Toshio Yamano

Reconversion of detergent- and sulfhydryl reagent-produced P-420 to P-450 by polyols and glutathione.

Abstract After treatment with detergents or sulfhydryl reagents, the cytochrome pigment P-420 can be converted back to P-450 by treatment with polyols or reduced glutathione. This reconversion of P-420 to P-450 is highly dependent on temperature, pH, concentration of sodium cholate and incubation time, but ionic strength was of little influence. However, after treatment with alcohols or ketones, P-420 could no longer be reconverted to P-450 by polyols and reduced glutathione. The presence of 20% (v/v) glycerol stabilizes P-450 for more than 1 week.

Aldosterone biosynthesis by a reconstituted cytochrome P-45011β system

Abstract [ 3 H]Corticosterone was incubated with cytochrome P-450 11β purified to electrophoretic homogeneity from bovine adrenocortical mitochondria, and the reaction products were analyzed by high performance liquid chromatography. The production of aldosterone (21.2 pmol/nmol P-450/min) and 18-hydroxycorticosterone (1.17 nmol/nmol P-450/min) was observed. When lipidic extracts from mitochondria of bovine adrenocortical zona glomerulosa were added to the reaction mixture, the rate of production of aldosterone was increased 28-fold. When [ 3 H]18-hydroxycorticosterone was incubated with cytochrome P-450 11β , the amount of aldosterone produced was 55.7 pmol/nmol P-450/min in the absence of the lipidic extracts and the enhancing effect of the lipidic extracts was 4-fold.

Electron spin resonance of microsomal cytochromes: Correlation of the amount of CO-binding species with so-called microsomal Fex in microsomes of normal tissues and liver microsomes of Sudan III-treated animals

Abstract A correlation has been established on intact microsomes of various tissues between the amount of CO-binding pigment (P-450) and microsomal Fex, which was designated as a component of rabbit liver microsomes with an ESR signal of low-spin type hemoprotein. This correlation also holds with vertebrate microsomes in which the enzyme contents were raised by injection of Sudan III into the animals. The correlation seen with intact microsomes is no longer observed after treatment with deoxycholate or alkaline, which readily destroys P-450. In the microsomes of bovine adrenal medulla a hemoprotein exists with an α-band at 559 mμ but without an ESR signal or CO-binding character.

The obligatory requirement of cytochrome b5 in the p-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome b5

The role of cytochrome b5 in the p-nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome b5. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome b5 reductase, and Triton X-100 in addition to cytochrome b5. The omission of cytochrome b5 from the complete system entirely abolished the activity. These results clearly show that cytochrome b5 is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome b5 in such a way that the second electron is transferred from cytochrome b5 and thus exhibits the demethylase activity.

Purification and crystallization of NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria

Flavoprotein and iron sulfur protein as components of an electron transport system have been shown to form a complex under certain conditions [ 1,2]. Chu and Kimura [3] demonstrated that the complex formation between adrenodoxin reductase and adrenodoxin depends on the ionic strength of the environment. We have succeeded in showing the complex formation of adrenodoxin reductase with immobilized adrenodoxin on Sepharose gel and achieving a high purification in one step. By the novel purification procedure including the affinity chromatography, the enzyme was crystallized as rhombic plates with a yield of 24% and 200-fold purification from the crude extract.

Relationship between the interconversion of cytochrome P-450 and P-420 and its activities in hydroxylations and demethylations by P-450 oxidase systems

Abstract The relationship between the interconversion of microsomal cytochrome P-450 and P-420 and the hydroxylation and demethylation activities of the P-450 oxidase system was studied. The hydroxylation and demethylation activities could only be partly restored by complete reconversion of P-420 to P-450 by addition of polyols by dilution or by washing. The P-420 produced by detergents, monohydric alcohols or anilines did not retain hydroxylation and demethylation activities. Cytochrome P-450 is not rate limiting in the overall reaction for hydroxylation and demethylation of compounds such as anilines, nitroanisoles and aminopyrine. The substrate specificities of the hydroxylations and demethylations by the P-450 oxidase systems of different tissues differ. The rate of hydroxylation of anilines at the p-position was influenced by not only the hydrophobic character, but also by electronic and steric effects of the substrate. The magnitude of the spectral changes of the microsomes induced by the substrate corresponded to the ESR signal height at gm = 2.25 of microsomal Fex rather than to the absorption peak of the CO complex of P-450 at 450 mμ. The hydroxylation and demethylation activities were observed with either NADPH or NADH. Evidence is presented that microsomal NADPH-cytochrome c reductase is reduced by NADH and the Km for the reductase with NADH is greater than the Km for NADPH.

Localization of D-amino acid oxidase in Bergmann glial cells and astrocytes of rat cerebellum.

The localization of D-amino acid oxidase in rat cerebellum was systematically studied in serial fixed sections at the levels of both light and electron microscopy using a coupled peroxidation method based on the intensifying effect of nickel ions. Deposits were only seen in astrocytes and Bergmann glial cells, and not in neuronal components, endothelial cells or ependymal cells. In the molecular layer, heavy deposits were present in the profiles of Bergmann glial processes around the complexes of synapses where the parallel fiber varicosities form synapses with the thorns emerging from the spiny branchlets of Purkinje cell dendrites. In the Purkinje cell layer, the oxidase-containing processes of Bergmann glial cells enveloped basket cell axons, their terminals, the terminals of the recurrent collaterals of Purkinje cell axons and Purkinje cell bodies. In the granular layer, the cerebellar glomeruli were enveloped by the heavily stained processes of astrocytes. Based on this characteristic localization of the oxidase, we discussed the physiological role of the oxidase in connection with the function of glial cells.

The electron spin resonance and absorption spectra of microsomal cytochrome P-450 and its isocyanide complexes

Abstract The absorption spectra of oxidized P-450-isocyanide complexes were the same in difference spectra irrespective of the isocyanide derivative tested. However, with these reduced P-450-isocyanide complexes, absorption at 455 mμ increased, and that at 430 mμ decreased, with increasing carbon atom number of the isocyanide derivative at a definite pH. The same changes were seen with individual complexes with increasing pH. The dissociation constants of oxidized P-450-isocyanide complexes decreased with increase in carbon atom number of the isocyanide. These results were confirmed by electron spin resonance (ESR) spectroscopy. However, the dissociation constants of reduced P-450-isocyanide complexes were essentially identical and the dissociation constants of the oxidized and reduced P-450-isocyanide complexes were little affected by pH. The oxidized P-450-isocyanide complexes gave magnetically specific ESR signals. The orbital energy differences of d e orbitals of the heme iron of the complexes increased with increase in the carbon atom number of the isocyanide. Purified P-450 and its isocyanide complexes were rapidly reduced by a ferredoxin-NADP + reductase system.

The role of the hydrophobic bonding in P-450 and the effect of organic compounds on the conversion of P-450 to P-420

Abstract The conversion of cytochrome P-450 to P-420 by organic solvents was studied. The transition concentrations of ureas used for conversion of P-450 to P-420 were, in decreasing order: urea, N-methylurea, 1,1-dimethylurea, ethylurea, tetramethylurea, 1,3-diethylurea. Similar results were obtained with amides. The transition concentrations of anilines and phenols closely paralleled the π values of the organic compounds. To maintain the structure which gives P-450 its unusual properties in comparison with other known hemoproteins, the hydrophobic bonding in P-450 appears to play an important role in that it binds the heme plane to the apoprotein.

Immunochemical relatedness between secretory phospholipase A2 and intracellular·phospholipase A2

The immunochemical relationship between rat pancreatic phospholipase A2 and rat splenic phospholipase A2 was examined with the use of anti-rat pancreatic phospholipase A2 antibody as a probe. The immunoelectrophoretic patterns showed that the antibody cross-reacted with the splenic enzyme. The immuno-crossreactivity was also shown by counter immunoelectrophoresis. The splenic phospholipase A2, whether it was purified from the cytosolic fraction or the microsomal fraction, formed an immunoprecipitin band with the anti-pancreatic phospholipase A2 antibody. The antibody was shown to inhibit the activity of the pancreatic phospholipase A2 as well as that of the splenic phospholipase A2.