PubMed Health⌕ Search

Biomedical subjects

T Amachi

Publications and source records attributed to T Amachi.

At least 37 records · Page 2Linked to original sources
PubMed1999

Direct photoaffinity labeling of the Kir6.2 subunit of the ATP-sensitive K+ channel by 8-azido-ATP.

ATP-sensitive potassium channels are under complex regulation by intracellular ATP and ADP. The potentiating effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2. We determined whether ATP directly interacts with a binding site on the Kir6.2 subunit to mediate channel inhibition by analyzing binding of a photoaffinity analog of ATP (8-azido-[gamma-32P]ATP) to membranes from COS-7 cells transiently expressing Kir6.2. We demonstrate that Kir6.2 can be directly labeled by 8-azido-[gamma-32P]ATP but that the related subunit Kir4.1, which is not inhibited by ATP, is not labeled. Photoaffinity labeling of Kir6.2 is reduced by approximately 50% with 100 microM ATP. In addition, mutations in the NH2 terminus (R50G) and the COOH terminus (K185Q) of Kir6.2, which have both been shown to reduce the inhibitory effect of ATP upon Kir6.2 channel activity, reduced photoaffinity labeling by >50%. These results demonstrate that ATP binds directly to Kir6.2 and that both the NH2- and COOH-terminal intracellular domains may influence ATP binding.

Adenosine Triphosphate↗
PubMed1999

Vinexin: a novel vinculin-binding protein with multiple SH3 domains enhances actin cytoskeletal organization.

Using the yeast two-hybrid system and an in vitro binding assay, we have identified a novel protein termed vinexin as a vinculin-binding protein. By Northern blotting, we identified two types of vinexin mRNA that were 3 and 2 kb in length. Screening for full-length cDNA clones and sequencing indicated that the two mRNA encode 82- and 37-kD polypeptides termed vinexin alpha and beta, respectively. Both forms of vinexin share a common carboxyl-terminal sequence containing three SH3 domains. The larger vinexin alpha contains an additional amino-terminal sequence. The interaction between vinexin and vinculin was mediated by two SH3 domains of vinexin and the proline-rich region of vinculin. When expressed, vinexin alpha and beta localized to focal adhesions in NIH 3T3 fibroblasts, and to cell-cell junctions in epithelial LLC-PK1 cells. Furthermore, expression of vinexin increased focal adhesion size. Vinexin alpha also promoted upregulation of actin stress fiber formation. In addition, cell lines stably expressing vinexin beta showed enhanced cell spreading on fibronectin. These data identify vinexin as a novel focal adhesion and cell- cell adhesion protein that binds via SH3 domains to the hinge region of vinculin, which can enhance actin cytoskeletal organization and cell spreading.

3T3 Cells↗
PubMed1999

Activation process of dipteran-specific insecticidal protein produced by Bacillus thuringiensis subsp. israelensis.

Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp. israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST-20-kDa fusion protein (GST-20) nor the GST-45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST-60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.

Amino Acid Sequence↗
PubMed1999

Recent progress in P-glycoprotein research.

P-glycoprotein can extrude a variety of structurally diverse, toxic xenobiotic compounds from cells. It is believed to be one of key molecules which can cause multidrug resistance in cancer. This paper deals with recent progress in P-glycoprotein research, especially in its structure, mechanisms for substrate recognition and transport. The review also discusses specific modulators of multidrug resistance in cancer and gene therapy using the MDR1 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein.

To identify the roles of the two nucleotide-binding folds (NBFs) in the function of human P-glycoprotein, a multidrug transporter, we mutated the key lysine residues to methionines and the cysteine residues to alanines in the Walker A (WA) motifs (the core consensus sequence) in the NBFs. We examined the effects of these mutations on N-ethylmaleimide (NEM) and ATP binding, as well as on the vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP. Mutation of the WA lysine or NEM binding cysteine in either of the NBFs blocked vanadate-induced nucleotide trapping of P-glycoprotein. These results suggest that if one NBF is non-functional, there is no ATP hydrolysis even if the other functional NBF contains a bound nucleotide, further indicating the strong cooperation between the two NBFs of P-glycoprotein. However, we found that the effect of NEM modification at one NBF on ATP binding at the other NBF was not equivalent, suggesting a non-equivalency of the role of the two NBFs in P-glycoprotein function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Comparative studies of suidatrestin, a specific inhibitor of trehalases.

Suidatrestin, isolated from a Streptomyces strain, was characterized as a new trehalase inhibitor. Its inhibitory potential was 7 to 50-fold higher than that of validamycin when tested against insect, fungal and mammalian trehalases. The kinetic properties of suidatrestin were studied in vitro with trehalases from flight muscle mitochondria of the fly, Protophormia terraenovae, from larval midgut of the moth, Spodoptera littoralis, and from porcine kidney, as well as with maltase from yeast. Suidatrestin was inactive on maltase but inhibited all trehalases with IC50 values of 0.08-0.1 microM; Ki values ranged from 0.02 to 0.05 microM. The very low Ki/K(m) ratios (3.9 x 10(-6) -4.9 x 10(-6)) indicated excellent in vitro inhibitory action of suidatrestin. When injected into larvae of S. littoralis, suidatrestin required high and repetitive doses which lead to reversible inhibition of larval growth only. Consecutive omission of the inhibitor even stimulated weight increase above that of controls. Significant mortality was achieved at a rather high dose only. Injection of a growth-inhibiting dose of suidatrestin did not change hemolymph osmolality as a measure of sugar concentration. The discrepancy between in vitro and in vivo potency of suidatrestin may be understood once its chemical structure is fully known.

Animals↗
PubMed1998

Substrate specificities and kinetic properties of proteinase A from the yeast Saccharomyces cerevisiae and the development of a novel substrate.

The substrate specificities and kinetic properties of proteinase A, an intracellular aspartic proteinase from the yeast Saccharomyces cerevisiae, were determined using a series of synthetic chromogenic peptides with the general structure P5-P4-P3-P2-Phe-(NO2)Phe-P2'-P3' [P5, P4, P3, P2, P2', P3' are various amino acids; (NO2)Phe is p-nitro-L-phenylalanine]. The nature of the residues occupying the NH2-terminal region of the substrate had a strong influence on the kinetic constants. Among those tested, Ala-Pro-Ala-Lys-Phe-(NO2)-Phe-Arg-Leu had the best kinetic constants (Km = 0.012 mM, kcat = 14.4 s-1, kcat/Km = 1,200 M-1.s-1). Compared with such aspartic proteinases as pepsin, cathepsin D, and renin, the substrate specificity of proteinase A was unique. Based on these results, a novel fluorescent substrate, MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2, was developed for the sensitive measurement of proteinase A.

2,4-Dinitrophenol↗
PubMed1997

Construction of a starch-utilizing yeast by cell surface engineering.

We have engineered the cell surface of the yeast Saccharomyces cerevisiae by anchoring active glucoamylase protein on the cell wall, and we have endowed the yeast cells with the ability to utilize starch directly as the sole carbon source. The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The constructed plasmid containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The glucoamylase activity as not detected in the culture medium, but it was detected in the cell pellet fraction. The glucoamylase protein transferred to the soluble fraction from the cell wall fraction after glucanase treatment but not after sodium dodecyl sulfate treatment, indicating the covalent binding of the fusion protein to the cell wall. Display of the fused protein was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. The transformant cells could surely grow on starch as the sole carbon source. These results showed that the glucoamylase was anchored on the cell wall and displayed as its active form. This is the first example of an application of cell surface engineering to utilize and improve the metabolic ability of cells.

Cell Membrane↗
PubMed1994

Divalent metal ion requirements of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula.

To understand the roles of metal ions on the catalytic properties and thermostability of the thermostable beta-galactosidase of Saccharopolyspora rectivirgula, a thermophilic actinomycete, we have investigated the binding kinetics and requirements of divalent metal ions by equilibrium dialysis, titration, and metal ion buffer techniques. We found that the monomeric multimetal enzyme (M(r) 136,977) had eight specific binding sites for divalent metal ions. These sites were classified as follows: a very tight (class I) site for Ca2+, three tight (class II) sites consisting of two Ca(2+)-specific sites (class IICa) and one Mn(2+)-specific site (class IIMn; Kd for Mn2+, 2.0 nM), and four loose (class III) sites for Mn2+ (Kd, 1.2 microM) and Mg2+ (Kd, 2 microM). Removal of metal ions bound to class II and III sites of the holoenzyme (Ca3Mn5 species; relative Vmax (Vrel), 100%) by a chelating resin at 4 degrees C yielded a less thermostable Ca1 species (Vrel, 1.7%) with a class I Ca2+ ion, removal of which by a chelating resin at 50 degrees C caused a complete irreversible inactivation of the enzyme. Titration studies revealed that stoichiometric binding of Mn2+ to a class IIMn site of the Ca1 species caused a 33-fold activation whereas binding of Ca2+ to class IICa sites had no effect on enzyme activity. Ca1 species could be also activated 8-fold by heating at 60 degrees C for 20 min, suggesting that the catalytically important class II Mn2+ plays important roles in maintaining the native structure essential for activity. Occupation of class III sites by Mg2+ or Mn2+ was of physiological importance to attain sufficient thermostability by which this extracellular beta-galactosidase remained active for a prolonged time at elevated temperatures as was observed during growth of S. rectivirgula.

Binding Sites↗
PubMed1994

Oxidation-reduction properties of compounds I and II of Arthromyces ramosus peroxidase.

At neutral pH, compound I of Arthromyces ramosus peroxidase (ARP) was stable and was reduced to ferric ARP without apparent formation of compound II upon titration with ascorbate or hydroquinone. In the titration experiments, compound II was seen as an intermediate only at alkaline pH. However, measuring a difference spectrum in the Soret region by a stopped-flow method, we found that compound II was formed during the catalytic oxidation of ascorbate even at neutral pH. Using an EPR spectrometer with a microflow system, we measured the steady-state concentration of benzosemiquinone formed in the ARP-catalyzed oxidation of hydroquinone. The results clearly showed that ARP catalyzes the oxidation of hydroquinone by a one-electron-transfer mechanism, as does horseradish peroxidase. These observations led to the conclusion that compound I is reduced to compound II through a one-electron reduction by ascorbate or hydroquinone. Therefore, we concluded that ARP compound II is unusually unstable and is rapidly reduced to ferric enzyme without accumulation in the titration experiment. The unusual instability of ARP compound II is explained in terms of the high reduction potential of compound II. The reduction potentials (E0') of compounds I and II were measured at several pH values from redox equilibria with potassium hexachloroiridate on the basis of E0' = 0.90 V for the IrCL6(2)-IrCl6(3)- couple. These values were determined to be 0.915 and 0.982 V at pH 7, respectively, and decreased with increasing pH. This pH dependence was markedly changed by the buffer concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Ascorbic Acid↗
PubMed1994

Crystal structure of the fungal peroxidase from Arthromyces ramosus at 1.9 A resolution. Structural comparisons with the lignin and cytochrome c peroxidases.

The crystal structure of the peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7) from the hyphomycete Arthromyces ramosus (ARP) has been determined by the multiple isomorphous replacement method and refined by the simulated annealing method to a crystallographic R-factor of 17.4% for the 19,191 reflections with F > 2 sigma F between 7.0 and 1.9 A resolution. The model includes residues 9 to 344, the heme group, two N-acetylglucosamine residues, two calcium ions and 246 water molecules. The root-mean-square deviation of bond lengths from the ideal values is 0.02 A. The mean coordinate error is estimated as 0.2 A. The electron density of the glycine-rich region of the amino-terminal eight residues was invisible. ARP has ten major and two short alpha-helices and a few short beta-strands. The overall tertiary structure of ARP is similar to that of yeast cytochrome c peroxidase (CCP) and is particularly similar to that of the lignin peroxidase (LiP) from Phanerochaete chrysosporium. Relative to CCP, ARP and LiP each have an extension of approximately 40 residues at the carboxy terminus. All eight cysteine residues in ARP form disulfide bonds (C12:C24, C23:C293, C43:C129 and C257:C322). Two calcium sites are inaccessible to solvent. The four disulfide bonds and two calcium sites, which are lacking in CCP, are conserved in ARP and LiP. The bond from Asn304C to Ala305N in ARP is the site sensitive to proteases. An Asx turn present in the Asn303 to Ala305 segment appears to orient the side-chain of Asn304 to outward from the molecule, rendering it easily trappable by pockets of proteases. The proximal heme ligand is His184 in helix F (distance of N epsilon 2 ... Fe, 2.10 A), and one of several water molecules in the distal pocket of the heme bridges the iron atom and the N epsilon 2 of His56. The orientation of the imidazole ring of the distal histidine residue relative to the heme group in ARP differs significantly from that in LiP. The access channel to the distal side of the heme of ARP is markedly wider along the heme plane than that of LiP. Many of the amino acid residues that comprise the entrance of this channel differ for ARP and LiP. This may account for the differences in substrate specificity.

Acetylglucosamine↗
PubMed1993

Crystallization and preliminary X-ray diffraction studies of peroxidase from a fungus Arthromyces ramosus.

Peroxidase (donor: H2O2 oxidoreductase [EC 1.11.1.7]) was purified from the culture broth of the hyphomycete Arthromyces ramosus in the early log phase to show a single band on SDS-PAGE. The crystals of A. ramosus peroxidase (ARP) were formed by salting out with ammonium sulfate at room temperature and pH 7.5. The repeated seeding technique was employed to grow the crystals to the size large enough for X-ray diffraction study. The crystals were characterized as tetragonal, space group P4(2)2(1)2, with unit cell dimensions of a = b = 74.5 A, c = 117.6 A. The asymmetric unit contains one molecule of peroxidase. They diffract X-rays to at least 2.0 A resolution and are stable to X-rays.

Crystallization↗
PubMed1992

Reclassification of two strains of Arthrobacter oxydans and proposal of Arthrobacter nicotinovorans sp. nov.

Arthrobacter oxydans DSM 419 and DSM 420 have chemical and microbiological properties that are consistent with assignment to the genus Arthrobacter. Both organisms have the lysine-alanine-threonine-alanine peptidoglycan type. DNA-DNA pairing studies indicated that A. oxydans DSM 419 should be reclassified as Arthrobacter ureafaciens and that A. oxydans DSM 420T forms the nucleus of a distinct genomic species. We propose that A. oxydans DSM 420 should be reclassified as Arthrobacter nicotinovorans sp. nov. The type strain is strain DSM 420.

Amino Acid Sequence↗
PubMed1991

Expression of a hybrid Cu/Zn-type superoxide dismutase which has high affinity for heparin-like proteoglycans on vascular endothelial cells.

Since plasma levels of enzymes, such as superoxide dismutase (SOD), that scavenge reactive oxygen species are low, surface membranes of endothelial and parenchymal cells of various tissues are often exposed to oxidative stress. To dismutase superoxide radicals efficiently in and around vascular endothelial cells, we constructed a fusion gene encoding a hybrid SOD (HB-SOD) consisting of human Cu/Zn-SOD and a C-terminal basic peptide that binds to heparin-like proteoglycans. The fusion gene was expressed in yeast, and the resulting HB-SOD was highly purified. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, HB-SOD revealed a protein band with an apparent molecular weight of 20,000. HB-SOD bound to endothelial cells of aortic segments by a mechanism which was inhibited by heparin but not by antithrombin III. When injected intravenously to rats, 125I-labeled HB-SOD rapidly disappeared from the circulation; the rate of disappearance was decreased by heparin. Less than 1% of the injected HB-SOD was found in the urine 20 min after administration at which time more than 70% of SOD was excreted in its intact form. Immunohistochemical studies revealed that HB-SOD predominantly bound to heparin-like proteoglycans on endothelial cells of the artery and other tissues. HB-SOD might permit studies on pathophysiological roles of superoxide radicals in and around vascular endothelial cells in vivo.

Amino Acid Sequence↗
PubMed1991

Effects of oleic, arachidonic and 5,8,11,14-nonadecatetraenoic acids on lipid secretion and ketogenesis in perfused rat liver.

The effects of perfused oleic (18:1n-9), arachidonic (20:4n-6) and 5,8,11,14-nonadecatetraenoic (19:4n-5) acids on triglyceride and cholesterol secretion and ketone body production were studied in isolated rat liver. As compared to oleic and 19:4n-5 acids, both ketone body production and triglyceride secretion were significantly lowered when arachidonic acid was perfused. The concentration of triglyceride in the post-perfused liver was lower upon perfusion with arachidonic acid than upon perfusion with oleic acid or 19:4n-5 acid. Cholesterol secretion in the liver perfused with arachidonic acid or 19:4n-5 acid was significantly higher than with oleic acid. The concentration of cholesterol in the post-perfused liver was slightly but significantly higher with 19:4n-5 acid than with the other fatty acids. The results suggest that 19:4n-5 acid when compared with arachidonic acid affects lipid metabolism in liver differently.

Animals↗
PubMed1990

Inhibition of oxygen toxicity by targeting superoxide dismutase to endothelial cell surface.

Since enzymes that degrade reactive oxygens, such as superoxide dismutase (SOD), are significantly lower in plasma than in intracellular compartments, cell surface membranes should be protected against hazardous oxygens particularly when animals are challenged with oxidative stress. To minimize oxygen toxicity on endothelial cell surface, a fusion gene consisting of cDNA coding human Cu2+/Zn2(+)-SOD and heparin-binding peptide was constructed and expressed in yeast. The resulting enzyme (HB-SOD) bound to a heparin-Sepharose column and cultured endothelial cells; binding was inhibited either by high NaCl concentrations or heparin. When injected intravenously, HB-SOD predominantly bound to vascular endothelial cell surface. Carrageenin-induced paw edema and cold-induced brain edema of the rat were markedly inhibited by a single dose of HB-SOD. These results suggest that superoxide radical and/or its metabolite(s) occurring at or near the outer surface of vascular endothelial cells might play a critical role in the pathogenesis of vasogenic edema.

Animals↗
PubMed1990

Correct splicing of modified introns of a Rhizopus proteinase gene in Saccharomyces cerevisiae.

The intron of the Rhizopus aspartic proteinase gene (RNAP-I) was modified by in vitro mutagenesis and examined for its splicing efficiency in Saccharomyces cerevisiae. The wild-type intron of the RNAP-I gene was not spliced at all in spite of its structural similarity to introns of S. cerevisiae. The primary transcript of the RNAP-I gene was converted to correctly translatable mRNA only when the complete consensus sequence of S. cerevisiae introns (i.e. 5'-GTATGT-----TACTAAC-----TAG-3') was introduced into its intron, although the efficiency of splicing was low. It is also shown that transformants carrying the RNAP-I gene with the complete consensus sequence of S. cerevisiae introns produce active RNAP-I protein.

Aspartic Acid Endopeptidases↗