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T Fukui

Publications and source records attributed to T Fukui.

At least 55 records · Page 3Linked to original sources
PubMed1990

Affinity labeling of aminoacyl-tRNA synthetases with adenosine triphosphopyridoxal: probing the Lys-Met-Ser-Lys-Ser signature sequence as the ATP-binding site in Escherichia coli methionyl-and valyl-tRNA synthetases.

Pyridoxal 5'-triphospho-5'-adenosine (AP3-PL), the affinity labeling reagent specific for lysine residues in the nucleotide-binding site of several enzymes [Tagaya, M., & Fukui, T. (1986) Biochemistry 25, 2958-2964; Yagami, T., Tagaya, M., & Fukui, T. (1988) FEBS Lett. 229, 261-264], was used to identify the ATP-binding site of Escherichia coli methionyl-tRNA synthetase (MetRS). Incubation of this enzyme with AP3-PL followed by reduction with sodium borohydride resulted in a rapid inactivation of both the tRNA(Met) aminoacylation and the methionine-dependent ATP-PPi exchange activities. Complete inactivation corresponded to the incorporation of 0.98 mol of AP3-PL/mol of monomeric trypsin-modified MetRS. ATP or MgATP protected the enzyme from inactivation. The labeling with AP3-PL was also applied to E. coli valyl-tRNA synthetase (ValRS). Both the tRNA(Val) aminoacylation and the valine-dependent ATP-PPi exchange activities were abolished by the incorporation of 0.91 mol of AP3-PL/mol of monomeric ValRS. AP3-PL was found attached to lysine residues 335, 402, and 528 in the primary structure of MetRS. In the case of ValRS, the AP3-PL-labeled residues corresponded to lysines 557, 593, and 909. We therefore conclude that these lysines of MetRS and ValRS are directed toward the ATP-binding site of these synthetases, more specifically at or close to the subsite for the gamma-phosphate of ATP. AP3-PL-labeled Lys-335 of MetRS and Lys-557 of ValRS belong to the consensus tRNA CCA-binding Lys-Met-Ser-Lys-Ser sequence [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

The role of Leu-190 in the function and stability of adenylate kinase.

To elucidate the role of C-terminal region of chicken adenylate kinase (a single polypeptide consisting of 193 amino acid residues) in the catalysis and stability of the enzyme, a series of mutant proteins truncated in the C-terminal region has been prepared by successive replacements of the sense codons by a termination codon via site-directed mutagenesis. Removal of the three C-terminal residues did not affect the apparent Michaelis constants (Km values) for AMP and ATP, although the Vmax values decreased gradually in parallel with the length of the polypeptide chain. A sudden increase in Km values for substrates, in particular for ATP, was observed on removal of one additional residue (Leu-190), the Vmax value also being less than one-half of that of the mutant enzyme with 3 residues shorter than the wild-type enzyme. These results suggest the importance of the highly conservative Leu-190. Therefore, we further prepared the mutant enzymes through replacement of Leu-190 by a variety of other amino acid residues. They all had substantially lower Vmax values and decreased thermostabilities. Their apparent Km values for ATP also changed, whereas those for AMP were affected to a lesser extent. The hydrophobicity of amino acid residues at position 190 was found to positively correlate with the specificity constants (kcat/Km values) for ATP and also with the thermostability of the enzyme. The fluorescence emission of the Trp-190 mutant enzyme was quenched by the addition of ATP. It is suggested that the C-terminal residues, particularly those around Leu-190, are present in a hydrophobic region which may be involved in binding of ATP.

Adenosine Monophosphate↗
PubMed1990

cDNA-derived amino acid sequence of L-histidine decarboxylase from mouse mastocytoma P-815 cells.

The primary structure of L-histidine decarboxylase (HDC: L-histidine carboxy-lyase, EC 4.1.1.22) from mouse mastocytoma P-815 cells has been determined by parallel analysis of the amino acid sequence of the protein and the nucleotide sequence of the corresponding cDNA. HDC contains 662 amino acid residues with a molecular mass of 74017, which is larger by about 21,000 Da than that of the previously purified HDC subunit (53 kDa), suggesting that HDC might be posttranslationally processed. The HDC cDNA hybridized to a 2.7 kilobase mRNA of mastocytoma cells. Homology was found between the sequences of mouse mastocytoma HDC and fetal rat liver HDC.

Amino Acid Sequence↗
PubMed1990

Identification of lysine 15 at the active site in Escherichia coli glycogen synthase. Conservation of Lys-X-Gly-Gly sequence in the bacterial and mammalian enzymes.

Glycogen synthases from Escherichia coli and mammalian muscle differ in many respects including regulation, sugar nucleotide specificity, and primary sequence. To compare the structure of the active sites in these enzymes, the affinity-labeling study of the E. coli enzyme was carried out using adenosine diphosphopyridoxal as the reagent. The E. coli enzyme was inactivated in a time- and dose-dependent manner when incubated with the reagent followed by sodium borohydride reduction. The inactivation was markedly protected by ADP-glucose and ADP, suggesting that the reagent was bound to the substrate-binding site. The stoichiometry of the bound reagent to the enzyme was approximately 1:1. Sequence analysis of the labeled peptide isolated from a proteolytic digest of the modified protein revealed that Lys15 is labeled. Based on the geometry of the reagent, the epsilon-amino group of this residue might be located close to the pyrophosphate moiety of ADP-glucose bound to the E. coli enzyme, like that of Lys38 in the rabbit muscle enzyme, which is labeled by uridine diphosphopyridoxal (Tagaya, M., Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 260, 6670-6676; Mahrenholz, A. M., Wang, Y., and Roach, P. J. (1988) J. Biol. Chem. 263, 10561-10567). The importance of the conserved sequence of Lys-X-Gly-Gly is discussed in connection with the glycine-rich region found in many nucleotide-binding proteins.

Amino Acid Sequence↗
PubMed1990

Plasma immunoreactive endothelin in essential hypertension.

PURPOSE: Endothelin plays a role in the regulation of vascular tonus. Therefore, it has been hypothesized that increased production or release of endothelin or both may contribute to the pathogenesis of hypertension. To assess any changes in the plasma endothelin concentration in essential hypertension, plasma immunoreactive endothelin concentrations were measured in patients with essential hypertension. PATIENTS AND METHODS: We measured plasma immunoreactive endothelin concentrations in 42 subjects with essential hypertension, 12 subjects with borderline hypertension, and 25 normotensive control subjects. RESULTS: The concentrations were higher in hypertensive patients than in borderline hypertensive patients and normotensive subjects (both p less than 0.05), although values in normotensives and hypertensives overlapped. Reverse-phase high-performance liquid chromatography (HPLC) and radioimmuno-assay showed two components of plasma endothelin, one corresponding to synthetic endothelin-1 (1-21) and the other corresponding to synthetic big endothelin (human, 1-38). The HPLC profile of plasma endothelin of hypertensive patients was the same as that of normotensive subjects. Hypertensives with reduced glomerular filtration rates or increased serum creatinine levels had higher plasma endothelin concentrations than hypertensive patients as a whole (p less than 0.05). Mean blood pressure and serum creatinine levels were correlated to plasma endothelin in the hypertensives. Correlation was negative between glomerular filtration rate and the endothelin level in the hypertensives. CONCLUSION: Plasma endothelin was elevated in many hypertensive patients with severe hypertension or renal involvement. Its major components were endothelin-1 and big endothelin.

Blood Pressure↗
PubMed1990

Endothelin stimulates release of atrial natriuretic factor in anesthetized rats.

The present study was designed to examine the effect of synthetic rat endothelin, a novel potent vasoconstrictor isolated from endothelial cells, on the release of immunoreactive atrial natriuretic factor (ir-ANF) in anesthetized rats. Systemic blood pressure and plasma ir-ANF concentration were measured at 1, 3, 5, 15, 30, and 60 minutes after rat endothelin administration (0.01, 0.10, 1.00 nmol/kg body weight [BW]). Administration of rat endothelin caused a long-lasting increase in mean blood pressure and produced a dose-dependent increase in plasma ir-ANF that peaked 5 minutes after the injection and remained higher than the basal value 60 minutes after the injection. These data suggest that administration of pharmacological doses of rat endothelin produces a profound release of ir-ANF into the circulation, which may cause vasodilation and diuresis to antagonize the effect of endothelin.

Animals↗
PubMed1990

Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells.

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.

Animals↗
PubMed1990

UDP-glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing.

We have isolated a cDNA encoding UDP-glucose pyrophosphorylase from a cDNA library of immature potato tuber using oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,758-base-pair insert including the complete message for UDP-glucose pyrophosphorylase with 1,431 base pairs. The amino acid sequence of the enzyme inferred from the nucleotide sequence consists of 477 amino acid residues. All the partial amino acid sequences determined protein-chemically [Nakano et al. (1989) J. Biochem. 106, 528-532] confirmed the primary structure of the enzyme. An N-terminal-blocked peptide was isolated from the proteolytic digest of the enzyme protein, and the blocking group was deduced to be an acetyl group by fast atom bombardment-mass spectrometry. On the basis of the predicted amino acid sequence (477 residues minus the N-terminal Met plus an acetyl group), the molecular weight of the enzyme monomer is calculated to be 51,783, which agrees well with the value determined by polyacrylamide gel electrophoresis. In the cDNA structure, the open-reading frame is preceded by a 125-base-pair noncoding region, which contains a sequence being homologous with the consensus sequence for plant genes, and is followed by a 174-base-pair noncoding sequence including a polyadenylation signal. Amino acid sequence comparisons revealed that the potato UDP-glucose pyrophosphorylase is homologous to the enzyme from slime mold, Dictyostelium discoideum, but not to ADP-glucose pyrophosphorylases from rice seed and Escherichia coli.

Amino Acid Sequence↗
PubMed1990

Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha.

To determine the region of human tumor necrosis factor-alpha (TNF-alpha), essential for cytotoxic activity against mouse L-M cells, single amino-acid-substituted TNF-alpha mutant proteins (muteins) were produced in Escherichia coli by protein engineering techniques. An expression plasmid for TNF-alpha was mutagenized by passage through an E. coli mutD5 mutator strain and by oligonucleotide-directed mutagenesis. Approximately 100 single amino-acid-substituted TNF-alpha muteins were produced and assayed for cytotoxic activity. The cytotoxic activities of purified TNF-alpha muteins, e.g. TNF-31T, -32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were less than 1% of that of parent TNF-alpha. These results indicate that the integrity of at least four distinct regions of the TNF-alpha molecule is required for full biological activity. These regions are designated as follows: region I, from position 30 to 32; region II, from position 82 to 89; region III, from position 115 to 117; region IV, from position 141 to 146. In addition, TNF-141Y could not completely compete with parent TNF-alpha for binding to the receptor. This demonstrates that region IV, and at least aspartic acid at position 141, must be involved in the TNF receptor binding site.

Amino Acid Sequence↗
PubMed1990

Release of immunoreactive endothelin from porcine aortic strips.

The present study was designed to determine whether endothelin and "big endothelin" are released from aortic strips with endothelium and to examine the effect of thrombin by using a specific radioimmunoassay. Porcine aortic strips with endothelium released immunoreactive endothelin (ir-endothelin) and immunoreactive big endothelin (ir-big endothelin) into the medium in a time-dependent manner. These releases were markedly inhibited by 10 micrograms/ml cycloheximide. Expectedly, after removal of endothelium, aortic strips did not release a detectable amount of ir-endothelin and ir-big endothelin. In contrast, thrombin (10 units/ml) significantly stimulated the release of ir-endothelin and ir-big endothelin. Reverse-phase high-performance liquid chromatography coupled with radioimmunoassay revealed that the major component of ir-endothelin corresponds to standard endothelin-1 (1-21) and the major component of ir-big endothelin corresponds to standard big endothelin (porcine, 1-39). These results suggest that aortic strips with endothelium release endothelin and big endothelin slowly but continuously into the extracellular space and that these releases can be stimulated by thrombin.

Animals↗
PubMed1990

[Relative contribution of history-taking, physical examination, and stat laboratory test to diagnosis in chest pain patients].

To evaluate the relative importance of the medical history, physical examination, and stat laboratory tests in diagnosis, twelve general internists recorded their hypotheses (diseases) with subjective probabilities assigned to them after taking the history, and after performing the physical examination, and again after obtaining stat laboratory test results. The resultant hypotheses generated for chest pain patients were compared with the final diagnoses which were determined an average of 7.6 months later. A hypothesis subjectively determined to have the highest probability was shown to agree with the final diagnosis in 71.1% of the patients after taking the history: the physical examination was useful in raising this proportion only by 5.0%. Stat laboratory tests, however, raised the proportion from 63.6% to 81.4% for patients where stat laboratory tests were done. When subjective probabilities were used as a measuring index, earlier hypotheses were correct with an average predictive value of 0.57 after the history-taking, 0.62 after the physical examination, and 0.73 after stat laboratory tests. The average subjective probability after physical examination for a group of patients who subsequently underwent stat laboratory tests was 0.59, while that for a group of patients who did not undergo stat laboratory tests was 0.78. Based on these results test-treatment threshold was estimated to be approximately 0.69. The number of hypotheses were, on the average, 2.48 after the history-taking, 2.35 after the physical examination, and 1.90 after stat laboratory tests. From the stand point of social responsibility and individual patient perspectives quantitative measurement of physicians' predictions should be provided as the basis for assessment of the benefit of high medical technologies.

Chest Pain↗
PubMed1990

[Fifteen-year experience with 24 pregnancies associated with prosthetic valve replacements].

During the past 15 years, we followed 21 patients with prosthetic heart valves who experienced a total of 24 pregnancies at mean age of 31.3 +/- 3.6 years. The course of these patients and their pregnancies were reviewed to evaluate the problems associated with prosthetic heart valves and anticoagulation. Among the 21 patients, the aortic valve (AV) had been replaced in 10 (12 pregnancies), the mitral valve (MV) in nine (10 pregnancies), AV + MV in one (one pregnancy), and the tricuspid valve (TV) in one (one pregnancy). The implanted prosthetic valves were mechanical type in 16 cases (Björk-Shiley 15, Starr-Edwards 1) and bioprosthetic type in six (Hancock 5, Ionescu-Shiley 1). With the exception of one case of intra-uterine fetal death probably related with warfarin therapy, all the patients with bioprosthetic valves underwent successful deliveries. Anticoagulant therapy was employed for 11 pregnancies; warfarin for 10 and subcutaneous heparin for one. No anticoagulant therapy was performed for 13 pregnancies. Ten of the 21 mothers had atrial fibrillation. Eighteen pregnancies (67%) culminated in uneventful deliveries for both mothers and infants. Three mothers (13%) died of thromboembolic complications; two of cerebrovascular accidents and one of acute heart failure caused by thrombus on the replaced valve. All of them had Björk-Shiley valves. Oral warfarin was administered in one of the three, heparin in one and no anticoagulant in the remaining one. Massive maternal bleeding occurred in two cases (8%). There were three cases (12%) of intra-uterine fetal death which were caused by intracranial hemorrhages.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities, Drug-Induced↗
PubMed1990

[Medical decision making: its history and technical terms].

Medical decision making, an application of decision sciences to medicine, has been increasingly recognized as an important clinical armamentarium in the past several decades. Clinical medicine abounds in uncertainties arising from the very nature of clinical data and physicians' judgments. These uncertainties provide room for decision sciences to be widely used in clinical medicine. There are two major research areas in medical decision making, i.e., prescriptive and descriptive approaches. The prescriptive approach, an endeavor to explore how decisions should be made, is quantitative and probabilistic, and employs decision analytical procedures which are designed to maximize expected value or utility. An example of a decision tree is shown as well as such essential technical terms as decision node, chance node, averaging out, folding back, sensitivity, specificity, predictive values, Bayes' theorem, and expected value of clinical information. The descriptive approach, on the other hand, is related to how people behave. In this context, strict meaning of judgment and decision making, some heuristics and psychological pitfalls, and the necessity of elucidating patients' preferences, though difficult, are recapitulated.

Clinical Laboratory Techniques↗
PubMed1990

[Hemodynamic studies on the vertebral artery system during the vertebral arterial surgery].

Few hemodynamic studies on the vertebral artery system in the human can be seen. The authors measured the vertebral arterial blood flow (VAF) with an electromagnetic flow meter in 45 patients who obtained vertebral arterial surgeries. The patients showing vertebrobasilar insufficiency such as vertigo and drop attack had serious kinking and stenosis at the first portion of the vertebral artery. The effects of induced hypotension by trimethaphan camsilate, induced hypertension by phenylephrine, cervical epidural anesthesia and induced hypertension under epidural anesthesia on the VAF were investigated. During the control state, mean systemic arterial blood pressure (SABP), mean VAF were 97 mmHg and 54 ml/min, respectively. The effects of varied SPBP were analyzed by (delta mean VAF/mean VAF)/(delta mean SABP/mean SABP), (delta V/delta S). The delta mean VAF and delta mean SABP indicated varied mean values of VAF and SABP, respectively. Mean SABP was varied significantly by about 25% in each method. The delta V/delta S in induced hypotension, induced hypertension, epidural anesthesia and induced hypertension under epidural anesthesia were -0.05, 0.07, 0.90 and 0.61, respectively, on the average. Induced hypotension by epidural anesthesia and induced hypertension under epidural anesthesia presented significant changes in mean VAF.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Pressure↗
PubMed1989

Polyphosphoinositides produced by phosphatidylinositol 3-kinase are poor substrates for phospholipases C from rat liver and bovine brain.

The ability of three pure types of bovine brain phospholipase C (PLC) and one pure rat liver PLC to utilize as substrates the recently discovered phosphatidylinositol 3-phosphate (PI-3-P), a putative phosphatidylinositol 3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3) was investigated. PI-3-P, PI-3,4-P2, and PIP3 are the products of phosphorylation of PI, PI-4-P, and PI-4,5-P2, respectively, by phosphoinositide 3-kinase activities that are associated with certain protein-tyrosine kinases. Although these new phospholipids have been found in intact cells, PI-3,4-P2 and PIP3 appear only after stimulation of quiescent cells with growth factors such as platelet-derived growth factor (Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175) and after transformation by certain oncoproteins (L. A. Serunian, K. R. Auger, T. M. Roberts, and L. C. Cantley, manuscript in preparation). Mixtures of [3H]PI-4-P plus [32P]PI-3-P or [3H]PI-4,5-P2 plus [32P]PI-3,4-P2 or PIP3 alone were used as substrates for PLCs in vitro. After incubation with enzyme followed by extraction with chloroform/methanol/HCl, the ratio of 3H/32P in the aqueous layer revealed the selective hydrolysis of PI-4-P and PI-4,5-P2 over PI-3-P and PI-3,4-P2. High performance liquid chromatography analysis of the aqueous layer containing reaction products confirmed that only PI-4-P and PI-4,5-P2, were hydrolyzed to inositol 1,4-P2 and inositol 1,4,5-P3, respectively. These findings suggest that the turnover of PI-3-P, PI-3,4-P2, and PIP3 occurs independently of the turnover of PI-4-P and PI-4,5-P2.

Animals↗
PubMed1989

Kinetic studies of the pyridoxal kinase from pig liver: slow-binding inhibition by adenosine tetraphosphopyridoxal.

Pyridoxal kinase from pig liver has been purified 10,000-fold to apparent homogeneity. The enzyme is a dimer of subunits of Mr 32,000. The enzyme is strongly inhibited by the product pyridoxal 5'-phosphate. Liver pyridoxamine phosphate oxidase, another enzyme involved in the biosynthesis of pyridoxal 5'-phosphate, is also strongly inhibited by this compound [Wada, H., & Snell, E. E. (1961) J. Biol. Chem. 236, 2089-2095]. Thus, the biosynthesis of pyridoxal 5'-phosphate in the liver might be regulated by the product inhibition of both pyridoxamine phosphate oxidase and pyridoxal kinase. Kinetic studies revealed that the catalytic reaction of liver pyridoxal kinase follows an ordered mechanism in which pyridoxal and ATP bind to the enzyme and ADP and pyridoxal 5'-phosphate are released from the enzyme, in this order. Adenosine tetraphosphopyridoxal was found to be a slow-binding inhibitor of pyridoxal kinase. Pre-steady-state kinetics of the inhibition revealed that the inhibitor and the enzyme form an initial weak complex prior to the formation of a tighter and slowly reversing complex. The overall inhibition constant was 2.4 microM. ATP markedly protects the enzyme against time-dependent inhibition by the inhibitor, whereas another substrate pyridoxal affords no protection. By contrast, adenosine triphosphopyridoxal is not a slow-binding inhibitor of this enzyme.

Adenine Nucleotides↗