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Biomedical subjects

H Kihara

Publications and source records attributed to H Kihara.

At least 109 records · Page 6Linked to original sources
PubMed1989

Kinetic study on the dimer-tetramer interconversion of phosphorylase b by a stopped-flow X-ray scattering method.

The dimer-tetramer interconversion of phosphorylase b induced by the binding of AMP and Mg2+ was monitored using a stopped-flow X-ray scattering method. The rate constants of this second-order reaction have been determined by a nonlinear least-squares method. Burst phases in both radii of gyration and zero-angle intensities were detected at the initial step of the reaction. This suggests that rapid association might take place, followed by a slow association process of which the kinetics were measured in the present study. The radius of gyration of tetrameric phosphorylase b was determined and found to be in excellent agreement with that of phosphorylase a, but different from that of phosphorylase b reported elsewhere (G. Puchwein, O. Kratky, C. F. Golker and E. Helmreich, Biochemistry 9 (1970) 4691). The reason for this inconsistency is discussed.

Animals↗
PubMed1989

Dissociation and auto-oxidation of hemerythrin induced by SH-modification: a kinetic study.

Hemerythrin from Siphonosoma cumanense has a trimeric structure consisting of identical subunits, which have no cooperativity nor Bohr effect on oxygen-binding. The trimer was dissociated into its monomers by the modification of the SH group of its cysteines with p-chloromercuriphenylsulfonic acid (PCMPS), which was monitored by stopped-flow of both spectrophotomeric and small angle X-ray scattering methods. The results showed that the process involved sequential modification of the SH groups, dissociation into monomers, and auto-oxidation of ferrous iron in the active center. The modification of the SH groups with PCMPS followed second-order kinetics with a rate constant of 1.8 M-1.s-1. The dissociation and auto-oxidation followed first-order kinetics with rate constants of 4 X 10(-3) s-1 and 5 X 10(-4) s-1, respectively. The obtained rate of auto-oxidation was much faster than that in the native state. These findings lead to the conclusion that the trimeric state of S. cumanense hemerythrin is necessary to prevent auto-oxidation.

Animals↗
PubMed1989

Negative cooperativity of chicken ovotransferrin on Al(III)-binding.

Chicken ovotransferrin, an iron binding protein, has two metal binding sites (amino (N) and carboxy (C) terminal sites). It binds Cu(II), Al(III), Co(II), and other metals, as well as Fe(III). In this study, the selectivity and cooperativity of the N and C sites on Al(III), Co(II), and Tb(III) binding were investigated. Metals were classified into two groups according to their site preference. Co(II) and Al(III) bound to the N site more preferably than to the C site, whereas Tb(III) bound to the C site more preferably. On Fe(III) binding, the binding constant of Fe(III) becomes larger when the other site is already occupied. Thus, positive cooperativity is seen. In the present study, the binding cooperativities of Co(II), Tb(III), and Al(III) as to the N and C sites were investigated. On Co(II) and Tb(III) binding, no cooperativity was observed, as in the case of Cu(II) [Yamamura, T. et al. (1985) in Proteins of Iron Storage and Transport (Spik, G., Montreuil, J., Crichton, R.R., & Mazurier, J., eds.) pp. 53-56, Elsevier Science Publ. B.V., Amsterdam]. In contrast, negative cooperativity was observed on Al(III) binding. Based on a model proposed by Yamamura et al. [Yamamura, T. et al. (1985) ibid.], the ratio of the binding constants, KC/KN, and the stacking coefficient, Kst, were estimated. KC/KN is 2.2 +/- 0.4 for the Tb(III) ion, 0.5 +/- 0.1 for the Co(II) ion, and 0.12 +/- 0.02 for the Al(III) ion. Kst (= 1 in a non-cooperative case) is 0.98 +/- 0.02 for the Tb(III) ion, 1.03 +/- 0.02 for the Co(II) ion, and 0.55 +/- 0.22 for the Al(III) ion.

Aluminum↗
PubMed1989

Investigation of an active site histidine-aspartate couple in Trimeresurus flavoviridis phospholipase A2.

When Trimeresurus flavoviridis phospholipase A2 was reacted with methyl p-nitrobenzenesulfonate, its activity decreased following first-order kinetics. The pH dependence of the rate constants of inactivation showed that His-48 with an apparent pKa of 6.5 controls the reaction. In the pH region below 6.5, N1-methylhistidine was predominantly formed. On the other hand, N1,N3-dimethylhistidine was almost exclusively produced in the pH region above 6.5. No N3-methylhistidine was detected at any pH tested. Such observations suggested that the first methylation occurred at the N1-position of the imidazole ring followed by a second methylation at the N3-position, and that His-48 couples the carboxylate of Asp-99 at the N3-position of the imidazole ring, in accord with the interaction observed in the crystal structure of homologous Crotalus atrox phospholipase A2. As it has been reported that, in the reaction of chymotrypsin with methyl p-nitrobenzenesulfonate at pH 7.8, only monomethylation occurred at the N1-position of the His-57 imidazole group (Nakagawa, Y. & Bender, M.L. (1970) Biochemistry 9, 259-267), the nature of the active site histidine-aspartate couple of T. flavoviridis phospholipase A2 seems not to be identical with that of chymotrypsin.

Amino Acid Sequence↗
PubMed1989

Auto-oxidation of Lingula unguis and Siphonosoma cumanense hemerythrins induced by some perturbants.

Auto-oxidation of human adult hemoglobin and Siphonosoma cumanense and Lingula unguis hemerythrins were investigated in the presence and absence of perturbants. In the presence of urea, there were no drastic increases in auto-oxidation rates for the three proteins. In contrast, thiocyanate and laurate enhanced the auto-oxidation rates remarkably. In the presence of thiol reagents such as PCMB and NEM, auto-oxidation rates were enhanced with dissociation into subunit, but not enhanced so much with SH-modification only. Thus oligomerization is probably necessary to protect auto-oxidation.

Animals↗
PubMed1988

Proton NMR resonance assignments and surface accessibility of tryptophan residues of a dimeric phospholipase A2 from Trimeresurus flavoviridis.

Proton NMR spectra of a dimeric phospholipase A2 from Trimeresurus flavoviridis have been recorded. N-1 proton resonances of the tryptophan indole rings have been detected and assigned to specific positions, Trp-3/Trp-30, Trp-68 and Trp-108, by comparing the spectra of the enzyme derivatives with tryptophans oxidized to differing extents. Photo-CIDNP experiments have revealed that Trp-68 and Trp-108 are exposed while Trp-3 and Trp-30 are buried in the molecule. This is consistent with the X-ray crystal structure of a homologous phospholipase A2 from Crotalus atrox where residues 3 and 30 are located at a dimer interface, but inconsistent with the results of stepwise oxidation of tryptophan residues.

Macromolecular Substances↗
PubMed1988

Reconstitution of myoglobin from apoprotein and heme, monitored by stopped-flow absorption, fluorescence and circular dichroism.

The reconstitution reaction of ferric cyanomyoglobin from apomyoglobin and hemin dicyanide was investigated with a stopped-flow apparatus by the use of five kinds of probes; (a) Soret absorption, (b) fluorescence quenching of tryptophan, (c) far-ultraviolet CD, (d) near-ultraviolet CD, and (e) Soret CD. After mixing of apomyoglobulin with equimolar amounts of hemin dicyanide, the Soret absorption band was shifted to longer wavelengths within 10 ms. The shifted band kept its shape for a few seconds, and then gradually shifted to shorter wavelengths. A rate constant of the slow reaction was 1.1 x 10(-2) s-1. Time courses of fluorescence quenching followed a second-order reaction with a rate constant of 9 x 10(7) M-1 s-1. Far-ultraviolet CD recovered to the level of native state within the response time of an apparatus (= 64 ms). Near-ultraviolet CD and Soret CD changed with first-order rate constants of 5-30 s-1 and 5 x 10(-3) s-1 respectively. On the basis of the kinetic results we propose the following reconstitution pathway of myoglobin. Apomyoglobin has essentially a highly folded structure similar to myoglobin, but there are some differences in the secondary structure between them. In the first step, heme enters the pocket-like site of apomyoglobin and interacts with surrounding hydrophobic residues in the pocket, and then the interaction may give a complete ordered structure to the protein. Second, the tertiary structure of the heme pocket is partly constructed. Third, the iron-proximal His bond occurs, followed by the attainment of the final conformation. This sequence of the events shows that the polypeptide chain is entirely folded before the completion of three-dimensional structure of the heme pocket. The reconstitution pathway is fairly different from that of the alpha subunit of hemoglobin reported by Leutzinger and Beychok [Proc. Natl Acad. Sci. USA (1981) 78, 780-784], which described how a drastic recovery in helicity was observed on the heme-binding, and that the recovery is introduced by the formation of the heme pocket structure. The difference in the results found for the alpha subunit and myoglobin suggests a difference in conformation: in apomyoglobin most of the helices are arranged and folded around a helix core to form a compact structure as a whole, while in apo-alpha subunit some helices are not folded around the helix core. Helix D, which is absent in the alpha subunit, may play an important role in folding of the helices.

Apoproteins↗
PubMed1988

Transition of environmental polarity of tyrosine-76 of Trimeresurus flavoviridis phospholipase A2 upon active site ligand binding.

Modification of Trimeresurus flavoviridis phospholipase A2 with a 5-fold molar excess of tetranitromethane produced 40% active mononitrotyrosyl phospholipase A2 in which Tyr-76 was specifically nitrated. This is in contrast to the case of mammalian pancreatic phospholipases A2 where Tyr-70 but not Tyr-76 was nitrated. When Ca2+ was bound to T. flavoviridis mononitrotyrosyl phospholipase A2, nitrated tyrosine (Tyr(NO2))-76 moved from a less polar site to a polar site with the decrease of the pKa value of its hydroxyl group. Nitration of Tyr-76 did not influence the binding affinity to Ca2+. Addition of laurylphosphorylcholine to mononitrotyrosyl phospholipase A2 in the presence of Ca2+ caused the movement of Tyr(NO2)-76 from a polar environment to a less polar environment with the rise in the pKa value. Tyrosine-76 is located in the site whose environmental polarity is affected by the binding of the ligands to the active site. As Tyr-76 is located in the site not proximal to the active site, it could be assumed that the conformational change induced by the binding of the ligands extends to the region remote from the active site in T. flavoviridis phospholipase A2. This might provide evidence of long-range diffusional coupling between remote sites in the noncooperative globular protein.

Amino Acid Sequence↗
PubMed1988

Tryptophan residue essential for activity of Naja naja atra phospholipase A2.

When Naja naja atra phospholipase A2, which contains three tryptophan residues at the 18th, 19th, and 61st positions, was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased in a convex manner with increase in the extent of oxidation of tryptophan residues. The curve shape showed that the tryptophan residue oxidized last is most responsible for the activity. The order of accessibilities of the three tryptophan residues, which was analyzed according to the method reported previously (Mohri et al. (1876) J. Biochem. 100, 883-893), was Trp-61 greater than Trp-19 greater than Trp-18. Thus, Trp-18 was evaluated to be essential for activity. Difference spectra of phospholipase A2 produced by titrating with laurylphosphorylcholine in the presence of Ca2+, which are due in large part to perturbation of the tryptophan residue(s), were retained with phospholipase A2 derivatives containing 1.2 and 2.0 mol of tryptophan residues oxidized but not with the derivative containing 3.0 mol of tryptophan residues oxidized. Such observations led us to assume that Trp-18 is involved in the specific site that interacts with phospholipid.

Amino Acid Sequence↗
PubMed1988

Amino acid sequence of a coagulant enzyme, flavoxobin, from Trimeresurus flavoviridis venom.

The amino acid sequence of a coagulant enzyme, named flavoxobin, isolated from the venom of Trimeresurus flavoviridis (the habu snake) was determined by sequencing the S-pyridylethylated derivative of the protein and its peptides generated by chemical (cyanogen bromide and hydroxylamine) and enzymatic (clostripain, Staphylococcus aureus V8 protease, Achromobacter protease I, and elastase) cleavages. Hydrazinolysis was also employed to determine the C-terminal amino acid. The enzyme consisted of 236 amino acids and had a calculated molecular weight of 25,744. Flavoxobin was found to be highly (69%) homologous in sequence to batroxobin, a coagulant enzyme from the venom of Bothrops atrox, and 27, 39, and 31% homologous to bovine thrombin, bovine trypsin, and human kallikrein, respectively. The sequence around the active site serine residue deduced from the homology relationship was Phe-Asp-Ser-Gly-Thr, which is different from the common sequence, Gly-Asp-Ser-Gly-Gly, for most serine proteases. Flavoxobin appears to be similar in secondary structure composition to batroxobin.

Amino Acid Sequence↗
PubMed1988

Tetrodotoxin-unaffected depolarization of frog muscles induced by the venom of jellyfish (Genus aurelia).

In the isolated frog muscle, the proteinaceous venom extracted from jellyfish (genus Aurelia) produced 1) a complete and irreversible block of indirectly and directly elicited muscle twitch and 2) an irreversible depolarization of the muscle membrane. This venom-induced depolarization was effectively reversed or prevented by the substitution of choline for sodium in Ringer solution, but not by the introduction of tetrodotoxin (TTX), a sodium channel blocker. The mechanism of muscle membrane depolarization appears to involve probably an increase in membrane permeability to sodium ion as shown by the decrease in membrane resistance. These results suggest that the venom forms a pore which has sodium selectivity or activates a TTX-insensitive sodium channel which is different from the known sodium channel.

Aminopyridines↗
PubMed1987

Kinetics of structure and activity changes during the allosteric transition of aspartate transcarbamylase.

For the first time, the structural change associated with an allosteric transition has been monitored by X-ray solution scattering. The kinetics of the quaternary structure change of aspartate transcarbamylase were first slowed by using acetyl phosphate instead of carbamyl phosphate, and by the presence of 10% or 30% ethylene glycol. At 6.5 degrees C, the quaternary structure change was found to have a time constant of about 11 seconds. This appears to be larger than that obtained for the switching of homotropic co-operativity, measured by chemical quench under the same conditions.

Allosteric Site↗
PubMed1987

Purification and characterization of phospholipase A2 from Trimeresurus gramineus venom.

Phospholipase A2 was purified to homogeneity from the venom of Trimeresurus gramineus (the Green Habu snake) via three steps consisting of Sephadex G-75, DEAE-cellulose, and DEAE-Toyopearl 650M column chromatographies. Molecular weight determinations showed that the enzyme consists of a single polypeptide chain with a molecular weight of approximately 14,000. The isoelectric point was 4.5. The enzyme is characterized by high contents of acidic amino acids, glycine, and half-cystine. Calcium ion was essential for eliciting activity. The enzyme was inactivated by alkylation of a single histidine residue with p-bromophenacyl bromide following pseudo first-order kinetics, and the rate of the inactivation was depressed in the presence of Ca2+. The N-terminal sequence of this enzyme determined to the 40th residue was found to be highly homologous to that of Trimeresurus okinavensis phospholipase A2 but not to that of Trimeresurus flavoviridis phospholipase A2. The phenylalanine residue at the 27th position of T. gramineus phospholipase A2 is noteworthy because all other phospholipases A2, with only two exceptions, contain a tyrosine residue at this position.

Acetophenones↗
PubMed1987

[Clinical trial of forphenicinol for the lung infection with Mycobacterium avium, Mycobacterium intracellulare complex].

Clinical evaluation of forphenicinol, a low molecular weight immunomodulator, in patients with Mycobacterium avium, Mycobacterium intracellulare complex pulmonary infections has been conducted in a multicenter trial participated by 9 institutions in Kyushu during a period of 12 months from July 1982 to August 1983. Forphenicinol was administered for 6 months without changing the regimen of antituberculous drugs used previously. The following results were obtained. 1. Ten out of 33 eligible patients were evaluated as showing good responses; In 5 of them, elimination of M. avium, M. intracellulare complex from sputum was observed. In the other 5 patients, number of bacilli excreted was decreased significantly after treatment. 2. All of the cases with good responses were those which were simultaneously administered with antituberculous drugs. 3. Patients with thick-walled cavities, following cured tuberculosis, which was superimposed with M. avium, M. intracellulare complex, were poorly responded to forphenicinol. 4. Side effects were observed in 3 out of 41 patients; 1 case each with fever, abdominal distension and anorexia. No abnormalities in laboratory test values were observed.

Adult↗