Search PubMed⌕ Search

Biomedical subjects

T Kitagawa

Publications and source records attributed to T Kitagawa.

At least 163 records · Page 9Linked to original sources
PubMed1997

Extracellular adenosine 5'-triphosphate induces Ca2+ efflux from freshly isolated adult rat cardiomyocytes: possible involvement of Na+/Ca2+ exchange mechanism.

In the present study, we examined the effect of extracellular adenosine 5'-triphosphate (ATP) on Ca2+ efflux from freshly isolated adult rat cardiomyocytes. ATP at 1 mM caused a release of 3.6+/-0.08% of the total cellular content. The 45Ca2+ efflux from the cells was also stimulated by adenosine-5'-O-(3-thiotriphosphate) (ATP-gamma s), alpha, beta-methylene-ATP and adenosine 5'-diphosphate (ADP), but not by adenosine 5'-monophosphate (AMP) or adenosine. The effect of ATP was inhibited by a known purinergic P2-receptor antagonist, but not by a P1-receptor antagonist. From these results, it is conceivable that the effect of ATP on Ca2+ efflux from cardiomyocytes is mediated through P2-purinoceptors. It was also observed that ATP caused a rise in [Ca2+]i to almost 200 nM. The ATP-stimulated 45Ca2+ efflux was not affected by removal of extracellular Ca2+, but was dependent on the presence of extracellular Na+. Moreover, ATP caused a 22Na+ influx into the cells of about 2.0-fold over the basal value. These result suggest that ATP stimulates extracellular Na+-dependent 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on plasma membrane P2-purinoceptors which may couple to Na+/Ca2+ exchange.

Adenosine Triphosphate↗
PubMed1997

Purification of bovine soluble guanylate cyclase and ADP-ribosylation on its small subunit by bacterial toxins.

Soluble guanylate cyclase (sGC) consisting of two different subunits (alpha: Mr = 74,000, beta: Mr = 69,000) was purified more than 12,000-fold in terms of specific activity from the supernatant of bovine lung homogenates and characterized. The heme content determined with the pyridine hemochromogen method and Bradford's protein assay was 0.8 heme per dimer. Cholera, pertussis, and botulinum C3 toxins modified exclusively the beta-subunit of sGC, yielding the ADP-ribose-bound compound with 1:1 stoichiometry, and Vmax for the cyclase reaction was increased 10 times by this modification. When the ADP-ribosylation of sGC was performed simultaneously with two or three bacterial toxins which have distinct amino acid specificities, the resultant enzyme had only one ADP-ribose, and the activity was the same as that of the enzyme modified with one toxin. When NO was incorporated into the reaction mixture containing the ADP-ribosylated sGC, the cyclase activity noticeably increased by approximately the same amount as that seen for the unmodified enzyme. Such effects were not seen with CO. When ADP-ribosylated sGC was incubated with Mn2+, the enzyme activity was synergistically increased. The heme-deleted sGC was also ADP-ribosylated by bacterial toxins and its activity was raised. These findings suggest that sGC has an ADP-ribosylation site near the GTP binding site, like other GTP-binding proteins, and that the beta-subunit regulates the activity.

Adenosine Diphosphate↗
PubMed1997

Development and application of a sandwich enzyme immunoassay for Glycyrrhizae Radix protein (GRP) using monoclonal antibodies.

We have developed mouse monoclonal antibodies (anti-GRP mAb-1-5, all IgG1 sub-isotype mAbs) against Glycyrrhizae Radix protein (GRP), which was recently determined to be a marker protein of Glycyrrhizae Radix (GR). Among these, anti-GRP mAb-1 and 2 were found to recognize different epitopes on the GRP molecule, as demonstrated by ELISA analysis, and were used for the development of a sandwich enzyme immunoassay (SEIA) for GRP in traditional Chinese medicines (TCMs). The SEIA was based on the principle of binding an analyte to anti-GRP mAb2 coated on polystyrene microtiter wells, followed by immunoreaction with biotinylated anti-GRP mAb1 and horseradish peroxidase-streptavidin. The SEIA was specific to GRP in GRs, and showed no cross-reaction with any Leguminosae crude drugs other than GRs. This SEIA detected GRP with excellent reproducibility (coefficient of variation=5.9%), an EC50 of 11.5 ng/well and a detection limit of 0.1 ng/well. The present SEIA was about 10-times more sensitive in detecting GRP than the selected antibody enzyme immunoassay (SAEIA) for GRP previously developed using an antiserum to GR itself. Also, the SEIA has such a low assay background that it allowed us to detect a low concentration of GRP in Kyuki-tyoketsu-in-daiichi-kagen (KTIDK), a TCM consisting of only 2.7% GR. The GRP SEIA was simple, accurate, reproducible and may provide a general analytical method for the quality control of GR-based TCMs.

Animals↗
PubMed1997

A general enzyme immunoassay for the licorice root component contained in traditional Chinese medicines.

The development and application of a new enzyme immunoassay for general assay of the Glycyrrhizae radix (GR) component in Chinese traditional medicines is described. Three commercial GR-based medicines, Tohoku kanzo (GRTK), Seihoku Kanzo (GRSEK) and Sinkyo kanzo (GRSK) were used as GR specimens. Anti-GRSK serum was elicited from rabbits immunized with GRSK fragments. The presence of common proteins as specific antigens of GR was first established by Western blot analysis of extracts of GRSK, GRSEK or GRTK using anti-GRSK. The specific antigens were applied to develop an ELISA for the assay of GR extract. Anti-GRSK was put in competition with a sample or standard GR extract and immobilized GRSK components in microtiter plate wells. The proportion of antibody binding to the solid-phase GRSK component was detected using an enzyme-labeled second antibody. The ELISA method was specific to GRSK extract and showed low sensitivity for the assay of GRTK extract. The technique of selected antibody enzyme immunoassay (SAEIA) was applied to develop a sensitive general assay method. Solid-phase GRTK extract, rather than immobilized GRSK extract, was used in the SAEIA. The SAEIA possessed the same quantitative working range of between 1 and 100 micrograms/ml for the assay of each extract of GRTK, GRSK and GRSEK. The SAEIA was successful in the detection and quantitative measurement of GR component contents in Chinese traditional medicines.

Animals↗
PubMed1997

Pharmacokinetic analysis of enterohepatic circulation of etodolac and effect of hepatic and renal injury on the pharmacokinetics.

This study was designed to evaluate the enterohepatic circulation of racemic etodolac in rats. Additionally, the effect of hepatic and renal failure on the pharmacokinetics was estimated. The biliary excretion and the reabsorption of the drug excreted in bile were examined in order to clarify the effect of enterohepatic circulation on the disposition, and a pharmacokinetics model was applied to describe the enterohepatic circulation. The relatively rapid elimination of etodolac was seen in the bile-exteriorized rats (BE rat) compared with that in control rats. Total biliary excretion of etodolac, mainly as glucuronides, after intravenous administration was about 45% of the dose, indicating extensive enterohepatic circulation of the drug. The plasma concentrations of the drug in bile duct-linked rats approximately agreed with the simulation curve by the model, with the peak concentration 6-7 h after dosing. The elimination of the drug was markedly retarded in rats with hepatic (CCI4-induced) and renal (uranyl acetate-induced) failure, and high plasma levels were maintained over the longer times, due to greatly decreased distribution volume. The biliary excretion of etodolac enantiomers was not significantly different between the control and CCI4-groups, suggesting that hepatic glucuronyl transferase activity was preserved in rats impaired by CCI4.

Acute Kidney Injury↗
PubMed1997

Application of a licorice root specific protein to a general method for the assay of licorice root components in traditional Chinese medicines.

A new fuzzy immunoassay method generally applied to ten Glycyrrhizae Radix (GR) preparations of four different botanical origins was studied. Four kinds of antisera were elicited in rabbits immunized with GRs of different botanical origins. The presence of the characteristic GR protein (GRP) was shown using Western blot analyses and selected antibody enzyme immunoassay (SAEIA) methods. A GRP was isolated from one of the GR specimens which was selected using SAEIA methods. The isolated GRP was heated to reduce its binding activity to an anti-GR serum. A new fuzzy SAEIA method generally applicable for assay of the extract of the ten GR specimens was developed using heat-treated GRP as the solid-phase antigen. The fuzzy SAEIA method was successfully applied for the detection and quantitative analysis of the GR component contained in traditional Chinese medicines.

Animals↗
PubMed1997

Transcranial assessment of maternal cerebral blood flow velocity in patients with pre-eclampsia.

BACKGROUND: To clarify the cerebral hemodynamics in pre-eclamptic pregnant women, we investigated the blood flow velocity of the cerebral arteries. METHODS: The mean blood flow velocity and pulsatility index (PI) of the middle cerebral artery (MCA) and internal carotid artery (ICA) in normal pregnant women (n = 35) and pre-eclamptic patients (n = 18) were examined transcranially using pulsed-wave Doppler technique with a 2 MHz probe. In two pre-eclamptic women with post-partum visual disturbance, we examined the mean blood flow velocity and PI of the MCA and ICA every day. RESULTS: The mean blood flow velocity of the MCA in the pre-eclamptic patients (89.7 +/- 20.5 cm/s) was significantly higher than that in the normal pregnant women (53.6 +/- 16.9 cm/s) (p < 0.05). PI of the MCA in the former group (0.67 +/- 0.13) was significantly lower than that in the latter (0.78 +/- 0.02) (p < 0.05). There was no significant difference between these two groups in these variables of the ICA. In the two patients with visual disturbance, the mean blood flow velocity of the MCA was increased before the onset of visual disturbance and decreased gradually following the disappearance of this symptom. In these patients, spasm of the MCA was confirmed by magnetic resonance angiography. CONCLUSIONS: In pre-eclamptic patients, we found increased MCA mean velocity before the onset of visual disturbance. Transcranial Doppler may be useful for the evaluation of cerebral hemodynamics and the prediction of eclampsia.

Adult↗
PubMed1997

Loss of heterozygosity at loci on chromosome 4, a common genetic event during the spontaneous immortalization of mouse embryonic fibroblasts.

Spontaneously immortalized fibroblast cell lines derived from embryonic tissues of C3D2F1, mice were analyzed for loss of heterozygosity (LOH) at multiple chromosomal loci to identify candidate suppressor loci for immortalization. Among 47 simple sequence repeat (SSR) loci selected for screening, those on chromosome 4 exhibited an exceptionally high LDH incidence of up to 89%. Only four other chromosomes (8, 11, 12, and 18) showed LOH, with the highest incidence being 33%. To further localize candidate suppressor genes on mouse chromosome 4, detailed deletion mapping was performed with 18 cell lines and 14 SSR markers. The greatest LOH incidence (94%) was observed at the D4Mit14 locus located on distal chromosome 4, indicating that a major suppressor gene resides in this region. On the other hand, at the D4Mit77 locus, 30 cM proximal to the D4Mit14 locus, we found the SSR to be homozygously lost in 39% of the cell lines. Because the D4Mit77 is tightly linked to the tumor suppressor gene p16, for which homozygous deletion has been reported in various human tumor cell lines, we also examined our fibroblast cell lines for gross aberrations of the p16 gene by using the Southern blot method. The p16 gene was found to be homozygously deleted in 56% of the cell lines. Although this result implies that the p16 gene plays a role as a suppressor gene for immortalization, the combined incidence of LOH and homozygous deletion at the D4Mit77 locus was 72%, which is significantly lower than the observed incidence at the D4Mit14 locus. Consequently, we concluded that immortalization of mouse embryonic fibroblasts may involve more than one suppressor gene on chromosome 4.

Animals↗
PubMed1997

Effect of evodiamine on catecholamine secretion from bovine adrenal medulla.

The effect of evodiamine on catecholamine secretion from bovine adrenal medulla was investigated. Evodiamine, a bioactive component isolated from dry unripened fruit of Evodia rutaecarpa Bentham, was found to stimulate the secretion of catecholamine from perfused bovine adrenal medulla at a concentration of 10 microM and its effect persisted for at least 30 min. This stimulatory effect of evodiamine was abolished by omission of Ca2+ from the perfusion fluid. Evodiamine (0.1-10 microM) markedly enhanced the secretion of catecholamine from the adrenal medulla induced by acetylcholine (100 microM or high K+(56 mM). The secretion of catecholamine was promptly enhanced by acetylcholine or high K+, but returned to the control level on treatment for 20 min. However, when evodiamine was added to the perfusion fluid after acetylcholine or high K+ stimulation for 10 min, the secretion of catecholamine again increased greatly. These results indicate that evodiamine not only stimulated the secretion of catecholamine from bovine adrenal medulla but also reversed insensitivity of these cells to acetylcholine or high K+ stimulation.

Adrenal Medulla↗
PubMed1996

Induction of recombination activating gene expression in a human lymphoid progenitor cell line: requirement of two separate signals from stromal cells and cytokines.

The activation and expression of recombination activating genes (RAGs) plays critical roles in V(D)J gene recombination machinery and lymphocyte development. We showed that RAG gene expression was induced in freshly isolated human bone marrow cells and a human lymphoid progenitor cell line, FL8.2.4.4, by coculture on a monolayer of a murine bone marrow-derived stromal cell line, PA6, in the presence of a mixture of recombinant cytokines. The RAG transcripts were detected 12 hours after initiation of culture, and the increased level was sustained at 24 hours. Among recombinant cytokines, interleukin-3 (IL-3), IL-6, and IL-7, but not IL-2, IL-4, stem cell factor (SCF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) could induce RAG-1 activation in FL8.2.4.4 cells, and a significant synergistic effect between IL-3, IL-6, and IL-7 was observed. Using a double chamber culture technique, it was shown that a cognate interaction between FL8.2.4.4 cells and PA6 stromal cells was a prerequisite for RAG-1 activation. Furthermore, RAG-1 transcripts were induced in FL8.2.4.4 cells when they were cocultured on paraformaldehyde-fixed PA6 stromal cells in the presence of cytokines. These results suggest that two separate signals are both required for induction of RAG-1 activation in lymphoid progenitors; one from the cell surface molecule(s) on stromal cells, and the other from the recombinant cytokine(s). Finally, we showed that expression of RAG mRNA in FL8.2.4.4 cells was concomitant with induction of recombinase activity. This system may provide useful means for further understanding the mechanisms controlling RAG activation and lymphocyte development in the human system.

Bone Marrow Cells↗
PubMed1996

Chromatin structure and transcriptional regulation of human RAG-1 gene.

The recombination activating genes (RAGs) play a critical role in V(D)J recombination machinery and lymphocyte development. Their expression is strictly regulated during lymphocyte ontogeny, with expression being rapidly lost as the lymphoid precursors differentiate into their progeny. To elucidate molecular mechanisms of regulation of human RAG-1 gene expression, we examined a chromatin structure of a approximately 24-kb DNA segment adjacent to a human RAG-1 promoter region in various cell lines by analyzing DNase I hypersensitive (DNase I HS) sites. In a RAG-1-expressing human pre-B-cell line, at least four DNase I HS sites (HS1, HS2, HS3, and HS4) were identified. Among these HS sites, one HS site (HS1) was ubiquitously detected in all cell lines examined, but the other three HS sites (HS2, HS3, and HS4) were associated only with RAG-1-expressing lymphoid cell lines. Using transient expression assays, we showed that the 5' upstream region of the major transcription start site showed low but significant promoter activity and that a DNA segment within HS3 located in the promoter region was indispensable to its basal promoter activity. Importantly, this promoter region was shown to be active in both RAG-1-expressing and RAG-1-nonexpressing cell lines. These results suggest that alteration of chromatin structure in the promoter region, in addition to other control elements outside of the promoter region, is one of the mechanisms regulating tissue- and stage-specific expression of human RAG-1 gene.

B-Lymphocytes↗
PubMed1996

Angiotensin II as a stimulator of Na(+)-dependent Ca2+ efflux from freshly isolated adult rat cardiomyocytes.

In cardiac tissues, angiotensin II causes inotropic and chronotropic effects on the heart. It is indicated that the mechanism of the inotropic effect of angiotensin II is attributed to an increase in cytosolic free calcium ([Ca2+]i) in cardiomyocytes. However, increased [Ca2+]i should be restored to a physiological level because cumulative elevation in [Ca2+]i leads to irreversible injury in cardiomyocytes. Whereas it is known that angiotensin II causes the increase in [Ca2+]i in cardiac cells, little is known about the mechanisms of decrease in [Ca2+]i in cardiomyocytes upon angiotensin II stimulation. In the present study, we examined the effect of angiotensin II on Ca2+ efflux from freshly isolated adult rat cardiomyocytes. Angiotensin II stimulated the efflux of 45Ca2+ from the cells in a concentration-dependent manner (10(-7)-10(-5) M). The 45Ca2+ efflux from the cells was inhibited by type 1 angiotensin II receptor inhibitor. The angiotensin II-stimulated 45Ca2+ efflux was not affected by deprivation of the extracellular Ca2+, but was dependent on the presence of extracellular Na+. These results indicate that angiotensin II stimulates extracellular Na(+)-dependent 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on the plasma membrane type 1 angiotensin II receptors which may couple to Na+/Ca2+ exchange.

Angiotensin II↗
PubMed1996

Resonance Raman/absorption characterization of the oxo intermediates of cytochrome c oxidase generated in its reaction with hydrogen peroxide: pH and H2O2 concentration dependence.

Effects of pH and H2O2 concentration on the reaction of cytochrome c oxidase (CcO) with H2O2 were studied with the high-performance Raman/absorption simultaneous determination technique reported previously (Proshlyakov et al., 1996). This reaction generates two intermediates called 607- and 580-nm forms, and we found that they show the same oxygen-isotope-sensitive RR bands as those of the intermediates in O2 reduction by CcO. In transient absorption spectra obtained under single turnover conditions, the 607-nm form appeared as the primary intermediate and subsequently the 580-nm and resting forms, suggesting that H2O2 serves as an oxidant for the resting enzyme but as a reductant for both the 607- and 580-nm forms in the peroxide cycle. The rise rate of absorption at 607 nm was insensitive to the H2O/D2O exchange, but the decay was significantly slower in D2O than in H2O. With the microcirculating system, each intermediate was maintained at a constant level under steady-state conditions by supplying H2O2 continuously. In the pH range between 7.4 and 10.0, the population of the 607-nm form decreased at higher pH and at higher concentrations of H2O2. The Fe=O stretching (VFe=O) frequencies of the oxo heme of the 607-nm form, observed at 804/769 cm-1 for their H2(16)O2/H2(18)O2 derivatives, were unaltered in this pH range and exhibited a D2O/H2O shift even at pH 10.0. This indicates that the iron-bound oxygen is hydrogen-bonded to a distal residue in this pH range. When the 580-nm form is dominant under the nonsaturating level of H2O2, two other oxygen-isotope-sensitive Raman bands have been observed at 785/750 cm-1 and 355/340 cm-1 at neutral pH, but the former disappeared above pH 8.5 and the latter above pH 9.0 without significant changes of absorption spectra, suggesting the presence of two separate species in the name of the 580-nm form. However, under the saturating concentration of H2O2, these Raman bands were unaltered between pH 7.4 and 10.0. In contrast, in the absence of excess peroxide, no oxygen-isotope-sensitive RR bands were observed despite dominance of the 580-nm form. The disappearance of these Raman bands demonstrates the occurrence of oxygen exchange between the oxo heme and bulk water, whose rate surpasses the formation rate of the 580-nm form at alkaline pH and/or at low H2O2 concentration. Such an oxygen exchange did not take place in the 607-nm form. Under the identical experimental conditions for generating a particular steady state, the exchange of H2O with D2O caused significant depopulation of the 580-nm form and concomitant increase of the 607-nm form. This was satisfactorily interpreted in terms of the difference in the decay rate of the 607-nm form between H2O and D2O. Thus, the reduction of the 607-nm form to the 580-nm form is likely to be a key step of the redox-linked proton pumping in the O2 reduction.

Animals↗
PubMed1996

Lectin affinity electrophoretic demonstration of tissue specificity and malignant alteration of human alpha-fetoprotein isoforms produced in transgenic mice.

Transgenic mouse which produces human alpha-fetoprotein (AFP) ubiquitously was used to study carbohydrate structures of AFP produced by various tissues as well as that in the serum. A series of tissues from the transgenic mouse were cultured in vitro and the AFP produced was analyzed by affinity electrophoreses with 4 kinds of lectins. Variable electrophoretic profiles of them suggested that the fine structures of the carbohydrate of human AFPs expressed in the mouse tissues were different. The characterization of human AFP produced by mouse hepatoma which developed in offspring between the AFP transgenic mouse and hepatoma-developing transgenic mouse indicated that the hepatoma AFP was distinct from the liver AFP and that the malignancy-specific phenotypic alteration was common to human and mouse.

Animals↗
PubMed1996

Two amine oxidases from Aspergillus niger AKU 3302 contain topa quinone as the cofactor: unusual cofactor link to the glutamyl residue occurs only at one of the enzymes.

Amine oxidases (EC 1.4.3.6) from Aspergillus niger, AO-I (2 x 75 kDa) and AO-II (80 kDa), were examined to determine the cofactor structure. Inactivated with p-nitrophenylhydrazine, they showed absorption and fluorescence spectra similar to those published for other copper amine oxidases and to topa hydantoin p-nitrophenylhydrazone. After digestion by thermolysin and pronase, cofactor peptides were purified by HPLC and sequenced. For thermolytic peptides, a typical topa consensus sequence, Asn-X-Glu-Tyr, was obtained for AO-II, although in case of AO-I it overlapped with Val-Val-Ile-Glu-Pro-Tyr-Gly. For pronase peptides of AO-I, only the latter sequence was obtained. NMR and mass spectroscopy confirmed the residue X as topa p-nitrophenylhydrazone in AO-II and revealed the presence of a residue Z attached to the Glu in the peptide Val-Val-Ile-Glu(Z)-Pro of AO-I. This residue was separated from the peptide by hydrolysis and identified as a product derived from topa quinone. The data, together with amino-acid sequence of AO-I, confer strong evidence for topa quinone as the cofactor, bound in the typical consensus sequence. Raman spectra of the p-nitrophenylhydrazone derivative of AO-I and its pronase peptide showed essentially the same peaks matching to a model compound for topa p-nitrophenylhydrazone. However, there may exist an unusual ester link between the topa-404 and Glu-145 in the native enzyme.

Amine Oxidase (Copper-Containing)↗
PubMed1996

Activation of glutathione transferase P gene by lead requires glutathione transferase P enhancer I.

Glutathione transferase P (GST-P) is specifically induced in rat liver and kidney by lead cation. The increase of GST-P mRNA after lead administration is blocked by actinomycin D, suggesting that GST-P production by lead is regulated at the transcriptional level. To further determine which part of the flanking region of the GST-P gene has the lead-responsive cis-element in vivo, we utilized transgenic rats with five different constructs having GST-P and/or chloramphenicol acetyl-transferase coding sequence. We studied the effect of lead on these transgenic rats and on transfected NRK (normal rat kidney) cells and found that GST-P induction by lead is indeed regulated at the transcriptional level and that the GST-P enhancer I (GPEI) enhancer is an essential cis-element required for the activation of the GST-P gene by lead. GPEI consists of two AP-1 (c-Jun/c-Fos heterodimer) site-like sequences that are palindromically arranged and can bind AP-1, c-jun mRNA in the liver increased after lead administration and GST-P, and c-Jun had patchy expression in the same hepatocytes 24 h after lead exposure. These results suggest that activation of the GST-P gene by lead is mediated in major part by enhancer GPEI and that AP-1 may be involved at least partially. GPEI has been shown to have essential sequence information for the trans-activation of the GST-P gene during chemical hepatocarcinogenesis of the rat (Morimura, S., Suzuki, T., Hochi, S., Yuki, A., Nomura, K., Kitagawa, T., Nagatsu, I., Imagawa, M., and Muramatsu, M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2065-2068; Suzuki, T., Imagawa, M., Hirabayashi, M., Yuki, A., Hisatake, K., Nomura, K., Kitagawa, T., and Muramatsu, M. (1995) Cancer Res. 55, 2651-2655). The present study establishes that the same enhancer element does operate in the activation of the GST-P gene by lead regardless of the trans-activators involved.

Animals↗
PubMed1996

Microcirculating system for simultaneous determination of Raman and absorption spectra of enzymatic reaction intermediates and its application to the reaction of cytochrome c oxidase with hydrogen peroxide.

A new high-performance device for Raman/absorption simultaneous determination was developed. This was combined with a newly designed microcirculating system and was successfully applied to study intermediates in the reaction of bovine oxidized cytochrome c oxidase (CcO) with hydrogen peroxide under steady state conditions at ambient temperatures. Measurements with this device made it possible to correlate directly the species defined in terms of the visible absorption characteristics with specific Raman bands. The "607 nm" form of the enzyme obtained with H2(16)O2 gave an oxygen isotope sensitive band at 804 cm-1 (769 cm-1 with H2(18)O2) in the Soret excited resonance Raman (RR) spectrum. Its frequency and isotope frequency shifts are exactly the same as those observed previously with 607 nm excitation in nonsimultaneous measurements for the 607 nm form, for which the presence of an oxoiron heme was demonstrated. The so-called " 580 nm" form of the enzyme obtained with H2(16)O2 gave the main oxygen isotope sensitive band at 785 cm-1 (750 cm-1 with H2(18)O2) but appeared to consist of multiple species. This band was assigned to the FeIV = O stretching mode of ferryloxo heme on the basis of its isotopic frequency shift. Another oxygen isotope sensitive band was found at 355 cm-1 (340 cm-1 for H2(18)O2), similar to the case of dioxygen reaction. Temporal behavior of this band did not agree with either that of the 804 cm-1 band or that of the 785 cm-1 band but seemed to grow between the two species. The RR spectra in the higher frequency region of the 607 nm and 580 nm forms excited at 427 nm were quite alike and did not support the formation of a porphyrin pi-cation radical.

Animals↗