Retsu Miura

NADPH-dependent melanin pigment formation from 5-hydroxyindolealkylamines by hepatic and cerebral microsomes

Abstract Incubation of 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxytryptophol and 5-hydroxyindoleacetic acid with rat liver or brain microsomes in the presence of an NADPH-generating system and molecular oxygen caused the formation of microsomal protein-bound melanin-like pigment. The free radical property of this pigment was proved by electron spin resonance spectroscopy. The formation of the pigment was inhibited by superoxide dismutase which indicates the participation of superoxide anion radical in this type of melanogenesis.

A reversible spin conversion of cytochrome b5 at high temperatures

Summary The thermodynamics for the reversible spin conversion of cytochrome b 5 have been studied by absorption and circular dichroic spectroscopy in the temperature range from 15 to as high as 83°C. The optical absorption spectrum of the oxidized cytochrome b 5 below 45°C, predominantly of low-spin type, changes to an essentially high-spin type on increasing the temperature. This spectral transition of cytochrome b 5 was found to take place in a relatively limited range from 45°C to 65°C. The circular dichroic spectrum of cytochrome b 5 at high temperatures is that of partially unfolded polypeptide with an exposed heme moiety. All these spectral changes were reversible.

Microsomal methoxymelanin formation from 6-hydroxymelatonin

In [l] we demonstrated that the superoxide anion (0;)dependent formation of microsomal proteinbound rnetabolite(s) of S-HT or S-HTP was accompanied by the consumption of NADPH and molecular oxygen. In addition to 5hydroxyindoles, 6-HT was found to be as active as 5-HT in stimulating the microsomal NADPH oxidase activity and oxygen consumption [l]. ln [2], we reported the Oi-dependent microsomal melanogenesis from 5-HT and related 5hydroxyindoles by demonstrating a free radical signal derived from a protein-bound metabolite(s) of 5-hydroxyindoles. The formation of 5-methoxyd-hydroxyindole from 5,6-DHI mainly by the action of HIOMT may not be important in melanogenesis whereas 5-hydroxy6-methoxyindoles derived from 5,6-DHI by the action of COMT can be converted spontaneously to’methylated melanins [3]. Here, we present data showing that 6-HM is a good substrate for the formation of methylated melanin, when incubated with microsomes in the presence of an NADPH-generating system and molecular oxygen. Thus, the position of a free hydroxylgroup at either 5 or 6 on the benzene ring of the indole nucleus is not of major importance, at least, in the O;-dependent microsomal melanogenesis.

Bovine adrenal cytochrome P-450(11β)-mediated conversion of 11-deoxycortisol to 18- and 19-hydroxy derivatives; structural analysis by 1H-NMR

Incubation of 11-deoxycortisol with a cytochrome P-450(11 beta)-reconstituted system yielded, in addition to cortisol, several new steroid products. In this study, structures of the three steroid products were elucidated. Retention time of the first product (Peak 2 substance) coincided with that of authentic 18-hydroxycortisol on reverse phase HPLC. To further confirm the chemical identity of this product, the purified sample was subjected to 1H-NMR analysis. The spectrum was essentially identical to that of 18-hydroxycortisol. The retention time of the second product (Peak 3 substance) did not coincide with those of commonly occurring steroids. The one- and two-dimension 1H-NMR spectra provided strong evidence for its structure of 19-hydroxy-11-deoxycortisol. The retention time of the third product (Peak 4 substance) did not coincide with those of commonly occurring steroids. The 1H-NMR spectrum showed the presence of signals of 19-CH3 and 18-CH2 protons. There was also evidence that this product is not hydroxylated at the 11-position. Further analysis of the COSY spectra identified its structure as 18-hydroxy-11-deoxycortisol. From these results, we conclude that bovine P-450(11 beta) can catalyze the hydroxylation of 11-deoxycortisol at 11 beta-, 18- and 19-positions and produce cortisol, 18-hydroxy-11-deoxycortisol, 18-hydroxycortisol and 19-hydroxy-11-deoxycortisol.

Purification and properties of cytochrome P-450 from adrenocortical mitochondria and its interaction with adrenodoxin.

Cytochrome P-450s from mammalian sources have been purified to a homogeneous state by several groups, and highly purified cytochrome P-450 from bacterial origin has been obtained In a crystalline form. Among the mammalian P-450s, the one from adrenal mitochondria (Harding et al., 1964) is the easiest to solubilize and the hydroxylating system can be reconstituted with purified components.

Rate enhancement of the electron transfer of the adrenodoxin‐adrenodoxin reductase system by inorganic and nucleotide phosphates

Phosphate and pyrophosphate increased the rate of reduction of adrenodoxin by NADPH‐adrenodoxin reductase and NADPH, pyrophosphate being one order more effective than the former. However, the cytochrome c reduction by the electron transport system was inhibited in the presence of inorganic (pyro)ptiosphate. On the other hand, ADP and ATP enhanced the rates of reduction of both adrenodoxin and cytochrome c through adrenodoxin by the electron transport system. GTP also enhanced the rate of reduction of cytochrome c by this system, whereas AMP showed no appreciable enhancement. These inorganic and nucleotide phosphates did not affect the rate of ferricyanide reduction by the reductase.

Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by dicarboxylic acids

The rate of electron transport in the cytochrome P-450 system in adrenocortical mitochondria was studied with purified adrenodoxin reductase, adrenodoxin and cytochrome c. Oxaloacetate enhanced the rate at concentrations of less than 1 mM; malate, succinate and fumarate enhanced the rate to a lesser extent; and pyruvate and alpha-ketoglutarate had no appreciable effect. The rate enhancement was observed when the reagents were preincubated with adrenodoxin, but not with adrenodoxin reductase. Rate enhancement was also evident when the rate limiting step was at adrenodoxin in the electron transport system.

An NADPH-induced change in lipid bilayer of rat liver microsomes as observed by spin-labeled phosphatidylcholine

Abstract Lipid bilayer of rat liver microsomes was spin-labeled by incubating with liposomes of 1-acyl 2-(12-doxylstearoyl) glycero-3-phosphorylcholine. When NADPH was added to the labeled microsomes, there appeared a rapidly tumbling component of spin label in the EPR spectrum. NADH was less effective than NADPH. The appearance of the sharp signal was prevented under anaerobic conditions or in the presence of either carbon monoxide, phenyl isocyanide or cytochrome c . The appearance of the rapidly tumbling component in the EPR spectrum was found to be due to the release of spin moiety from the membrane into the aqueous phase. That the release was associated with superoxide anion formation or with lipid peroxidation is unlikely, since 1) superoxide dismutase had little effect, 2) addition of either α-tocopherol or EDTA did not inhibit the release. These observations suggest that electron transfer from NADPH to oxygen via cytochrome P-450 system induces a physical perturbation in the lipid bilayer resulting in the release of its component into the aqueous phase.