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Biomedical subjects

T Fukui

Publications and source records attributed to T Fukui.

At least 361 records · Page 20Linked to original sources
PubMed1988

Affinity labeling of ras oncogene product p21 with guanosine diphospho- and triphosphopyridoxals.

Purified v-rasH p21 overproduced in Escherichia coli was treated with guanosine diphospho- and triphosphopyridoxals (GP2- and GP3-PL), affinity labeling reagents specific to a lysyl residue located in the guanine nucleotide binding site. GP2-PL and GP3-PL inhibited [3H]GDP binding to p21 competitively. Incubation of p21 with GP2-PL and GP3-PL followed by reduction with NaBH4 resulted in 40 and 50% loss of [3H]GDP binding activity, respectively, whereas the addition of excess GDP completely protected p21 from the inactivation. The tryptic digest of p21 which was modified with GP2-PL or GP3-PL in the presence or absence of protective GDP and subsequently reduced by NaBH4 was analyzed by reverse phase high performance liquid chromatography. The profile of the effluent monitored by the fluorescence from the pyridoxyl moiety showed the existence of peptides which were specifically labeled only in the absence of GDP. Structural analyses of these peptides allowed us to identify the labeled residue as Lys-16. These results suggest that Lys-16 is located in the guanine nucleotide binding site, close to the beta- or gamma-phosphate group of the nucleotide.

Affinity Labels↗
PubMed1988

Alpha-glucan phosphorylase from Escherichia coli. Cloning of the gene, and purification and characterization of the protein.

By using a synthetic oligonucleotide probe identical to a part of the gene for the Escherichia coli major outer membrane lipoprotein, we have cloned a gene from E. coli chromosomal DNA. However, the cloned gene was not one of the lipoprotein genes. The amino acid sequence deduced from its nucleotide sequence shows extensive similarities instead to alpha-glucan phosphorylase (EC 2.4.1.1). The gene, glgP, is located immediately downstream from glgA, the gene for glycogen synthase. The glgP gene was inserted into pUC9 vector and expressed in the presence of the lac inducer. The gene product was purified to apparent homogeneity as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In all chromatographies, the protein was eluted accompanied by a low phosphorylase activity. The final preparation showed phosphorolytic activity to various alpha-glucans, although the specific activity was extremely low compared to other alpha-glucan phosphorylases under the standard assay conditions. Its enzymatic activity, however, increased almost linearly as the concentration of glucan increased, reaching a value comparable with those of other phosphorylases. The amino acid sequence deduced was compared with those of alpha-glucan phosphorylases from other sources.

Amino Acid Sequence↗
PubMed1988

The ATP-binding site in gamma subunit of phosphorylase kinase.

To reveal the structure of the ATP-binding site(s) in rabbit muscle phosphorylase kinase, we modified the enzyme with adenosine polyphosphopyridoxals. Adenosine tri- and tetraphosphopyridoxals at micromolar concentrations effectively inactivated the enzyme in a time-dependent manner. Inactivation of the enzyme was accelerated by the addition of Ca2+ and Mg2+. Protection from inactivation was afforded by adenylyl beta,gamma-imidodiphosphate and ADP. In reversible inhibition kinetics, adenosine polyphosphopyridoxals as well as their reduced compounds (adenosine polyphosphopyridoxines) competed with ATP. These results suggest that adenosine polyphosphopyridoxals bind to the ATP-binding site(s) in phosphorylase kinase. When phosphorylase kinase was incubated with adenosine triphosphopyridoxal in the presence of Ca2+ and Mg2+, incorporation of the label into alpha, beta, and gamma subunits was observed. In the absence of both cations, larger amounts of the label were incorporated into all the subunits. Structural study on adenosine triphosphopyridoxal-modified sites in the gamma subunit (having a catalytic site) revealed that Lys-151 is mainly labeled. Based on the results of the present and other studies, it is suggested that the site around Lys-151 is involved in recognition of the substrate protein.

Adenosine Triphosphate↗
PubMed1988

Identification of alpha-subunit Lys201 and beta-subunit Lys155 at the ATP-binding sites in Escherichia coli F1-ATPase.

Binding of about 1 mol of adenosine triphosphopyridoxal to Escherichia coli F1-ATPase resulted in the nearly complete inactivation of the enzyme [(1987) J. Biol. Chem. 262, 7686-7692]. About two thirds of the label was bound to the alpha-subunit, and the rest to the beta-subunit. The present study revealed that Lys201 in the alpha-subunit and Lys155 in the glycine-rich region of the beta-subunit are the major sites labeled with this reagent. Thus, these two residues might be located close to the gamma-phosphate of the bound ATP.

Adenosine Triphosphate↗
PubMed1988

Base-catalyzed reactivation of glycogen phosphorylase reconstituted with a coenzyme-substrate conjugate and its analogues.

Glycogen phosphorylase reconstituted with pyridoxal (5')diphospho(1)-alpha-D-glucose (PLDP-Glc) is catalytically inactive but slowly converted to the active enzyme through the cleavage of the pyrophosphate linkage. A similar reaction occurs more rapidly on PLDP-Gal and -Xyl but not on PLDP-Man. Values of pKa for all the reactions are about 8.3, suggesting the participation of a common basic residue in these reactions. Based on the present and other results, it is presumed that Tyr-573 or Lys-574 acts as the base abstracting the proton from 2-hydroxyl group of the glucosyl moiety of PLDP-Glc.

Animals↗
PubMed1988

Adenosine di-, tri- and tetraphosphopyridoxals modify the same lysyl residue at the ATP-binding site in adenylate kinase.

Adenosine diphosphopyridoxal modifies Lys-21 in adenylate kinase which is located in a glycine-rich loop [(1987) J. Biol. Chem. 262, 8257-8261]. We presently report that adenosine tri- and tetraphosphopyridoxals modify the same lysyl residue more rapidly than the diphospho compound does. However, susceptibilities of the Schiff bases between the labels and the lysyl residue to sodium borohydride considerably differ in the modifications with the three reagents. These observations seem to be ascribable to the mobility of the epsilon-amino group of Lys-21 in the active-site region of the enzyme.

Adenine Nucleotides↗
PubMed1988

Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity.

The extracellular poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis T1, which hydrolyzes both hydrophobic poly(3-hydroxybutyrate) and water-soluble oligomers of D(-)-3-hydroxybutyrate, lost its hydrolyzing activity toward the hydrophobic substrate on mile trypsin treatment, but retained its activity toward water-soluble oligomers. The molecular mass of the trypsin-treated enzyme was 44 kDa, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, which was 6 kDa smaller than that of the native enzyme (50 kDa). The trypsin-treated enzyme seemed to be less hydrophobic than the native one, because it was rather weakly adsorbed to a hydrophobic butyl-Toyopearl column compared with the native enzyme, and showed no ability to bind to poly(3-hydroxybutyrate), to which the native enzyme tightly bound. These results suggest that, in addition to a catalytic site, the enzyme has a hydrophobic site, which is not essential for the hydrolysis of water-soluble oligomers, but is necessary for the hydrolysis of hydrophobic substrates, and this hydrophobic site is removed from the enzyme by the action of trypsin.

Alcaligenes↗
PubMed1988

Affinity labeling of the ATP-binding site of Ca2+-transporting ATPase of sarcoplasmic reticulum by adenosine triphosphopyridoxal: identification of the reactive lysyl residue.

Adenosine triphosphopyridoxal (AP3PL) was used as an affinity label directed toward the ATP binding site of the Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum (SR). The reagent inhibited the ATPase activity competitively with ATP, Ki = 20 microM. Incubation of SR membranes with 100 microM AP3PL followed by treatment with NaBH4 resulted in 90% inactivation of the E-P forming activity as well as of the Ca2+-transporting activity. Adenosine di- and tetraphosphopyridoxals had similar but less pronounced effects on the Ca2+-transport system. AP3PL was bound to ATPase in a one-to-one stoichiometry in parallel with the loss of the enzymatic activities. ATP and ADP prevented the binding of AP3PL and thereby protected the enzyme from inactivation. The SR membranes were labeled with [3H]AP3PL and then digested with thermolysin in order to identify the attachment site of the affinity label. A 3H-labeled peptide (Val-Glu-Pro-Ser-His-Lys* 684-Ser-Lys) was purified to homogeneity by Sephadex LH-20 chromatography and C18-reversed phase HPLC (Lys* denotes the binding site of [3H]AP3PL). These results indicate that the SR-ATPase peptide is folded in such a manner that Lys684 and Asp351, the phosphorylation site, are located very close to each other, since the distance between the 4-formyl group reacting with Lys684 and the gamma-phosphoryl group of the ATP moiety of AP3PL is rather small.

Adenosine Triphosphate↗
PubMed1988

Characterization of an ATPase Associated with the Inner Envelope Membrane of Amyloplasts from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

Amyloplast envelope membranes isolated from cultured, white-wild cells of sycamore (Acer pseudoplatanus L.) have been found to contain a Mg(2+)-ATPase, ranging in specific activity from 5 to 30 nanomoles per minute per milligram protein. This ATPase hydrolyzes a broad range of nucleoside triphosphates, whereas it hydrolyzes nucleoside mono- and diphosphates poorly, if at all. The ATPase activity was stimulated by several divalent cations, including Mg(2+), Mn(2+) and Ca(2+), whereas it was not affected by Sr(2+), K(+), or Na(+). The K(m) for total ATP was 0.6 millimolar, and the activity showed a broad pH optimum between 7.5 and 8.0. The ATPase was insensitive to N,N'-dicyclohexylcarbodiimide and oligomycin, but it was inhibited by vanadate. All these characteristics are basically similar to those reported previously for the Mg(2+)-ATPase of the chloroplast inner-envelope membrane. Likewise, the amyloplast envelope enzyme was shown to be located specifically on the inner envelope membrane. The amyloplast envelope membranes were chemically modified with a series of unique affinity labeling reagents, the adenosine polyphosphopyridoxals (M Tagaya, T Fukui 1986 Biochemistry 25: 2958-2964). About 90% of the ATPase activity was lost when the envelope membranes were preincubated with 0.1 millimolar adenosine triphosphopyridoxal. Notably, the enzyme was protected completely from inactivation in the presence of its substrate, ATP. In contrast, both adenosine diphosphopyridoxal and pyridoxal phosphate caused much less of an inhibitory effect. This greater relative reactivity of the triphosphopyridoxal analog is similar to that reported previously with Escherichia coli F(1) ATPase (T Noumi et al. 1987 J Biol Chem 262: 7686-7692).

Journal Article↗
PubMed1988

Pharmacological studies on anethole trithione.

Pharmacological studies on trithio-p-methoxy-phenylpropene (anethole trithione, ANTT, Felviten) were performed. ANTT at a dose of 100 mg/kg, p.o. lowered the increased serum transaminases GOT and GPT, and protected the liver from injuries caused by CCl4 in mice. In other studies in vivo, ANTT at a dose of 1000 mg/kg showed no effect on the central nervous system or the autonomic nervous system. In vitro experiments with smooth muscle preparations showed no significant effects of ANTT.

Anethole Trithione↗
PubMed1987

Inhibition of acetoacetyl-CoA synthetase from rat liver by fatty acyl-CoAs.

The activity of acetoacetyl-CoA synthetase from rat liver was found to be negatively regulated by coenzyme A, fatty acyl-CoAs and acetoacetyl-CoA in vitro. With increasing concentrations of coenzyme A (substrate inhibition occurring at concentrations higher than 50 microM) the pH optimum shifted toward the acidic side (7.5-8.5 with 5 microM coenzyme A and 6.5-7.0 with 500 microM coenzyme A), in parallel with progressively decreasing enzyme activity. Fatty acyl-CoAs of various chain lengths dose-dependently inhibited acetoacetyl-CoA synthetase from rat liver, but much less effectively a similar enzyme from a bacterium, Zoogloea ramigera I-16-M. Palmitoyl-CoA, the most potent inhibitor of the rat liver enzyme, with an apparent Ki value of 9.8 microM, apparently inhibited the enzyme below its critical micellar concentration, not due to its detergent action. Acetoacetyl-CoA showed product inhibition with a Ki value of 15 microM. These results suggest a possible physiological regulation mechanism for this enzyme with respect to fatty acid biosynthesis.

Acyl Coenzyme A↗
PubMed1987

Reversed-phase ion-pair chromatography of oligodeoxyribonucleotides.

The separation of large oligodeoxyribonucleotides, synthesized chemically and subsequently deblocked, was carried out by reversed-phase ion-pair chromatography (RP-IPC) with a linear gradient of acetonitrile concentration. It was found that tetrabutylammonium phosphate is suitable as an ion-pairing reagent and produces a linear relationship between the base numbers of the samples and their elution volumes. It was also verified that various factors effective in reversed-phase chromatography, such as temperature, end-capping of the columns, differences in the type of C18 alkylating reagents and in the base silica, and the pore size of the base silica have little effect on the resulting separation by RP-IPC.

Chromatography, High Pressure Liquid↗
PubMed1987

Affinity labeling of adenylate kinase with adenosine diphosphopyridoxal. Presence of Lys21 in the ATP-binding site.

Adenosine diphosphopyridoxal, the affinity labeling reagent specific for a lysyl residue in the nucleotide-binding site of several enzymes (Tagaya, M., and Fukui, T. (1986) Biochemistry 25, 2958-2964; Tamura, J. K., Rakov, R. D., and Cross R. L. (1986) J. Biol. Chem. 261, 4126-4133) was applied to adenylate kinase from rabbit muscle. Incubation of the enzyme with a low concentration of the reagent at 25 degrees C for 20 min followed by reduction by sodium borohydride resulted in rapid inactivation of the enzyme. Extrapolation to 100% loss of enzyme activity gave a value of 1.0 mol of the reagent per mol of enzyme. ADP, ATP, and MgATP almost completely protected the enzyme from inactivation, whereas AMP offered little retardation of the inactivation. Dilution of the inactivated enzyme which had not been treated with the reducing reagent led to restoration of enzyme activity. This reactivation was accelerated by ATP but not by AMP. Structural study of the labeled peptide showed that Lys21 is exclusively labeled by adenosine diphosphopyridoxal. These results suggest that the epsilon-amino group of Lys21 is located in the ATP-binding site of the enzyme, more specifically at or close to the subsite for the gamma-phosphate of the nucleotide.

Adenosine Diphosphate↗