Biomedical subjects

Y Shiro

Publications and source records attributed to Y Shiro.

At least 73 records · Page 4Linked to original sources

PubMed1996

Effect of chronic ipsapirone treatment on the density of 5-HT1A receptors and 5-HT1A receptor mRNA in discrete regions of the rat brain.

There is increasing evidence that the 5-hydroxytryptamine (HT)1A partial agonist ipsapirone is an effective anxiolytic/antidepressant agent, although its mechanism of action is not clear. In this study, we investigated the effects of chronic ipsapirone treatment (5 or 10 mg/kg; twice daily, 3 weeks) on 5-HT1A receptor density 8-hydroxy-2-(di-n-propyl amino) tetralin (8O H-DPAT) binding and the level of its mRNA (in situ hybridization) in various regions of the rat (male Wistar 250 g) brain. Receptor density was reduced in the frontal cortex, but did not change significantly in the hippocampus and dorsal raphe nucleus. The level of receptor mRNA was unchanged in each of these brain regions. The present results suggest that the clinical anxiolytic effects of ipsapirone may be mediated partly by postsynaptic action on serotonergic transmission in the frontal cortex, but not in the hippocampus or dorsal raphe nuclei.

Animals↗

PubMed1995

Iron-ligand structure and iron redox property of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum: relevance to its NO reduction activity.

We studied the nitric oxide reductase, cytochrome P450nor, purified from a denitrifying fungus Fusarium oxysporum with electron paramagnetic resonance spectral and redox potential measurements. The EPR spectral features of P450nor in the ferric resting, the ferric cyanide-bound, and the ferrous NO-bound forms were the same as the corresponding ones of other general P450s such as Pseudomonas putida P450cam. In contrast, the metyrapone complex of ferric P450nor gave an EPR spectrum with significantly different g values from that of P450cam. The EPR results were explained in terms of similarity in the immediate configuration of the S(-)-Fe-ligand (H2O, CN-, NO) structure between P450nor and P450cam but a structural difference at the heme distal pocket, especially in the substrate binding domain; P450cam has a camphor binding domain, while P450nor does not. In spite of the same S(-)-Fe-H2O configuration, the redox potential of P450nor in the ferric/ferrous couple was measured to be -307 mV, which is much lower than those of the camphor-bound (-140 mV) and -free (-250 mV) P450cam. The lower redox potential could be attributable to the different electrostatic interaction of the heme with its surroundings; e.g., the heme environment of P450nor is charged either more negatively or less positively than P450cam.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1995

Novel ligand binding properties of the myoglobin substituted with monoazahemin.

The iron complex of alpha-azamesoporphyrin XIII was combined with apomyoglobin to investigate influence of the meso nitrogen on ligand binding properties in the reconstituted protein. Stoichiometric complex formation between the two components was confirmed, and conservation of the native coordination structures in the resultant myoglobin was established with spectroscopic criteria and apparently normal ligand binding. The visible absorption spectra of various ferric and ferrous derivatives are characteristic with less intense Soret peaks and enhanced visible bands. The electron paramagnetic resonance spectrum with g = 5.2 suggests an anomalous intermediate spin (S = 3/2) character for the aquomet protein. The oxygen affinity of reduced azaheme myoglobin, 0.010 mm Hg, is 50 times larger than that of the native myoglobin. In addition, azaheme myoglobin forms stable complexes with imidazole, pyridine, or cyanide in ferrous state. All of these new properties were consistently explained in terms of stronger equatorial ligand field of the heme iron in a narrower coordination cavity. Similarities of azaheme to verdoheme were also pointed out.

Carbon Dioxide↗

PubMed1995

The mechanism of electron donation to molecular oxygen by phagocytic cytochrome b558.

Phagocytic cytochrome b558 is a unique heme-containing enzyme, which catalyzes one electron reduction of molecular oxygen to produce a superoxide anion with a six-coordinated heme iron. To clarify the mechanism of the superoxide production, we have analyzed oxidation-reduction kinetics of cytochrome b558 purified from porcine neutrophils by stopped-flow and rapid-scanning spectroscopy. Reduced cytochrome b558 was rapidly reoxidized by O2 showing spectral changes with clear isosbestic points, which were also observed during the reduction of ferric cytochrome b558 with Na2S2O4 under anaerobic conditions. The single turnover rate for the reaction with O2 linearly depended on the O2 concentration but was not affected by addition of CO. The rate of the reaction decreased with an increase of pH giving a pKa of 9.7. Under complete anaerobic conditions, ferrous cytochrome b558 was oxidized by ferricyanide at a rate faster than by O2. The thermodynamic analysis shows that the enthalpic energy barriers for the reactions of cytochrome b558 are significantly lower when compared to the autoxidation of native and modified myoglobins through the formation of the iron-O2 complex. These findings are most consistent with the electron transfer from the heme to O2 by an outer-sphere mechanism.

Animals↗

PubMed1995

Spectroscopic and kinetic studies on reaction of cytochrome P450nor with nitric oxide. Implication for its nitric oxide reduction mechanism.

Cytochrome P450 purified from Fusarium oxysporum (P450nor) is a unique heme enzyme that catalyzes the reduction of nitric oxide to nitrous oxide with electrons directly transferred from NADH (2NO + NADH + H+--> N2O + H2O + NAD+). We studied the reaction of P450nor with NO and NADH using stopped-flow rapid scan and low temperature spectroscopic methods. The NO ligand can bind to the ferric enzyme to form the stable NO bound complex, P450nor(Fe3+NO). Reduction of P450nor(Fe3+NO) with NADH yielded an intermediate, which transiently formed (tau = approximately 100 ms) and spontaneously decomposed to the Fe3+ state. The optical absorption spectrum of the intermediate was different from that of P450nor(Fe2+NO), which was formed by either a one-electron reduction of P450nor(Fe3+NO) with Na2S2O4 or NO binding to P450nor(Fe2+). On the basis of these observations, we suggested that the intermediate is presumably a two-electron reduced product of P450nor(Fe3+NO) by NADH, formally the (Fe3+NO)2-complex. We determined the rate constants of these reactions at 10 degrees C for the NO binding to P450nor(Fe3+) (2.6 x 10(7) M-1 s-1), the NADH reduction of P450nor(Fe3+NO) (0.9 x 10(6) M-1 s-1), and the spontaneous decomposition of the intermediate (0.027 s-1). In these kinetic measurements, it was found that the former two processes are fast enough, while the latter is extremely slow, compared with the fast turnover of the catalytic reaction (1200 s-1 at 10 degrees C), which we measured by monitoring the NADH consumption. Therefore, we suggested that in the catalytic cycle, decomposition of the intermediate is fairly accelerated by free NO, resulting in such a fast turnover. On the basis of several lines of the spectroscopic and the kinetic evidence, we proposed a possible mechanism of the NO reduction by P450nor.

Cytochrome P-450 Enzyme System↗

PubMed1994

Spectroscopic study of Ser92 mutants of human myoglobin: hydrogen bonding effect of Ser92 to proximal His93 on structure and property of myoglobin.

Neutron diffraction studies have demonstrated that the hydroxyl group oxygen of Ser92(F7) is hydrogen bonded to the proximal His93(48) N epsilon H proton in myoglobin (Mb) [Cheng, X., & Shoenborn, B. P. (1991) J. Mol. Biol. 220, 381-399]. In order to examine the importance of this hydrogen bond, Ser92 was replaced with Ala and Asp in human Mb. By comparing the optical, 1H-NMR, resonance Raman, and IR spectra of Mb(S92A) in several spin and oxidation states with those of wild-type Mb, it was found that the mutation causes a structural change on the heme proximal side but not on the distal side. Comparison of the NMR spectra of the cyanomet form of Mb(S92A) and Mb(WT) suggests that the imidazole plane of His93 rotates somewhat around the Fe-N delta (His93) bond upon loss of the hydrogen bond between His93 and Ser92. The 2D 1H-NMR measurements of the CO complexes show that mutation of Ser92 to Ala changes the relative position of the His97 imidazole group to the heme plane, but the change is not so drastic as reported in the crystal data of Ser92 mutant of pig Mb [Smerdon et al. (1993) Biochemistry 32, 5132-5138]. On the other hand, ligand (CO, O2) binding is only slightly affected by this mutation. From these results, we conclude that the Ser92-His93 hydrogen bond maintains the protein structure of the proximal heme pocket, but it does not strongly affect the electronic structure of the heme as well as of the His93 imidazole ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Monoxide↗

PubMed1994

Consequence of rapid heme rotation to the oxygen binding of myoglobin.

The myoglobin complexed with octamethylhemin was prepared. The visible absorption profiles are typical of general alkylhemin-substituted myoglobins, suggesting normal insertion of the hemin into the hydrophobic cavity. The NMR spectra of various ferric proteins were anomalous, without clearly resolved signals from the prosthetic group, most probably reflecting rapid heme rotation about the iron-histidine bond. The equilibrium and kinetic oxygen bindings were measured to examine the functional significance of the heme motion. Functional comparison with the myoglobin having immobile hemin indicates that rotation of the octamethylheme negligibly affects the myoglobin function. The results suggest that neither the heme peripheral contacts nor the proximal imidazole orientation about the heme normal is the dominant factor to control myoglobin function.

Heme↗

PubMed1994

Kinetics and thermodynamics of CO binding to cytochrome P450nor.

The CO-binding reaction of cytochrome P450nor isolated from denitrifying fungus, Fusarium oxysporum, has been studied by using a flash photolysis method in the millisecond time domain. We obtained the CO on- and off-rate constants in the bimolecular reaction, and determined the activation free energy, enthalpy, and entropy from the temperature dependence of these rate constants. To discuss the structural characteristics of P450nor, these parameters were compared with those of other cytochrome P450s, such as cytochrome P450cam from Pseudomonas putida and myoglobin. The on-rate constant (k(on)) for P450nor is larger than those of camphor-bound P450cam [P450cam(+)], suggesting that ligand entry to the heme pocket of P450nor is sterically less restricted than that of P450cam(+). In the P450norCO complex, the IR stretching band of the iron-bound CO is observed at 1942 cm-1, which is the same position as in P450cam(+)CO. This result suggests that the heme pocket immediate to the ligand-binding site is the same size in the two enzymes, in good agreement with the observation that the equilibrium constant (K = k(on)/k(off)) is identical in P450nor and P450cam(+). On the other hand, the entropy changes in the equilibrium and the off-activation processes are smaller in P450nor than in P450cam(+). This feature could reflect the lack of a bound substrate at the active site of P450nor. These structural characteristics of P450nor are discussed in relation to its unique catalytic property, rapid NO reduction to yield N2O.

Carbon Monoxide↗

PubMed1994

Crystallization and preliminary X-ray diffraction studies of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum.

Crystals of cytochrome P450nor (P450nor), nitric oxide reductase from Fusarium oxysporum, were obtained by means of the hanging-drop vapour diffusion technique in the presence of 1.88 M ammonium sulphate (pH 7.2). They belong to the monoclinic space group P2(1), with unit cell dimensions of a = 74.7 A, b = 86.7 A, c = 62.0 A and beta = 97 degrees. There are two molecules of P450nor in the asymmetric unit. The crystal diffracted to beyond 2.5 A resolution and is suitable for X-ray crystallographic study.

Crystallization↗

PubMed1994

Hepatic encephalopathy with increased cerebral blood flow in SPECT and MRI abnormalities in the basal ganglia.

A case of hepatic encephalopathy with increased cerebral blood flow in single-photon emission computed tomography (SPECT) and abnormalities upon magnetic resonance imaging (MRI) is reported. SPECT showed increased blood flow exceeding 70 ml/100 g/min in the cerebellum, basal ganglia and cerebral cortex, with hyperammonemia and abnormal electroencephalographic signs showing severe generalized slowing. SPECT data and MRI might reflect abnormal metabolism and neuropathological changes in hepatic encephalopathy.

Antiparkinson Agents↗

PubMed1993

Specific modification of structure and property of myoglobin by the formation of tetrazolylhistidine 64(E7). Reaction of the modified myoglobin with molecular oxygen.

Tetrazole-myoglobin (Tet-Mb), a site selectively modified myoglobin with tetrazole anion (-CN4-) covalently attached to the imidazole N epsilon of the distal histidine 64(E7) (see Fig. 1; Kamiya, N., Shiro, Y., Iwata, T., Iizuka, T., and Iwasaki, H. (1991) J. Am. Chem. Soc. 113, 1826-1829), exhibited unique properties in the reduction from ferric to ferrous states and in the reaction of its deoxy form with O2. The redox potential of Tet-Mb is obtained to be -193 mV, which is much lower than that of unmodified (native) myoglobin (50 mV), possibly due to the electrostatic interaction between the heme iron and the tetrazole group. The ferrous deoxy form of Tet-Mb was rapidly oxidized to its ferric form in the reaction with O2 at room temperature through an intermediary formation of its oxy form and with the generation of O2-. The oxy form of Tet-Mb can be detected by the optical spectral measurement at -12 degrees C, the rapid scan measurement at room temperature, and the electron spin resonance measurement of its cobalt-substituted derivative (Tet-Mb(Co2+)) at 77 K. In the kinetic measurement of the O2 binding reaction to Tet-Mb, its association and dissociation rate constants in the bimolecular reaction were 6.1 x 10(7) M-1 s-1 and 2200 s-1, respectively, showing that the tetrazole modification of His-64 extremely accelerates its association and dissociation rates. Taken together with the extremely fast autoxidation rate (53 h-1) obtained, these kinetic results suggested that the channel of O2 from the solvent region to the protein interior is open enough to pass the external ligand. The structure is discussed in relation to those of some genetic mutants. Taking these properties, we demonstrated that Tet-Mb can catalyze O2 consumption to generate O2-, coupled with the NADH-supported enzymatic reduction system of cytochrome P-450cam under an aerobic condition.

Amino Acid Sequence↗

PubMed1992

Effect of NaCl addition on nanosecond O2 escaping reaction of myoglobin: evidences for the transition of myoglobin dynamic structure at 20 degrees C.

We studied the nanosecond (ns) geminate O2 escape reaction from the protein interior of myoglobin (Mb) to the solvent phase in the temperature range of 5-40 degrees C containing 0-0.1 M NaCl. In the flash photolysis experiments, we found that both the rate constant, kout, and its Arrhenius plot changed upon the variation of the NaCl concentration. In particular, it was noteworthy that the Arrhenius plot of kout dramatically changed in its slope, keeping the break at 20 degrees C, upon the addition of NaCl, indicating that the thermodynamic parameters such as an enthalpy of activation (delta H not equal to) and an entropy of activation (delta S not equal to) are different between above and below 20 degrees C, and that they are further altered upon the NaCl addition to the sample solution. From these results, we suggested that the Mb dynamic structure in the ns geminate O2 escape reaction is sensitively regulated by the interaction of the protein surface and the salt. The present study also showed that an inconsistency of the Arrhenius plot of kout between Chatfield et al. ((1990) J. Am. Chem. Soc. 112, 4680-4687) and us ((1990) J. Biol. Chem. 265, 18823-18828) is probably due to the difference in the solution condition.

Myoglobin↗

PubMed1992

Kinetic studies on CO binding to reconstituted myoglobins with four synthetic hemes; structural control in ligand binding to myoglobin.

Examination was made of CO binding reactions to four kinds of modified sperm whale myoglobin (Mb), whose heme was reconstituted by iron complexes of synthetic porphyrins such as porphine (Por), meso-tetramethylporphyrin (TMeP), meso-tetraethylporphyrin (TEtP) and meso-tetra(n-propyl)porphyrin (TnPrP), using flash photolysis and stopped-flow methods. The CO association rate was found to be 5- to 20-times and dissociation rate 10- to 36-times accelerated by replacement with synthetic hemes. These features could be explained based on characteristic structures of modified Mbs indicated by X-ray crystallography. The side chain of Arg-45 protruded from the heme vicinity into the solvent region and heme was tilted by interactions of meso-alkyl side chains with surrounding peptides, resulting in the formation of widely opened channels and pockets for ligand passage. These structural features indicate the CO ligand to more easily enter or exit from heme pockets of reconstituted myoglobins, compared to native Mb.

Animals↗

PubMed1992

[A case of ornithine transcarbamylase deficiency presenting severe symptoms in adulthood].

An adult female case of ornithine transcarbamylase (OTC) deficiency is presented in the following. The patient had had past episodes of drowsiness with a duration less than a few minutes several times a year during childhood. She suddenly became comatose at 25 years of age, and died after 13 months of persistent vegetative state. Blood chemistry showed hyperammonemia with no liver cirrhosis or portal-systemic shunt. Plasma amino acid analysis indicated elevated glutamate and glycine levels, and plasma levels of citrulline and arginine to be low. The urinary orotic acid level was high. OTC activity of a liver specimen was 65 percent of the normal level. This is a rare case demonstrating hyperglycinemia and an elevated level of serum OTC. The importance of ruling out defective ureagenesis in adults with disturbed consciousness should be emphasized.

Adult↗

PubMed1991

Structural and electronic characterization of heme moiety in oxygenated hemoproteins by using XANES spectroscopy.

Iron K-edge X-ray absorption near edge structure (XANES) spectra were measured for oxy-forms of cytochrome P-450cam (P-450cam), horseradish peroxidase (HRP) and myoglobin (Mb) by using Synchrotoron Radiation of Photon Factory (Tsukuba). A pronounced 1s-4p transition and some fine structures were well-resolved in the spectra obtained. Comparing the spectra, the features at the fine structures termed P, C and D, were similar among the three hemoproteins, suggesting a similar site-symmetry around the heme iron and the same Fe-O-O bond angle (about 115 degrees). On the other hand, absorption features at the edge region (7115-7135 eV) were slightly but significantly different from one another; the absorption intensity at 7115-7125 eV region increased in the order of Mb, HRP and P-450cam, while that at 7125-7135 eV decreased in the same order. A similar absorption feature was also obtained with their deoxy (ferrous high spin) forms. We assumed that the absorption at the lower energy region (7115-7125 eV) reflects the pi-character in the Fe-ligand bond, whereas that at the higher energy region (7125-7135 eV) does the sigma-character, on the basis of the previous and comprehensive studies of the XANES spectroscopy of the adsorbed molecules on the metal surface (McGovern et al. (1989) Handbook on Synchrotoron Radiation, Vol. 2, pp. 467-539). According to our assumption, our XANES results indicated that the pi-character of the Fe-ligand bond increases in the order of Mb, HRP and P-450cam, and that the pi-electron of the thiolate S- in P-450cam is donated to the Fe-O-O moiety, most probably to the antibonding pi* orbital of O2. Such an interpretation is consistent with the experimental findings or data accumulated so far by other methods, such as the resonance Raman spectroscopy.

Animals↗

PubMed1991

Superoxide production by cytochrome b558 purified from neutrophils in a reconstituted system with an exogenous reductase.

Cytochrome b558, which is considered to be an essential component of the phagocytic superoxide (O2-)-generating system, was highly purified from porcine neutrophils. The isolated cytochrome was resolved into two polypeptides with molecular masses of 60-90 and 19 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For enzymatic reduction of purified cytochrome b558, we utilized hepatic NADPH-cytochrome P450 reductase purified from rat liver microsomes. More than 80% of the cytochrome was reduced by incubation with the reductase and NADPH under the anaerobic condition, and was quickly reoxidized by the air. As indicated by measurement of oxygen consumption, the purified cytochrome catalytically reduced oxygen at a rate equal to approximately 30% of the activity of the phorbol myristate acetate-activated cells on the basis of cytochrome b558 content. Electron paramagnetic resonance study with a spin trapping agent 5, 5-dimethyl-1-pyrroline-1-oxide demonstrated that O2- is the exclusive primary product in the reduction of oxygen by the cytochrome. This gives direct evidence that cytochrome b558 functions as the terminal oxidizing enzyme in the O2- -generating system of neutrophils. This also establishes a new functional class of heme proteins that catalyzes one-electron reduction of molecular oxygen.

Animals↗

PubMed1990

Thermodynamic study of protein dynamic structure in the oxygen binding reaction of myoglobin.

We examined the flash photolysis of oxy complexes of sperm whale myoglobin (Mb) on the nanosecond time scale at ambient temperatures. In this time range, we can observe the geminate reaction of Mb with the O2 ligand existing in the protein matrix after the photodissociation from the heme iron. We found that the fraction of the geminate component to the total O2 photodissociation exhibited temperature dependences. The geminate fraction decreased with rising temperature, indicating that the protein fluctuation is enhanced at high temperature because of thermal agitation. However, the temperature-dependent behavior showed a break at 20 degrees C. Concerning the geminate O2 escaping reaction from the protein matrix to the solvent region, the activation energy above 20 degrees C (0.4 +/- 0.4 kcal/mol) is significantly lower than that below 20 degrees C (5.1 +/- 0.4 kcal/mol). Thermodynamic analysis on the basis of the transition state theory indicated that the O2 escaping reaction above 20 degrees C is entropy dominated whereas that below 20 degrees C is enthalpy dominated. The results were qualitatively compatible with the theoretical prediction by J. Kottalam and D. A. Case [1988) J. Am. Chem. Soc. 110, 7690-7697). Comparing the kinetic and thermodynamic process of the O2 geminate reaction among several Mbs, we concluded that the geminate O2 reaction with Mb is governed by the dynamic motion of the protein which is sensitively controlled by the static interaction of the heme moiety with the surroundings.

Animals↗

PubMed1990

New transient species of sperm whale myoglobin in photodissociation of dioxygen from oxymyoglobin.

We carried out the flash photolysis of oxy complexes of sperm whale myoglobin, cobalt-substituted sperm whale myoglobin, and Aplysia myoglobin. When the optical absorption spectral changes associated with the O2 rebinding were monitored on the nanosecond to millisecond time scale, we found that the transient spectra of the O2 photoproduct of sperm whale myoglobin were significantly different from the static spectra of deoxy form. This was sharply contrasted with the observations that the spectra of the CO photoproduct of sperm whale myoglobin and of the O2 photoproducts of cobalt-substituted sperm whale myoglobin and Aplysia myoglobin are identical to the corresponding spectra of their deoxy forms. These results led us to suggest the presence of a fairly stable transient species in the O2 photodissociation from the oxy complex of sperm whale myoglobin, which has a protein structure different from the deoxy form. We denoted the O2 photo-product to be Mb*. In the time-resolved resonance Raman measurements, the nu Fe-His mode of Mb* gave the same value as that of the deoxy form, indicating that the difference in the optical absorption spectra is possibly due to the structural difference at the heme distal side rather than those of the proximal side. The structure of Mb* is discussed in relation to the dynamic motion of myoglobin in the O2 entry to or exit from the heme pocket. Comparing the structural characteristics of several myoglobins employed, we suggested that the formation of Mb* relates to the following two factors: a hydrogen bonding of O2 with the distal histidine, and the movement of iron upon the ligation of O2.

Animals↗