Biomedical subjects

T Fukui

Publications and source records attributed to T Fukui.

At least 271 records · Page 15Linked to original sources

PubMed1993

Use of adenosine (5')polyphospho(5')pyridoxals to study the substrate-binding region of glutathione synthetase from Escherichia coli B.

Adenosine(5')polyphospho(5')pyridoxals (APn-PLs, n = 2, 3, 4) were examined for affinity labeling of glutathione synthetase (EC 6.3.2.3) from Escherichia coli B. When the enzyme was incubated with an APn-PL or pyridoxal phosphate in the presence of Mg2+ and then reduced with sodium borohydride, it was most rapidly inactivated by AP4-PL. AP4-PL had a high affinity to the enzyme. The dissociation constant of AP4-PL in the inactivation process was 23 microM. The enzyme was almost completely protected from inactivation by addition of either ATP or gamma-glutamylcysteine. Complete inactivation corresponded to the incorporation of 1 mol of AP4-PL/mol of subunit of the tetrameric enzyme. Proteolytic digestion and sequence analysis of the AP4-PL-labeled enzyme revealed that only Lys-18 was modified. In contrast, the less efficient AP3-PL was found attached to Lys-17, Lys-18, Lys-144, and Lys-148. In the three-dimensional structure of the enzyme, Lys-18 is located close to the putative gamma-glutamylcysteine-binding site, but Lys-17, Lys-144, and Lys-148 are in the mouth of the inner-solvent region, at the bottom of which is the active-site cleft. Furthermore, difference Fourier analysis with the AP4-PL-soaked crystal of the enzyme showed that the adenosine moiety of the bound AP4-PL was in the crevice, which is the ATP-binding site of the enzyme. These results demonstrate the bivalent binding of AP4-PL lying across the gamma-glutamylcysteine- and ATP-binding sites.

Adenine Nucleotides↗

PubMed1993

Nucleotide sequence of the cDNA encoding nucleoside diphosphate kinase II from spinach leaves.

The primary structure of nucleoside diphosphate (NDP) kinase II, one of the two isozymes found in spinach leaves, has been deduced from its cDNA sequence. NDP kinase II comprises 233 amino acid residues and has a molecular mass of 26,107 Da, which is larger than that of the purified NDP kinase II subunits (18 kDa) by about 8 kDa, suggesting that NDP kinase II might be post-translationally processed. Homology was found between the sequence of spinach NDP kinase II, and the sequences of spinach NDP kinase I, rat NDP kinases alpha and beta, Dictyostelium discoideum NDP kinase, the human Nm23-H1 and Nm23-H2 proteins and the awd protein of Drosophila melanogaster.

Amino Acid Sequence↗

PubMed1993

Probing the pyrophosphate-binding site in potato tuber UDP-glucose pyrophosphorylase with pyridoxal diphosphate.

Potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9) catalyzes the reversible uridylyl transfer from UDP-glucose to MgPPi forming glucose 1-phosphate and MgUTP, according to an ordered bi-bi mechanism in which UDP-glucose and MgPPi bind in this order. To probe the active site of this enzyme, we have applied pyridoxal 5'-diphosphate, a reactive PPi analogue. The enzyme was rapidly inactivated when incubated with the reagent in the presence of Mg2+ followed by sodium borohydride reduction. The degree of the inactivation was decreased by MgUTP, MgPPi, and glucose 1-phosphate, but enhanced by UDP-glucose. The enhancement was prevented by co-addition of Pi, the competitive inhibitor with respect to PPi. The complete inactivation corresponded to the incorporation of 0.9-1.1 mol of reagent/mol of enzyme monomer. In the presence of UDP-glucose, labels were almost exclusively incorporated into Lys-329. Thus, this residue may be located near the bound MgPPi and its modification is promoted, probably through conformational changes, by the binding of UDP-glucose to the enzyme. The results of the modification by the same reagent of the mutant enzymes in which Lys-329 and Lys-263 are individually replaced by Gln suggest the roles of these lysyl residues in the binding of MgPPi and in the UDP-glucose-induced conformational changes, respectively.

Affinity Labels↗

PubMed1993

Engineered plant phosphorylase showing extraordinarily high affinity for various alpha-glucan molecules.

alpha-Glucan phosphorylases are characterized by considerable difference in substrate specificities, even though the primary structures are well conserved among the enzymes from microorganisms, plants, and animals. The higher plant phosphorylase isozyme designated as type L exhibits low affinity for a large, highly branched glucan (glycogen), presumably due to steric hindrance caused by a unique 78-residue insertion located beside the mouth of the active-site cleft, whereas another isozyme without the insertion (designated as type H) shows very high affinity for both linear and branched glucans. Using the recombinant type L isozyme from potato tuber as a starting framework and aiming at altering its substrate specificity, we have genetically engineered the 78-residue insertion and its flanking regions. Firstly, removal of the insertion and connection of the newly formed C- and N-terminals yielded a totally inactive enzyme, although the protein was produced in Escherichia coli cells in a soluble form. Secondly, a chimeric phosphorylase, in which the 78-residue insertion and its flanking regions are replaced by the corresponding region of the type H isozyme, has been shown to exhibit high affinity for branched glucans (Mori, H., Tanizawa, K., & Fukui, T., 1993, J. Biol. Chem. 268, 5574-5581), but when two and four unconserved residues in the N-terminal flanking region of the chimeric phosphorylase were mutated back to those of the type L isozyme, the resulting mutants showed significantly lowered affinity for substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Serial changes in atrial and brain natriuretic peptides in patients with acute myocardial infarction treated with early coronary angioplasty.

To examine the role of brain natriuretic peptide (BNP) in acute myocardial infarction (AMI), we measured the plasma concentration of immunoreactive (ir) BNP together with that of atrial natriuretic peptide (ANP) over the 4-week course of AMI in 16 patients treated with early coronary angioplasty. Both the plasma ir-ANP and ir-BNP levels were increased on the first day of the infarction compared with the values in normal subjects. During the clinical course of the infarction, the plasma ir-ANP concentration soon decreased, while the plasma ir-BNP level remained elevated at 2 weeks after the infarction, also exhibiting a high level at 4 weeks. Plasma ir-BNP levels on day 1 or days 14 and 28 were inversely correlated with left ventricular ejection fraction obtained by left ventriculography at the acute or chronic phase, respectively. Plasma ir-BNP concentrations on days 14 and 28 were positively correlated with the maximal myosin light chain I level, an indicator of infarct size. These observations suggest that the plasma ir-BNP level increased to compensate for the ventricular dysfunction associated with the size of the infarct in AMI. BNP may act as a cardiac hormone in AMI, differing somewhat from ANP in its synthetic, secretory, or clearance behavior.

Angioplasty, Balloon, Coronary↗

PubMed1993

Stimulation of endothelin-1 release by low density and very low density lipoproteins in cultured human endothelial cells.

To examine the effects of lipoproteins on the secretion of endothelin-1 from endothelial cells, we measured immunoreactive (ir) endothelin-1 release from cultured human umbilical vein endothelial cells in the presence or absence of various concentrations of native low density lipoprotein (LDL), oxidized LDL, and very low density lipoprotein (VLDL). Cultured endothelial cells secreted ir-endothelin-1 into serum-free medium in a time-dependent manner, and the secretion was clearly stimulated following a 15-24-h incubation with 10 micrograms/ml oxidized LDL. The secretion of ir-endothelin-1 increased in a dose-dependent manner after a 24-h incubation with oxidized LDL, while only a high dose of native LDL and VLDL significantly increased ir-endothelin-1 secretion. The release of ir-endothelin-1 stimulated by 20 micrograms/ml oxidized LDL was reproduced by the same concentration of acetylated LDL but not native LDL. These observations indicate that the release of ir-endothelin-1 from endothelial cells is stimulated by lipoproteins, in particular by oxidized LDL, probably through the endothelial scavenger receptor. This increase in ir-endothelin-1 release induced by oxidized LDL may contribute to the development of atherosclerotic vascular lesions.

Cells, Cultured↗

PubMed1993

The effects of biotin deficiency on organic acid metabolism: increase in propionyl coenzyme A-related organic acids in biotin-deficient rats.

Volatile organic acid levels in plasma and tissues and nonvolatile organic acid levels in urine of biotin-deficient (BD) rats were measured and compared with other factors of biotin deficiency. Biotin levels and the activities of propionyl coenzyme A (CoA) carboxylase (PCC) in the livers of these rats were decreased, respectively, to 22% +/- 3% and 3.6% +/- 0.3% of the average values of pair-fed controls. Plasma concentrations of propionate were higher (15 to 223 micrograms/mL) than those of controls (5 to 7 micrograms/mL), whereas plasma levels of 3-methylcrotonate were only minimally increased as compared with those of controls. Concentrations of these volatile acids in the tissues were similarly increased, although those in brain showed less remarkable increases as compared with levels in other tissues. In the urine of BD rats, large amounts of organic acids derived from propionyl CoA, as well as those from 3-methylcrotonyl CoA, were excreted. Plasma propionate levels were not apparently related to the severity of clinical symptoms, biotin levels, or carboxylase activities, but were related to the amounts of urinary ketone bodies, lactate, and some of the organic acids derived from branched-chain amino acids, including those from propionyl CoA.

Acyl Coenzyme A↗

PubMed1993

Analysis of stress-strain curves in the rat molar periodontal ligament after application of orthodontic force.

Previous studies have shown that the mechanical strength of the periodontal ligament decreased markedly after application of an orthodontic force to the rat molars. However, an analysis of stress-strain curves obtained from transverse sections of the rat molars has not been made. The present study analyzed the stress-strain curves obtained from the mesial root of the rat mandibular first molar to evaluate the changes of the mechanical properties of the ligament after application of an orthodontic force. An elastic band was inserted between the mandibular first and second molars for 7 days. The maximum shear stress, elastic stiffness, and failure strain energy density were significantly less in the experimental group than in the control group, but the maximum strain was significantly greater. Histologic examinations of the transverse section after mechanical testing showed that the area of the ligament adhering to the mesial surface (compression side) of the socket bone was significantly less in the experimental group. It is suggested that orthodontic forces may cause changes in the constitution of the periodontal collagen, in osteoclastic activity in alveolar bone, and in mineralization patterns of Sharpey's fibers followed by reductions of the mechanical strength of the ligament, particularly on the compression side of alveolar bone.

Alveolar Process↗

PubMed1993

Molecular cloning, nucleotide sequencing, and affinity labeling of bovine liver UDP-glucose pyrophosphorylase.

A bovine liver cDNA encoding UDP-glucose pyrophosphorylase [EC 2.7.7.9], which catalyzes the reversible uridylyl transfer between glucose 1-phosphate and MgUTP, has been cloned by the use of oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,689 base-pair insert including the complete message for the subunit polypeptide (508 amino acid residues) of the octameric enzyme. The bovine liver enzyme shows significant sequence similarities with the enzymes from potato tuber and a slime mold, Dictyostelium discoideum, but not with the enzyme from Escherichia coli, or ADP-glucose pyrophosphorylases from rice seed and E. coli. To probe the substrate-binding site in the bovine liver enzyme, the purified enzyme was incubated with an affinity labeling reagent, uridine triphosphopyridoxal, and then reduced with sodium borohydride. The enzyme was inactivated rapidly and irreversibly by the reagent at low concentrations. The inactivation was almost completely retarded by UDP-glucose and MgUTP. Structural analysis of the labeled enzyme revealed that three lysyl residues, Lys291, Lys357, and Lys396, were modified by the reagent. The three lysyl residues are conserved at the corresponding positions in the sequence of the potato tuber enzyme, in which they have catalytically important functions. These results show that the active-site structure of bovine liver UDP-glucose pyrophosphorylase is very similar to that of the potato tuber enzyme.

Affinity Labels↗

PubMed1993

Site-directed random mutagenesis of AMP-binding residues in adenylate kinase.

Two highly conservative residues, Val67 and Gln101, in adenylate kinase are located in the hydrophobic region putatively involved in binding of the adenine ring of AMP. We have performed polymerase chain reaction-based random mutagenesis of the two residues using recombinant chicken muscle adenylate kinase cDNA as a template. The synthetic oligonucleotide primers contained A,G,C,T-mixed bases in the codons corresponding to those for Val67 and Gln101. The amplified fragments were ligated with the expression plasmid pKK223-3, and the mutant proteins expressed were identified by immunoblotting. Enzymatically active mutant proteins were selected on the basis of the growth at 45 degrees C of the temperature sensitive Escherichia coli mutant for adenylate kinase. At position 67, various amino acid residues other than Val have been found to restore the growth at 45 degrees C of the ts mutant. In contrast, only Gln, His, and Met could be present at position 101. These results are compatible with the proposal that Val67 contributes to the AMP binding through hydrophobic interactions and Gln101 by forming a hydrogen bond with the adenine ring. Indeed, several purified Val67 and Gln101 mutant enzymes exhibited markedly high Km values for AMP, whereas the Km values for MgATP were comparable to those of the wild-type enzyme. Substrate specificity for nucleoside monophosphates was changed significantly by the mutagenesis of the two residues.

Adenosine Monophosphate↗

PubMed1993

Natriuretic peptides inhibit mesangial cell production of endothelin induced by arginine vasopressin.

The present study examined the effects of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively) on endothelin-1 (ET-1) secretion after stimulation with arginine vasopressin (AVP), using cultured rat glomerular mesangial cells. AVP stimulated immunoreactive (ir) ET-1 secretion in a concentration-dependent manner via a receptor-mediated process. Rat ANP-(1-28) and rat BNP-45 potently inhibited this stimulated secretion in a concentration-dependent manner. Inhibition by ANP and BNP of AVP-stimulated ET-1 secretion was paralleled by an increase in the medium level of guanosine 3',5'-cyclic monophosphate (cGMP). The addition of a cGMP analogue, 8-bromo-cGMP, reduced the stimulated ET-1 secretion. CNP was much less effective than rat ANP-(1-28) or rat BNP-45 with respect to inhibiting irET-1 secretion and increasing cGMP levels. High-performance liquid chromatography indicated that the major component of irET-1 in the culture medium corresponds to ET-1-(1-21). These findings indicate that AVP stimulates ET-1 secretion in cultured rat mesangial cells and that rat ANP and BNP inhibit this stimulated secretion, probably through a cGMP-dependent process.

Animals↗

PubMed1993

Utilization behavior and concomitant motor neglect by bilateral frontal lobe damage.

We reported on a patient who had bilateral mesial frontal lesions, presumably of a primary cerebral malignant lymphoma. The patient presented with a combination of unusual behavioral disorders of the upper extremities, which has not yet been documented in the literature: bilateral utilization and imitation behaviors and motor neglect of the left arm. Utilization behavior was closely associated with bilateral manual grasping behavior and was caused by the bifrontal lesions. Damage to the right supplementary motor area resulted most likely in concomitant motor neglect of the left hand. Discussion includes differentiation from other related behavioral disorders.

Aged↗

PubMed1993

Biotinidase activity in patients with liver disease.

To investigate whether biotinidase deficiency may occur in liver disease, we determined biotinidase activity, biotin levels, and organic acids in patients with liver disease. Serum biotinidase activity in patients with liver disease (2.63 +/- 1.40 nmol/min/ml) was significantly lower than in the control group (5.43 +/- 1.06 nmol/min/ml). Serum biotinidase activity in decompensated liver cirrhosis (LC) and hepatoma was significantly lower than in acute viral hepatitis (AVH), chronic viral hepatitis (CVH), and compensated LC. The mean serum level of biotin in decompensated LC (1.8 +/- 0.6 microgram/ml) and hepatoma (1.7 +/- 0.8 microgram/ml) was significantly lower than in the control group (2.5 +/- 1.0 microgram/ml), and urinary excretion of biotin was increased in patients with liver disease, particularly in decompensated LC. Biotinidase activity correlated positively with serum biotin level and correlated negatively with urinary biotin level. Moreover, in four of five patients with severe liver disease the excretion of propionate, lactate, and 3-hydroxybutyrate decreased after biotin supplementation. The data for patients with severe liver disease so resembled those for late-onset multiple carboxylase deficiency that biotinidase deficiency is likely in patients with severe liver disease.

3-Hydroxybutyric Acid↗

PubMed1993

Deep breathing test (DBT) in predicting white coat hypertension.

We have evaluated the diagnostic value of deep breathing test (DBT) in detecting the patients with white coat hypertension on the outpatient basis. Thirty patients with untreated mild to moderate hypertension underwent 24-hour ambulatory blood pressure monitoring. Those who had a mean 24-hour blood pressure less than 135/80 mmHg were categorized as having white coat hypertension and the remainder were categorized as sustained hypertension. These two groups were compared with regard to the differences of office blood pressures before and after DBT (i.e., deep breathing 5 times for a minute in sitting position). Although the office systolic pressure significantly fell after DBT in both groups (p < 0.001 vs p < 0.05), there was no significant difference (p = 0.27) between the two groups. On the other hand, the office diastolic blood pressure significantly fell in the white coat hypertensives in contrast with no meaningful fall of it in the sustained hypertensives (p < 0.01 vs p = 0.66). At the cutoff level of -3%, -5% and -10% of the differences in office diastolic blood pressure before and after DBT, the sensitivity for the presence of white coat hypertension were 64.7%, 58.8% and 29.4%, respectively, and the specificity were 61.5%, 84.6% and 100%, respectively. These findings demonstrate that the deep breathing test is useful for identifying white coat hypertension in the outpatient clinic.

Female↗

PubMed1993

Exploring the active site in UDP-glucose pyrophosphorylase by affinity labelling and site-directed mutagenesis.

UDP-glucose pyrophosphorylase catalyses the reversible uridylyl transfer from UDP-glucose to MgPPi, forming glucose 1-phosphate and MgUTP. We have identified, by using uridine di- and tri-phosphopyridoxals, five lysyl residues located at or near the UDP-glucose-binding site. Another reactive analogue, pyridoxal diphosphoglucose, also modified the same set of lysyl residues. Based on the amounts of labels incorporated into each lysyl residue, we have provided a hypothetical model for possible locations of the five lysyl residues around the substrates bound to the enzyme. This model was consistent with the results of the mutagenesis studies. Lys367 is essential for the catalysis, whereas Lys263 may participate in the binding of PPi and/or glucose 1-phosphate in the binary complex. On the other hand, the results of affinity labelling with pyridoxal 5'-diphosphate suggest the role of Lys329 in the direct interaction with PPi in the enzyme-UDP-glucose complex.

Affinity Labels↗

PubMed1993

Hemiballism-hemichorea induced by subcortical ischemia.

Four patients presented with hemiballism-hemichorea as a clinical manifestation of white matter ischemia. These patients illustrate "positive" motor phenomena rather than limb weakness as a consequence of cerebral ischemia. In each patient, the involuntary movements disappeared following worsening of paresis. Subcortical white matter infarction in three patients and hemodynamic hypo-perfusion in the cerebral hemisphere contralateral to dyskinetic movements were possible causes. Neuroradiologically, none had pathological changes in the vicinity of the subthalamic nucleus. We presume from these observations that ischemia of the subcortical white matter, without involvement of the basal ganglia or the subthalamic nucleus, may cause hemiballism-hemichorea.

Aged↗

PubMed1992

Glycyrrhetinic acid bound to 11 beta-hydroxysteroid dehydrogenase in rat liver microsomes.

A binding protein which exhibits high affinity to [3H]glycyrrhetinic-acid in the rat liver microsomal fraction was solubilized with 0.2% Triton DF-18 and then purified to homogeneity. The equilibrium dissociation constant of the [3H]glycyrrhetinic-acid binding reaction and the maximal concentration for the binding of the purified protein, as determined by Scatchard plot analysis, were 27.6 nM and 7.79 nmol/mg protein, respectively. The molecular mass of the subunit (34 kDa) and 30 amino acids of N-terminal sequence of the purified protein were entirely the same as those of the reported 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). In each purification step, the recovery and purification (fold) of the glycyrrhetinic-acid binding activity corresponded to the values of 11 beta-HSD activity. These results show that the purified [3H]glycyrrhetinic-acid binding protein is 11 beta-HSD. From the molecular mass of 11 beta-HSD (135 kDa) and the maximal concentration of the binding site, it was calculated that one glycyrrhetinic acid molecule binds to one 11 beta-HSD molecule. The inhibitory effects of various glycyrrhetinic-acid derivatives on [3H]glycyrrhetinic acid binding and 11 beta-HSD activity indicate that the C30-carboxyl and C11-carbonyl groups of glycyrrhetinic acid are the principal structures for the 11 beta-HSD inhibition.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1992

The amino acid sequence of nucleoside diphosphate kinase I from spinach leaves, as deduced from the cDNA sequence.

The primary structure of nucleoside diphosphate (NDP) kinase from spinach leaves has been deduced from its cDNA sequence. A lambda gt 11 cDNA library derived from spinach leaves was screened using an antibody against NDP kinase I, which we previously purified to electrophoretic homogeneity (T. Nomura, T. Fukui, and A. Ichikawa, 1991, Biochim. Biophys. Acta 1077, 47-55). The cDNA sequences of positive clones contained the amino acid coding region (444 base pairs) for NDP kinase I as well as 5' and 3' noncoding regions of 33 and 361 base pairs, respectively. The cDNAs hybridized to a 1.1-kb mRNA. NDP kinase I contains 148 amino acid residues with a molecular mass of 16,305, which is in excellent agreement with that of the purified enzyme (16 kDa). Homology was found between the sequence of spinach NDP kinase I and those of the rat, Myxococcus xanthus, and Dictyostelium discoideum NDP kinases, as well as the human Nm23-gene product and the awd protein of Drosophila melanogaster.

Amino Acid Sequence↗