxy or amino group instead of imidazole, there was a less extensive increase in cytochrome P450 content. This inducing effect was lost completely by the lack of an imidazole moiety and imidazole itself. 1-Phenylethylimidazole and 1-benzylimidazole induced cytochrome P450 to a similar extent. All of these findings suggest that 1-substituted heteroaromatic compounds having two or more nitrogen atoms are likely to be required for inducing cytochrome P450. Immunoblot analysis revealed that clotrimazole and other various inducers found in this study increased cytochrome P450b/e content. These results could provide information on the effects of drugs and chemicals on cytochrome P450 induction.

Animals↗
PubMed1991

Species differences between rats and mice in the teratogenic action of ethylenethiourea: in vivo/in vitro tests and teratogenic activity of sera using an embryonic cell differentiation system.

In vivo/in vitro studies on rats showed that ethylenethiourea inhibited the differentiation of midbrain cells more severely than that of limb bud cells. In in vitro studies using midbrain cell cultures, ethylenethiourea concentrations inhibited the production of differentiated foci by 50% in mouse cells, a rate 11-fold higher than that in rat cells. Differentiation of rat midbrain cells was also inhibited by the serum samples prepared from rats or mice dosed with up to 200 mg/kg of ethylenethiourea. However, differentiation of mouse cells was not inhibited by these animal serum samples. The concentration of ethylenethiourea in rat sera was only 2-fold higher than that in mice sera at 2 hr after dosing with 200 mg/kg. Therefore, the different sensitivity of the midbrains of these two species may be one reason that ethylenethiourea is teratogenic in rats but not in mice.

Animals↗
PubMed1991

DNA damage in forestomach epithelium from male F344 rats following oral administration of tert-butylquinone, one of the forestomach metabolites of 3-BHA.

DNA damage in the forestomach epithelium of male F344 rats was tested by alkaline elution assay following oral administration of 3-tert-butyl-4-hydroxyanisole (3-BHA) and its free metabolites. Although 1% 3-BHA and 0.001% tert-butylhydroquinone (BHQ) caused no detectable DNA damage, a tert-butylquinone (BQ) treated group showed a significantly higher elution rate than the corn oil and 3-BHA group. Dose-dependent induction of DNA damage in forestomach epithelium by 3-BHA metabolites was compared. BQ showed DNA damaging activity from 0.0001% (1 microgram/ml). On the other hand, BHQ showed the activity at greater than 100 times higher concentration than that of BQ. Six other quinones related to the BQ have been synthesized and examined in vivo for their activity in single strand breaks in forestomach epithelium DNA. The order of the DNA damage was not always correlated with the order of the first reduction potentials of the quinones. 2-Benzylthio-5-tert-butyl-p-benzoquinone (BQSBn-2), 2-phenyl-1,4-benzoquinone and 2,5-di-tert-butyl-p-benzoquinone had higher DNA damaging activity than BQ. The effects of ethylacrylate (EA) pretreatment on the DNA damage by BQ and BQSBn-2 were compared. The DNA damage by BQ was decreased by increasing the EA concentrations. On the other hand, BQSBn-2 had a DNA damaging activity following the EA pretreatment. In order to examine whether 3-BHA is metabolized in forestomach, 3-BHA and BQ levels in the mucosa of forestomach were measured by HPLC with an electrochemical detector. The concentrations of BQ in mucosa correlated with the 3-BHA dose levels from 0.01% to 0.1% 3-BHA. The concentration of BQ in mucosa was approximately 1/4000 of the concentration of 3-BHA in mucosa at the dose level of 1% 3-BHA. The present results indicate that BQ and its thiol conjugates are the active metabolites from 3-BHA in forestomach and may play an important role in inducing carcinogenicity of 3-BHA in F344 rats.

Acrylates↗