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Biomedical subjects

Y Ishimura

Publications and source records attributed to Y Ishimura.

At least 163 records · Page 9Linked to original sources
PubMed1983

Proton coupling in the ligand-binding reaction of ferric cytochrome P-450 from Pseudomonas putida.

Effects of pH on the ligand-binding reactions of ferric heme in cytochrome P-450 from Pseudomonas putida (camphor 5-monooxygenase, EC 1.14.15.1) were studied by using cyanide, N-methylimidazole, pyridine, and ethylisocyanide as ligands. In all cases, affinity of the ferric heme for the ligand was found to increase as pH of the medium was raised from around 6 to 9. Depending on the ligand, the increase was 10- to 1000-fold and the shapes of their pH-affinity curves were remarkably different. Analyses such pH profiles disclosed the presence of a dissociable group in the enzyme with a pK value of approximately 9.5 and that its ionization greatly enhanced the affinity of the heme for ligands. When a dissociable ligand such as hydrogen cyanide and N-methylimidazole was used, the dissociated form of the ligand had a higher affinity toward the heme than the undissociated form. The shapes of the pH-affinity curves were successfully simulated as overlapping curves of ionization reactions of the ligand and the dissociable group. In addition, size of the ligand molecule was shown to be also important in the binding reaction: relatively large molecules such as pyridine, ethylisocyanide, and N-methylimidazole bound to the enzyme in a competitive manner against d-camphor concentration, whereas the binding of a smaller molecule such as cyanide was inhibited by the substrate in a noncompetitive manner. On the basis of these findings, control mechanisms for the ligand-binding reactions of the cytochrome P-450 from P. putida are discussed.

Bacterial Proteins↗
PubMed1983

Magnetic and natural circular dichroism of L-tryptophan 2,3-dioxygenases and indoleamine 2,3-dioxygenase. I. Spectra of ferric and ferrous high spin forms.

The ferric form of L-tryptophan 2,3-dioxygenases from both Pseudomonas acidovorans (ATCC 11299b) and rat liver showed magnetic CD spectra ascribable to a high spin protohemoprotein at neutral pH, whereas the ferric indoleamine 2,3-dioxygenase from rabbit intestine exhibited a spectrum due to a mixture of high and low spin states under comparable conditions. Upon addition of L-tryptophan, the spectra of the former enzymes changed to another type of high spin spectra, while the latter showed a marked increase in the low spin component. From these findings and effects of pH on the spectra, it is suggested that the sixth ligand of ferric L-tryptophan 2,3-dioxygenases is water at a neutral pH and that for the ferric indoleamine 2,3-dioxygenase is a strong field ligand such as an imidazole nitrogen. The mixed spin state observed for the latter enzyme was ascribable to a thermal equilibrium between high and low spin states as judged by low temperature spectroscopy. With the ferrous form, the Soret magnetic CD spectra of these enzymes were all similar, giving those of a typical high spin ferrous protohemoprotein, whereas visible spectra were different from one another, suggesting differences in the electronic structure of the heme and its vicinity. The natural CD spectra of both ferric and ferrous forms of each enzyme showed negative Cotton effects in the Soret region. Their intensities were different from one another, presumably due to some differences in the interaction of the heme with nearby aromatic amino acid residue(s).

Animals↗
PubMed1982

Regulation of cytochrome P450 activities in adrenocortical mitochondria from normal rats and human neoplastic tissues.

From our immunocytochemical studies, cytochrome P450scc and P45011 beta systems were localized on the matrix side of inner membrane of the mitochondria in the parenchymal cells of adrenal cortex. However, the degree of immunocytochemical staining varied from one mitochondrion to another within a single cell; some stained intensely along the entire inner membrane, some stained only along segments of the inner membrane, and some did not stain at all. The ratio of stained to unstained mitochondria was approximately unity in untreated rats, while stained mitochondria greatly increased upon ACTH administration suggesting that the population of cytochrome P450 system-containing mitochondria increased upon long-term ACTH action. By the combined use of flash photolysis and substrate difference spectroscopy, quantitative determination of substrate-bound and free forms of P450scc and P45011 beta in mitochondria became possible. Increases in total amounts of P450scc and cholesterol resulted from the long-term ACTH administration to rats. The ratio of cholesterol-bound P450scc to its free form was essentially unchanged under these conditions. On the other hand, amounts of P45011 beta were not increased significantly by long-term ACTH administration. The ratio of DOC-bound P45011 beta to the free form increased significantly, however, by long-term ACTH administration and also in the mitochondria from patients suffering from Cushing's syndrome.

Adrenal Cortex↗
PubMed1982

Cytochrome P-45011 beta and P-450scc in adrenal cortex: zonal distribution and intramitochondrial localization by the horseradish peroxidase-labeled antibody method.

In an attempt to elucidate the regulation mechanism(s) of adrenocortical steroidogenesis, cytochrome P-450scc and cytochrome P-45011 beta were localized in bovine adrenal glands by the direct peroxidase-labeled antibody method. At the light microscopic level, parenchymal cells of the zona fasciculata and the zona reticularis stained heavily for both cytochromes, while the parenchymal cells of zona glomerulosa stained lightly for both. At the electron microscopic level, these two cytochromes were associated with the matrix side of the inner mitochondrial membranes of parenchymal cells from all three zones of the adrenal cortex. The association of cytochrome P-450 with the inner mitochondrial membrane, in a manner similar to that previously reported for adrenodoxin and adrenodoxin reductase (F Mitani, Y Ishimura, S Izumi, K Watanabe, Acta Endocrinol 90:317, 1979), establishes that the steroid monooxygenase systems exist at this site. The degree of immunocytochemical staining within a single cell varied from one mitochondrion to another: some stained intensely along the entire inner membrane, including the cristae, some stained only along segments of the inner membrane, and some did not stain at all. This heterogeneity in staining was observed in mitochondria stained in situ as well as in isolated mitochondria. These findings suggest that there is a heterogeneity in steroidogenesis among mitochondria contained within a single cell of the adrenal cortex.

Adrenal Cortex↗
PubMed1981

Magnetic circular dichroism studies of cytochrome P-450cam. Characterization of axial ligands of ferric and ferrous low-spin complexes.

MCD was applied to ferric and ferrous low-spin complexes of cytochrome P-450 cam to elucidate the electronic states and the nature of the axial ligands of the heme in cytochrome P-450cam. (1) Low-spin complexes of ferric cytochrome P-450cam, produced either by ligation of external ligands such as pyridine and imidazole derivatives or by being freed of (-)-camphor, showed sinusoidal Soret and alpha-MCD bands. The magnitude ratio of the Soret vs. alpha-MCD bands was quite sensitive to the nature of axial ligands of the ferric low-spin complexes. The ratio (2.7) for the camphor-free form of cytochrome P-450cam, thus, was the smallest among those (2.7-9.0) for low-spin forms of cytochrome P-450cam and other corresponding low-spin hemoproteins (ratio 7.8-13.9). The ratio (4.2) for the alpha-picoline-bound form of cytochrome P-450cam, however, was the closest to that (2.7) for the camphor-free form of cytochrome P-450cam among those (4.2-9.0) for the external ligand-bound form of cytochrome P-450cam. The ratio for the 2-methylimidazole-bound form of cytochrome P-450cam was the smallest among those of cytochrome P-450cam bound with imidizole derivatives. Thus, among the nitrogen-bound low-spin forms, the low-spin form with a sterically hindered nitrogen ligand trans to the thiolate anion (-S-) most reproduced spectral characteristics of the native low-spin ferric form. Low-temperature absorption studies offered the same results. (2) It was found that MCD magnitudes of alpha-bands of ferrous low-spin complexes are intimately related to the electronic character of axial ligands. Thus, the CO, O2 and NO-bound forms of cytochrome P-450cam, which have two pi-type axial ligands, showed the smallest alpha-MCD bands ([theta]M = 5.2-7.5) among complexes, while ferrous cytochrome b5 and cytochrome c, which have two sigma-electron-donating axial ligands, showed the largest magnitude ([theta]M = 120-176). The data for the ferrous low-spin complexes of other hemoproteins so far available were well rationalized in consideration of the property of the axial ligands.

Animals↗
PubMed1981

Resonance Raman spectra of bovine adrenal cytochrome P-450SCC.

Resonance Raman spectra were observed for a mitochondria-type cytochrome p-450 (P-450SCC) for the first time. Reduced P-450SCC at pH 7.4 exhibited the V4 line at 1342 cm-1, which is an unusually low frequency compared with an ordinary protohemoprotein but is common to the family of cytochrome P-450, suggesting the coordination of a strong pi-donor such as thiolate anion at the fifth coordination position of the heme iron. The anomaly was preserved for the CO-complex of the reduced form. The V10 line of oxidized P-450SCC with a substrate was observed at 1617 cm-1. This frequency and those of other structure-sensitive bands implied that the heme iron of oxidized P-450SCC adopts the hexa-coordinate high-spin structure, in contrast with the high-spin type cytochrome P-450 purified from phenobarbital- or 3-methylcholanthrene-treated rabbit liver microsomes which presumably have a penta-coordinate structure. In the presence of 20alpha-hydroxycholesterol, oxidized P-450SCC gave the V10 line at 1637 cm-1, i.e., at a frequency similar to that of low-spin type cytochrome P-450. The alkaline-denatured P-420SCC preparation in the presence of both dithiothreitol and EDTA, but not the P-450SCC gave the V10 line at 1637 cm-1, i.e., at a frequency similar to that of low-spin type cytochrome P-450. The alkaline-denatured P-420SCC preparation in the presence of both dithiothreitol and EDTA, but not the P-450SCC.

Adrenal Cortex↗
PubMed1981

Magnetic and natural circular dichroism spectra of cytochgromes P-450(11) beta and P-450scc purified from bovine adrenal cortex.

Magnetic (MCD) and natural circular dichroism (CD) spectra various complexes of cytochrome P-450(11) beta (P-450(11) beta) and cytochrome P-450scc (P-450scc) from bovine adrenal cortex were measured from 250 nm to 700 nm. MCD and CD spectral contours of cytochromes P-450(11) beta and P-450scc in the Soret and visible regions were, as a whole, analogous to those of cytochromes P-450 from rabbit liver microsomes and also from Pseudomonas putida in their high-spin ferric, high-spin ferrous and ferrous-CO complexes. MCD spectrum of the low-spin ferric P-450scc free from the substrate, cholesterol, was very similar to that caused by addition of 20 alpha-hydroxycholesterol, a reaction intermediate. However, it was distinct from those of the low-spin ferric P-450(11) beta and P-450scc complexes caused by addition of external nitrogenous ligands. The electronic states of the heme in the low-spin ferric P-450 free from substrates seemed to be subtly different from those of low-spin complexes coordinated with external nitrogenous ligands. Soret CD spectra of ferric low-spin complexes were not so different from each other. Upon reduction of high-spin ferric P-450(11) beta or P-450scc, the Soret CD magnitudes increased significantly in contrast with those of other P-450s, the Soret CD magnitudes of which decrease upon reduction. This may reflect an increased proximity of the neighbouring aromatic groups upon reduction of high-spin P-450(11) beta or P-450scc. High substrate specificity of adrenal P-450s compared with liver P-450s can be explained in view of the above findings. THe CD spectra in the near ultraviolet region (250-350 nm) were found to be quite sensitive to the spin change for ferric P-450scc, while the MCD spectra in this region did not reflect substantially the spin state of the enzyme. MCD parameters of cytochrome P-450s were compared to those of other hemoproteins in diagrams describing selected MCD spectral values of hemoproteins so far available and were discussed in connection with the structures of the heme environment of P-450.

Adrenal Cortex↗