Biomedical subjects

T Fukui

Publications and source records attributed to T Fukui.

At least 235 records · Page 13Linked to original sources

PubMed1995

Effect of repeated cold stress on mouse stomach histidine decarboxylase.

The effect of repeated cold stress (RCS) on both mRNA level and enzyme activity of stomach histidine decarboxylase (HDC) was studied in ddY mice. Following 1-day treatment of RCS, stomach HDC activity, but not its mRNA level, increased two fold. Following 3-day treatment of RCS, which is the essential period for the induction of hyperalgesia in mice, HDC mRNA level and enzyme activity increased in the stomach. After cessation of RCS treatment, HDC mRNA level decreased and reached the level of non-RCS treated mice, but HDC activity did not. The stomach from the 1-day RCS-treated mouse contained proteolytic activity, which converts the in vitro-translated 74 kD HDC species into the 53 kD HDC species. These data demonstrate that RCS-treated mouse stomach induces both the de novo synthesis of the 74 kD HDC species and its proteolytic cleavage to 53 kD HDC species.

Animals↗

PubMed1994

Generation of the topa quinone cofactor in bacterial monoamine oxidase by cupric ion-dependent autooxidation of a specific tyrosyl residue.

The quinone of 2,4,5-trihydroxyphenylalanine (topa), recently identified as the covalently bound redox cofactor in copper amine oxidases, is encoded by a specific tyrosine codon. To elucidate the mechanism of its formation, the recombinant phenylethylamine oxidase of Arthrobacter globiformis has been overproduced in Escherichia coli and purified in a Cu(2+)-deficient form. The inactive precursor enzyme thus obtained was dramatically activated upon incubation with Cu2+, concomitantly with the formation of the topa quinone at the position corresponding to Tyr382, occurring in the tetrapeptide sequence highly conserved in this class of enzymes. The topa quinone was produced only under aerobic conditions, but its formation required no external enzymatic systems. These findings demonstrate the Cu(2+)-dependent autooxidation of a specific tyrosyl residue to generate the topa quinone cofactor.

Arthrobacter↗

PubMed1994

Association of N-ethylmaleimide-sensitive factor with synaptic vesicles.

N-Ethylmaleimide-sensitive factor (NSF) mediates docking and/or fusion of transport vesicles in the multi-pathways of vesicular transport. NSF was highly expressed in brain and adrenal gland. Immunostaining of cerebellum with an anti-NSF monoclonal antibody showed that NSF is predominantly localized in the molecular layers and the glomeruli of the granule cell layers. This distribution coincided well with that of synaptophysin, a marker protein of synaptic vesicles. Purification and immunoprecipitation revealed that NSF is associated with brain synaptic vesicles. The present results suggest that NSF is associated with synaptic vesicles without Ca2+ influx.

Animals↗

PubMed1994

Cloning and sequencing of phenylethylamine oxidase from Arthrobacter globiformis and implication of Tyr-382 as the precursor to its covalently bound quinone cofactor.

The gene of Arthrobacter globiformis encoding a quinoprotein, phenylethylamine oxidase, has been cloned and sequenced. In the deduced amino acid sequence comprising 638 residues is a tetrapeptide sequence, Asn-Tyr-Asp-Tyr, which has been found to be highly conserved in other copper amine oxidase. Mutation of the former Tyr (Tyr-382) of the recombinant enzyme into Phe resulted in the complete loss of catalytic activity and disappearance of the quinone compound that is specifically detected in the wild-type enzyme, suggesting that Tyr-382 is the precursor to the covalently-bound cofactor, most probably topa quinone. Furthermore, the expression of the active, quinone-containing enzyme in Escherichia coli cells was markedly dependent on the presence of Cu2+ ions in the culture medium, and the inactive, Cu2(+)-deficient enzyme produced without Cu2+ ions could be converted to the active quinone form by reconstitution with Cu2+ ions.

Amine Oxidase (Copper-Containing)↗

PubMed1994

Involvement of conserved lysine 68 of Bacillus stearothermophilus leucine dehydrogenase in substrate binding.

Lysine 68 of Bacillus stearothermophilus leucine dehydrogenase is highly conserved in the corresponding regions of NAD(P)+-dependent amino acid dehydrogenase sequences. To elucidate its functional role, the lysyl residue of the recombinant enzyme has been replaced with alanine or arginine by site-directed mutagenesis. Either mutation resulted in nearly complete loss of activity in the oxidative deamination, whereas only the mutation to alanine led to a marked increase in Michaelis constants for both amino and keto acid substrates. On the other hand, an ionizable group in the wild-type enzyme with a pKa value of 10.1-10.7, which must be protonated for binding of substrate and competitive inhibitor with an alpha-carboxyl group, was unobservable in both mutant enzymes. These results altogether led to the conclusion that Lys-68 is located at the active site of the enzyme and involved in binding of the alpha-carboxyl group of substrate through an ionic interaction. In addition, the alanine mutant enzyme that is almost inactive in the deamination but significantly active in the amination was greatly stimulated by exogenously added ammonia, suggesting that proper binding of the substrate alpha-carboxyl group at Lys-68 is essential for catalysis.

Alanine↗

PubMed1994

Pyridoxal 5'-phosphate probes at Lys-480 can sense the binding of ATP and the formation of phosphoenzymes in Na+,K(+)-ATPase.

The Lys-480 in the alpha-subunits of Na+,K(+)-ATPase from pig kidneys was specifically modified with pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (AP2PL) probes in the presence of NaCl. The site was shown to be the same as the ATP-protectable binding of these probes (Hinz, H.R., and Kirley, T.L. (1990) J. Biol. Chem. 265, 10260-10265). Modifications strongly reduced both Na+,K(+)-ATPase activity and the amount of Na(+)-dependent phosphoenzyme from [32P]ATP but not from [32P]acetyl-phosphate (AcP). Addition of AcP to the enzyme induced a slight decrease in the fluorescence of the PLP probe in the presence of 2 M NaCl and 4 mM MgCl2 but a single exponential increase in the presence of 16 mM NaCl and 4 mM MgCl2. The addition of ATP induced single exponential fluorescence increases at both Na+ concentrations. The data show that these probes can sense molecular events related to the formation of phosphoenzymes induced by AcP and presumably to the formation of Mg-Na-ATP-enzyme complex. The data also suggest that PLP or AP2PL probes at Lys-480 in the presence of Na+ and Mg2+ do not affect the transphosphorylation from AcP to Asp-369 to form phosphoenzymes but that they inhibit the transphosphorylation from the gamma-phosphoryl group of ATP and also ATP binding in the absence of Mg2+.

Adenosine Triphosphate↗

PubMed1994

Microheterogeneity in glycosylphosphatidylinositol anchor structures of bovine liver 5'-nucleotidase.

In our study, 5'-nucleotidase was released from bovine liver by the treatment with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and purified to a homogeneous state by concanavalin A-Sepharose and (diethylaminoethyl)-Toyopearl column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified 5'-nucleotidase were then cleaved by cyanogen bromide (CNBr), and then inositol phosphoglycan-containing C-terminal peptides (IPG peptides) were separated by C18 reverse-phase liquid chromatography and analyzed by peptide sequencer, amino acid analyzer, gas chromatography (GC), and GC-mass spectrometry (MS). Ser523 of the amino acid sequence deduced from 5'-nucleotidase cDNA [Suzuki et al. (1993) J. Biochem. (Tokyo) 113, 607-613] is revealed to be the C-terminal amino acid to which a glycosylphosphatidylinositol is anchored. Separated peaks of CNBr-cleaved IPG peptides were then analyzed by electron spray ionization (ESI)-MS. Eight different molecular weight (MW) species of CNBr-cleaved IPG peptides were detected. Three fractions of CNBr-cleaved IPG peptides were separately treated by trypsin, and trypsinized IPG peptides were purified by C18 reverse-phase liquid chromatography. Finally, five different MW species of trypsinized IPG peptides (1629.4, 1752.7, 1791.8, 1832.8, and 1994.5) were detected by ESI-MS. Together with sequential exoglycosidase treatment and quantitative analysis of sugar moieties by GC and GC-MS, microheterogeneity in the structures of these five glycosylphosphatidylinositol (GPI) anchor species was determined. The common core structure was ethanolamine phosphate-mannose-mannose-mannose(-ethanolamine phosphate)-glucosamine-myoinositol phosphate. Variations observed in additional mannose, N-acetylhexosamine, and ethanolamine phosphate moieties form this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗

PubMed1994

Identification of Lys277 at the active site of Escherichia coli glycogen synthase. Application of affinity labeling combined with site-directed mutagenesis.

Lys15 in Escherichia coli glycogen synthase, which is specifically labeled by adenosine diphosphopyridoxal, is mainly involved in binding of the substrate ADP-glucose (Furukawa, K., Tagaya, M., Tanizawa, K., and Fukui, T. (1993) J. Biol. Chem. 268, 23837-23842). We have found that the mutant glycogen synthase in which Lys15 is replaced by Gln via site-directed mutagenesis is inactivated by adenosine diphosphopyridoxal at concentrations higher than those required for the inactivation of the wild-type enzyme. ADP and ADP-glucose offered protective effects on inactivation, suggesting that the label binds to the ADP-glucose-binding site in the mutant enzyme. Sequence analysis of the labeled peptide revealed that the labeled residue is Lys277. This lysyl residue is conserved in maize starch synthase, which shows about 30% amino acid identity to E. coli glycogen synthase. Substitution of Gln for Lys277 by site-directed mutagenesis resulted in a 140-fold decrease in the kcat value with little changes in the Km values for ADP-glucose and glycogen. These results suggest that Lys277 at the active site participates in the catalytic reaction rather than binding of substrate. The present study shows the usefulness of the combined application of affinity labeling and site-directed mutagenesis.

Affinity Labels↗

PubMed1994

Clinical evaluation of biotin-binding immunoglobulin in patients with Graves' disease.

Biotin-binding immunoglobulin (BBI) was recently identified in human serum and has been suggested to have a significant association with allergic and autoimmune disorders. Attempts were made to evaluate the clinical significance of BBI in autoimmune thyroid disorders. Prevalence of BBI was significantly higher in Graves' disease (47%) than in Hashimoto's disease (8%) and healthy controls (10%). The BBI consisted of heterogeneous subtypes with respect to binding of several immunoglobulin classes. Sera in Graves' disease showed predominantly IgG-binding BBI, whereas healthy subjects had IgM-binding BBI. Thyroid stimulating hormone receptor antibody (TRAb) level was significantly higher in the BBI non-detected group than in the detected group. There was no significant relationship between BBI prevalence and thyroid hormone concentrations, anti-thyroglobulin antibody (TGAb) or anti-thyroid microsomal antibody (McAb) titers. In addition, biotin levels in peripheral blood and red blood cells and biotinidase activity did not differ in the BBI detected and non-detected groups. The present results suggest that BBI is associated with autoimmune dysfunction in Graves' disease.

Adult↗

PubMed1994

A detection method for point mutation in the precore region of human hepatitis B virus (HBV)-DNA using mutation-site-specific assay.

In the natural progression of acute active hepatitis and chronic active hepatitis in human hepatitis B virus (HBV)-infected patients, inactive hepatitis develops by seroconversion, which can be explained by the disappearance of HBe antigen. However, it has been found that in some patients with hepatitis, alanine aminotransferase levels undergo fluctuation even though their serum is negative for HBe antigen. In these patients, HBV-DNA has been detected in the serum and the HBV-DNA so detected has been considered a cause of worsening liver function. Most HBV-DNA detected in these cases has a point mutation from G to A at the 83rd base in the precore region. As a result of this point mutation, HBV is unable to produce HBe antigen. We have devised a sensitive polymerase chain reaction (PCR) method, a mutation-site-specific assay, for the detection of point mutations at the 83rd base in the precore region using a specific mutation-trapped oligonucleotide primer for the mutant HBV genome.

Base Sequence↗

PubMed1994

Overproduction and characterization of recombinant UDP-glucose pyrophosphorylase from Escherichia coli K-12.

Using oligonucleotide probes synthesized on the basis of partial amino acid sequences, we have cloned and sequenced the gene of Escherichia coli K-12 encoding UDP-glucose pyrophosphorylase. The gene consists of 906 base pairs and encodes a polypeptide of 302 amino acid residues with a calculated molecular weight of 32,941. Its nucleotide sequence was found to be identical with that recently registered (EMBL, X59940) for a gene coding for an unknown 33-kDa protein, which was later annotated as UDP-glucose pyrophosphorylase on the basis of genetic studies. The UDP-glucose pyrophosphorylase gene, mapped at 27.3 min in the E. coli chromosome, complemented the galU mutation, which renders the bacterium unable to ferment galactose. The recombinant enzyme overproduced in E. coli cells and purified to homogeneity catalyzed the synthesis and pyrophosphorolysis of UDP-glucose by a sequential mechanism. The enzyme required Mg2+ for maximal activity and was inhibited by free UTP and pyrophosphate. The E. coli enzyme shows significant sequence similarities with the enzymes from Acetobacter xylinum and Salmonella typhimurium. However, little or no similarity was found with the eukaryotic enzymes that are involved in the biosynthesis of storage carbohydrates, or with other enzymes acting on similar sugar nucleotides. Thus, UDP-glucose pyrophosphorylases participating in diverse metabolic pathways can be classified structurally into the prokaryotic and eukaryotic groups, even though they have almost identical catalytic properties.

Amino Acid Sequence↗

PubMed1994

Role of the conserved glycyl residues located at the active site of leucine dehydrogenase from Bacillus stearothermophilus.

A tetrapeptide sequence, Gly-Gly-(Gly/Ala)-Lys, containing a catalytically important lysyl residue, is highly conserved in NAD(P)+-dependent amino acid dehydrogenases. To elucidate functional roles of the glycyl residues in this conserved sequence Gly-77, Gly-78, and Gly-79 of the recombinant leucine dehydrogenase from Bacillus stearothermophilus have been individually replaced with Ala by site-directed mutagenesis. All of the mutant enzymes had Michaelis constants for alpha-keto-iso-caproate and ammonia several times larger than the wild-type enzyme while retaining considerable catalytic activities. However, inhibition constants for a substrate analog without an alpha-carbonyl group were unchanged by the mutations. On the other hand, the rate of inactivation by pyridoxal 5'-phosphate and the microenvironment of aromatic residues, in particular of the sole tryptophanyl residue (Trp-46) located in the vicinity of the active site, were affected by the mutations of the glycyl residues. All of these results suggest that the conserved glycyl residues are important for fine-tuning of the position and/or orientation of the epsilon-amino group of Lys-80 at the active site to function efficiently as a general-base catalyst. Furthermore, the Gly-77 and Gly-78 mutant enzymes had markedly decreased thermal stabilities, showing that these two glycyl residues are also critical for the conformational stability of this thermostable enzyme.

Amino Acid Oxidoreductases↗

PubMed1994

Affinity labeling of the two species of Escherichia coli lysyl-tRNA synthetase with adenosine di- and triphosphopyridoxals.

Lysyl-tRNA synthetase (LysRS), a representative of the class 2 aminoacyl-tRNA synthetases, occurs as two species in Escherichia coli: LysRSs and LysRSu. To identify the ATP-binding site in this enzyme, we have applied affinity labeling with reactive adenine nucleotide analogs. Incubation of either enzyme species with adenosine di- or triphosphopyridoxal, followed by borohydride reduction, resulted in a time-dependent incorporation of the reagent, accompanied with the loss of both tRNA(Lys) aminoacylation, and lysine-dependent isotopic ATP-PPi exchange activities. LysRSu appeared less sensitive to adenosine triphosphopyridoxal than LysRSs. Complete inactivation with either reagent corresponded to the incorporation of about 2 mol of reagent per mol of dimeric enzyme. MgATP and ATP protected both enzyme species against the inactivation, suggesting that the modification occurs at the ATP-binding site. Sequence analysis of the labeled peptide isolated from the inactivated LysRSs and LysRSu revealed that bulk of the label was distributed among six lysyl residues at positions 25, 82, 114, 156, 364, and 505, with preference for Lys-114 and Lys-156. In LysRSs, Lys-132 and Lys-185 were also modified by both reagents, although these residues are not conserved in LysRSu. It is concluded that the folding of the LysRSs and LysRSu polypeptides and the relative locations of the identified lysyl residues with respect to the binding site for the two labels are very similar.

Adenosine Diphosphate↗

PubMed1994

Affinity labeling of Escherichia coli lysyl-tRNA synthetase with pyridoxal mono- and diphosphate.

Pyridoxal 5'-phosphate (PLP) and pyridoxal 5'-diphosphate (PLDP) were used to identify lysyl residues at the phosphate-binding locus in the lysS-encoded and the lysU-encoded lysyl-tRNA synthetases (LysRSs and LysRSu, respectively) from Escherichia coli. Incubation of LysRSs with either reagent, followed by borohydride reduction, resulted in a time-dependent covalent incorporation of the reagent, accompanied with the loss of both tRNA(Lys) aminoacylation and lysine-dependent ATP-PPi exchange activities. By contrast, LysRSu activity was insensitive to prolonged incubation with either reagent, possibly reflecting a difference at the phosphate-binding locus in the two enzyme species. MgATP protected LysRSs against inactivation by PLP or PLDP. Complete inactivation of LysRSs corresponded to the incorporation of 2.6 +/- 0.1 mol of PLP or PLDP per mol of dimeric enzyme. Either reagent was found to label the same set of eight lysyl residues (Lys-25, Lys-82, Lys-114, Lys-132, Lys-156, Lys-185, Lys-364, and Lys-505) as adenosine di- or triphosphopyridoxal (see the preceding paper in this issue). These lysyl residues might represent the subsite for the phosphate moiety of ATP in LysRSs. None of the identified lysyl residues is located within the three sequence motifs considered as characteristic of the class 2 aminoacyl-tRNA synthetases. The present results are discussed on the basis of the crystalline structure of the closely related aspartyl-tRNA synthetase from Saccharomyces cerevisiae.

Affinity Labels↗

PubMed1994

Identification of active site lysyl residues of phenylalanine dehydrogenase by chemical modification with methyl acetyl phosphate combined with site-directed mutagenesis.

A monoanionic acetylation reagent, methyl acetyl phosphate, was used to acetylate lysyl residues of the recombinant thermostable phenylalanine dehydrogenase from Thermoactinomyces intermedius. The enzyme was irreversibly inactivated with the reagent in a time- and dose-dependent manner. Simultaneous addition of substrate and coenzyme markedly protected the enzyme from inactivation. Acetylated lysyl residues presumably occurring at the active site were determined by differential modification; the enzyme was first modified with a cold reagent in the presence of both substrate and coenzyme and, after removal of the added substances by gel filtration, was then labeled with a radioactive reagent. At least 7 lysyl residues per enzyme subunit were radiolabeled by this method. To further specify the lysyl residue(s) whose modification results in inactivation of the enzyme, 5 lysyl residues highly conserved in various amino acid dehydrogenase sequences were replaced with Ala by site-directed mutagenesis. Although all of the single mutant enzymes were inactivated with the reagent as effectively as the wild-type enzyme, a double mutant enzyme in which both Lys-69 and Lys-81 were replaced with Ala was found to be inactivated very slowly. These results suggest that the reagent can acetylate both of these lysyl residues and inactivate the enzyme. Kinetic analyses of the single Lys-69 and Lys-81 mutant enzymes revealed that they are involved in substrate binding and catalysis, respectively, like the corresponding residues in the homologous leucine dehydrogenase.

Amino Acid Oxidoreductases↗

PubMed1994

Cardiac hypertrophy and brain natriuretic peptide in experimental hypertension.

The blood pressure was decreased after chronic treatment with enalapril, MK-954, and hydralazine in deoxycorticosterone acetate (DOCA)-salt-induced malignant hypertension of spontaneously hypertensive rats (SHR); however, ventricular weight and plasma brain natriuretic peptide (BNP) concentration were decreased after enalapril and MK-954 but not after hydralazine. The BNP secretory rates from the ventricle in enalapril- and MK-954-treated DOCA-salt SHR were decreased to approximately 50% of those in untreated DOCA-salt SHR. The BNP secretory rate from the ventricle was positively correlated with ventricular weight in untreated and treated DOCA-salt SHR. In contrast, acute administration of captopril or MK-954 did not decrease the BNP secretory rate from the heart. Results suggest that the decrease in plasma BNP after enalapril and MK-954 is attributed to a decline in the secretion from the ventricle but not from the atrium. The reduction in ventricular mass appeared to be related to this decline.

Animals↗

PubMed1994

Human T lymphotropic virus type I associated myelopathy and myasthenia gravis: a possible association?

We report the first known patient with human T lymphotropic virus type I (HTLV-I) associated myelopathy (HAM) and myasthenia gravis (MG). A 50-year-old woman developed fluctuating muscle weakness with easy fatigability, transient bilateral blepharoptosis and double vision. Spastic paraparesis complicated these symptoms. Neurological assessments and specific laboratory findings revealed that the patient had definite HAM and MG. By inference from decreasing serum anti-HTLV-I antibody titers after thymectomy, the presence of antigenicity for HTLV-I in the thymic reticular cells, and a high incidence of various coexistent autoimmune diseases in HAM or MG, we suggested the possibility that these two diseases were associated with each other and with HTLV-I infection.

Adult↗

PubMed1994

Cognitive functions in subjects with incidental cerebral hyperintensities.

We investigated the association between incidental cerebral hyperintensities (CH) found by magnetic resonance imaging (MRI) and cognitive functions in neurologically normal, nondemented subjects. Semiquantitative scores for MRI lesions and those for brain atrophy were compared with the results of extensive cognitive examinations using multivariate analysis. There was no correlation between CH and cognition, except that periventricular hyperintensities, especially those in posterior locations, were associated with reduced performance in the Stroop test. Overall cognitive functions were associated with age, and age was a predominant factor in the prefrontal functions. Brain atrophy was associated more with decline of the posterior and dorsolateral frontal brain functions. We suggest that disturbances in attention and speed may initially result from incidental CH, while other cognitive functions remain unaffected.

Adult↗