Coupling efficiency in light-driven hybrid P450BM3 and CYP119 enzymes.

The light-driven hybrid P450 enzyme approach utilizing the photochemical properties of a covalently attached Ru(II)-diimine photosensitizer was extended to the archaeal Sulfolobus acidocaldarius CYP119 enzyme leading to high photocatalytic activity in the hydroxylation of the chromogenic substrate, 11-nitrophenoxyundecanoic acid. The determined kcat was greater than those reported with various natural redox partners. In addition, the sacrificial electron donor, diethyldithiocarbamate, used in the photocatalytic reaction is shown to play a dual role. It acts as an efficient quencher of the Ru(II) excited state leading to a highly reducing species necessary to inject electrons into the heme. It is also known for its antioxidant properties and is shown herein to be a useful probe to determine coupling efficiency in the light-driven hybrid enzymes.

Ultraviolet irradiation of human skin causes functional vitamin A deficiency, preventable by all-trans retinoic acid pre-treatment.

We report here that ultraviolet irradiation substantially reduced the mRNA and protein of the two major nuclear retinoid receptors, RAR-gamma and RXR-alpha, in human skin in vivo. Pre-treatment with retinoic acid mitigated this loss of nuclear retinoid receptors. Ultraviolet irradiation caused a near-total loss of retinoic acid induction of two RAR/RXR target genes, cellular retinoic acid binding protein-II and RA 4-hydroxylase, but did not affect 1,25-dihydroxyvitamin D3 induction of the vitamin D receptor/RXR-regulated gene vitamin D 24-hydroxylase. In effect, ultraviolet irradiation causes a functional vitamin A deficiency that may have deleterious effects on skin function, contributing to skin photo-aging and carcinogenesis.

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.

Substrate specificity of the carbon monoxide-dependent cytochrome P-450 kinetics.

The substrate-dependent kinetics of the carbon monoxide-inhibited cytochrome P-450 activity and its light reversibility is reinvestigated in microsomal preparations. In order to find out whether the substrate specificity is mediated by an isoenzyme-specific binding of carbon monoxide with different dissociation constants an experimental design has been chosen where it could be established that essentially the same isoenzyme component was involved in two different monooxygenase reactions, i.e., the O-dealkylation of 7-ethoxycoumarin and the 7-hydroxylation of coumarin. The dissociation constant kD(CO) of the ferrous cytochrome P-450 carbon monoxide complex is 6-fold higher in the presence of 7-ethoxycoumarin than in the presence of coumarin. But the light-induced relative changes of the Warburg partition coefficient for the 7-ethoxycoumarin deethylation and for coumarin 7-hydroxylation do not differ remarkably from each other. These relative changes are shown to represent the ratio of the photoinduced rate constant to the spontaneous rate constant of the dissociation for the ferrous cytochrome P-450 carbon monoxide complex. The differences in the dissociation constants are assigned to substrate specific effects on the carbon monoxide binding, indicating a substrate-specific change of the free binding enthalpy for carbon monoxide.

Target inactivation analysis applied to determination of molecular weights of rat liver proteins in the purified state and in microsomal membranes.

In principle, target inactivation analysis provides a means of determining the molecular weights (Mr) and states of aggregation of proteins in native environments where they are functionally active. We applied this irradiation technique to the rat liver microsomal membrane proteins: cytochrome b5, epoxide hydrolase, flavin-containing monooxygenase, NADH-ferricyanide reductase, NADPH-cytochrome P-450 reductase, and seven different forms of cytochrome P-450. Catalytic activities, spectral analysis of prosthetic groups, and sodium dodecyl sulfate-polyacrylamide electrophoresis/peroxidase-coupled immunoblotting were used to estimate apparent Mr values in rat liver microsomal membranes. Except in one case (cytochrome P-450PCN-E), the estimated Mr corresponded most closely to that of a monomer. Purified cytochrome P-450PB-B, NADPH-cytochrome P-450 reductase and epoxide hydrolase were also subjected to target inactivation analysis, and the results also suggested monomeric structures for all three proteins under these conditions. However, previous hydrodynamic and gel-exclusion results clearly indicate that all three of these proteins are oligomeric under these conditions. The discrepancy between target inactivation Mr estimates and hydrodynamic results is attributed to a lack of energy transfer between monomeric units. Thus, while P-450PCN-E may be oligomeric in microsomal membranes, target inactivation analysis does not appear to give conclusive results regarding the states of aggregation of these microsomal proteins.

Effects of extremely low frequency electromagnetic field (ELF-EMF) on catalase, cytochrome P450 and nitric oxide synthase in erythro-leukemic cells.

AIMS: Extremely low frequency electromagnetic fields (ELF-EMFs) are widely employed in electrical appliances and different equipment such as television sets, mobile phones, computers and microwaves. The molecular mechanism through which ELF-EMFs can influence cellular behavior is still unclear. A hypothesis is that ELF-EMFs could interfere with chemical reactions involving free radical production. Under physiologic conditions, cells maintain redox balance through production of ROS/RNS and antioxidant molecules. The altered balance between ROS generation and elimination plays a critical role in a variety of pathologic conditions including neurodegenerative diseases, aging and cancer. Actually, there is a disagreement as to whether there is a causal or coincidental relationship between ELF-EMF exposure and leukemia development. Increased ROS levels have been observed in several hematopoietic malignancies including acute and chronic myeloid leukemias. MAIN METHODS: In our study, the effect of ELF-EMF exposure on catalase, cytochrome P450 and inducible nitric oxide synthase activity and their expression by Western blot analysis in myelogenous leukemia cell line K562 was evaluated. KEY FINDINGS: A significant modulation of iNOS, CAT and Cyt P450 protein expression was recorded as a result of ELF-EMF exposure in both phorbol 12-myristate 13-acetate (PMA)-stimulated and non-stimulated cell lines. Modulation in kinetic parameters of CAT, CYP-450 and iNOS enzymes in response to ELF-EMF indicates an interaction between the ELF-EMF and the enzymological system. SIGNIFICANCE: These new insights might be important in establishing a mechanistic framework at the molecular level within which the possible effects of ELF-EMF on health can be understood.

Cytochrome P450-catalyzed brassinosteroid pathway activation through synthesis of castasterone and brassinolide in Phaseolus vulgaris.

The last reaction in the biosynthesis of brassinolide has been examined enzymatically. A microsomal enzyme preparation from cultured cells of Phaseolus vulgaris catalyzed a conversion from castasterone to brassinolide, indicating that castasterone 6-oxidase (brassinolide synthase) is membrane associated. This enzyme preparation also catalyzed the conversions of 6-deoxocastasterone and typhasterol to castasterone which have been reported to be catalyzed by cytochrome P450s, CYP85A1 of tomato and CYP92A6 of pea, respectively. The activities of these enzymes require molecular oxygen as well as NADPH as a cofactor. The enzyme activities were strongly inhibited by carbon monoxide, an inhibitor of cytochrome P450, and this inhibition was recovered by blue light irradiation in the presence of oxygen. Commercial cytochrome P450 inhibitors including cytochrome c, SKF 525A, 1-aminobenzotriazole and ketoconazole also inhibited the enzyme activities. The present work presents unanimous enzymological evidence that cytochrome P450s are responsible for the synthesis of brassinolide from castasterone as well as of castasterone from typhasterol and 6-deoxocastasterone, which have been deemed activation steps of BRs.

Porphyrin-induced photodynamic cross-linking of hepatic heme-binding proteins.

Three types of hepatic proteins, a heme-binding Z protein, a mixture of the glutathione S-transferases and a cytochrome P450 isozyme, were shown to be susceptible to photodynamic cross-linking and loss in antigenicity by naturally occurring porphyrins. At 50 microM, uroporphyrin caused the most and protoporphyrin the least photodecomposition. Hemopexin, a specific serum heme carrier, was photodecomposed but no cross-linking was detected. Heme and scavengers of singlet oxygen partially prevented protein photodecomposition.

Photoreactive cardiolipin analogues.

New photoreactive analogues of cardiolipin have been chemically synthesized. Photoreactive aryl azido acyl groups were placed at two different locations within the cardiolipin molecule: at the 2-sn position of the 2-sn glycerol of cardiolipin; at the 2-sn position of the 3-sn-phosphatidyl group; or at both locations to provide a dual labeled analogue. Thus three different cardiolipin analogues distinguished by the positions of the aryl azido acyl groups were prepared. Two different aryl azido acyl groups were employed in the above syntheses and the site of acylation was stereospecifically identified using several phospholipids of known specificity for cardiolipin. Acylation of cardiolipin with the symmetrical anhydride of either acyl aryl azido fatty acid analogue, 2-(N-4-azido-2-nitrophenyl)beta-alanine or 12-(N-4-azido-2-nitrophenyl)aminododecanoic acid provided 1-(3-sn-phosphatidyl)-2-(acyl aryl azido)-3-(3-sn-phosphatidyl)-sn-glycerol. Acylation of monolysocardiolipin (1-(3-sn-phosphatidyl)-3-(1-acyl-2-lyso-glycero(3)phospho)-sn-glyce++ + rol provided two products. 1-(3-sn-phosphatidyl)-3-(1-acyl-2-(acyl aryl azido)-glycero(3)phospho)-sn-glycerol and the doubly labeled 1-(3-sn-phosphatidyl)-2-(acyl aryl azido)-3-(1-acyl-2-(acyl aryl azido)glycero(3)phospho)-sn-glycerol. These are the first reported photoreactive analogues for cardiolipin. The analogues were positive effectors for cytochrome P-450sec, and as shown by SDS-PAGE, they labeled the single subunit of cytochrome P-450sec and the smallest subunits of cytochrome c oxidase from beef heart.

Influence of gamma-rays on the mouse liver cytochrome P450 system and its modulation by phenothiazine drugs.

PURPOSE: Phenothiazine drugs have been found to sensitize hypoxic cancer cells while offering protection to normal cells. Since phenothiazines are known to induce the cytochrome P450 system, its radiomodulation by phenothiazines has been examined. MATERIALS AND METHODS: Mice were administered phenothiazines intraperitoneally and irradiated with different doses of gamma-rays at 1.38 Gy/min. The activities of NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase, the content of cytochrome P450 and b5, the extent of lipid peroxidation as well as the activities of LDH, XO, SOD, GST and DTD were determined in the liver. RESULTS: The levels of different components of the cytochrome P450 system and antioxidant enzymes were enhanced up to 5 Gy and decreased thereafter. However, a progressive increase was noticed in peroxidative damage and the activities of LDH and XO. Administration of phenothiazines enhanced the radiation effect on components of the cytochrome P450 system (except NADH-cytochrome b5 reductase) and the activities of SOD, GST and DTD. Concomitantly, phenothiazines inhibited lipid peroxidation, LDH and XO. CONCLUSIONS: Activation of the cytochrome P450 system by phenothiazines leading to the enhancement of antioxidant potential of animals and free-radical scavenging are attributes of the radioprotective action of phenothiazines.

Tris(2,2'-bipyridyl)ruthenium(II)-mediated photoinduced electron transfer of engineered cytochrome P450 1A2.

In this study, it is shown that an electron from photoreduced tris(2,2'-bipyridyl)Ru2+ ion reaches the haem iron of engineered wild-type cytochrome P450 1A2 (P450 1A2) with an electron transfer rate of 6.04 x 10(-3) min(-1). The electron transfer rate, 4.05 x 10(-2) min(-1), of a His163Glu mutant, which has a redox potential 40 mV lower than that of the wild type, is more than sixfold faster than that of the wild type. The photoinduced electron transfer rates of the present system are strongly influenced by detergents, cholic acid and Emulgen 913. We discuss the intermolecular and intramolecular electron transfer mechanism of the P450 1A2 system based on the kinetic data.

Green tea catechins protect rats from microwave-induced oxidative damage to heart tissue.

We investigated the effects of green tea catechin on oxidative damage in microwave-exposed rats. The microwave-exposed rats received one of three diets: catechin-free (MW-0C), 0.25% catechin (MW-0.25C), or 0.5% catechin (MW-0.5C). Rats were sacrificed 6 days after microwave irradiation (2.45 GHz, 15 minutes). Cytochrome P(450) levels in the MW-0C group was increased by 85% compared with normal, but was 11% and 14% lower in the MW-0.25C and MW-0.5C groups than in the MW-0C group. NADPH-cytochrome P(450) reductase activity in the MW-0C group was increased by 29%, compared with the normal group, but was significantly less in the MW-0.25C and MW-0.5C groups. Superoxide dismutase activity in the MW-0C group was decreased by 34%, compared with the normal group, but in the MW-0.25C and MW-0.5C groups was 19% and 25% higher. The activity of glutathione peroxidase in the MW-0C group was decreased by 28% but remained near normal with catechin supplements. Superoxide radical concentrations in the MW-0C group were increased by 35%, compared with the normal group. However, superoxide radicals in the MW-0.25C and MW-0.5C groups were 11% and 12% lower, respectively, compared with the MW-0C group. Microwave irradiation significantly increased levels of thiobarbituric acid-reactive substances, carbonyl values, and lipofuscin contents, but green tea catechin partially overcame the effects of the microwave irradiation. In conclusion, the mixed function oxidase system was activated, the formation of superoxide radical, lipid peroxide, oxidized protein, and lipofuscin was increased, and the antioxidative defense system was weakened in heart tissue of microwave-exposed rats, but the oxidative damage was significantly reduced by catechin supplementation.

[Effects of different doses of x-ray irradiation on the contents of microsomal hemoproteins in the rat liver]. / Vliianie rentgenovskogo oblucheniia v raznykh dozakh na soderzhanie mikrosomnykh gemoproteidov v pecheni krys.

The content of cytochromes P-450 and b5 in microsomal fraction of rat liver was studied at 1, 3, 6, 12 and 24 hours after X-irradiation with doses of 4, 8 and 12 Gy. It was found that post-irradiation changes in the cytochromes content were already observed in the first hours after X-irradiation independently of a dose of ionizing radiation.

[An EPR study of the joint action of sodium nitrite and whole-body irradiation on animal tissues]. / Issledovanie metodom EPR sovmestnogo deistviia nitrita natriia i obshchego oblucheniia na tkani zhivotnykh.

Changes in metabolic paramagnetic centers of the liver, kidney, spleen, brain and heart tissues, and blood after sodium nitrite intraperitoneal administration irradiation, or combined action of sodium nitrite and irradiation on mice, were studied using the EPR method. It was shown that irradiation of mice after sodium nitrite administration enhanced the effects induced by sodium nitrite alone: the levels of nitrosyl complexes Heme-NO in the tissues, cytochrome P-450 inhibition, and MetHb were higher.

[The characteristics of the effect of centimeter-range microwaves on drug pharmacokinetics in the body of experimental animals]. / Osobennosti vliianiia mikrovoln santimetrovogo diapazona na farmakokinetiku medikamentov v organizme éksperimental'nykh zhivotnykh.

The experiments on 130 rat males have shown that the exposure of the animals' liver to centimeter microwaves enhances catalytic activity of P-450p cytochrome, an enzyme of drug microsomal metabolism. However, changes in pharmacokinetic parameters of elimination from the blood of the drugs based on microsomal substrates of hepatic enzymatic system are more dependent on the microwaves' impact on physiological factors modifying drug pharmacokinetics. This implies the necessity of pharmacokinetic investigations in each individual case of combining drugs with microwaves.

Blue light-promoted rice leaf bending and unrolling are due to up-regulated brassinosteroid biosynthesis genes accompanied by accumulation of castasterone.

In this study the relationship between blue light- and brassinosteroid-enhanced leaf lamina bending and unrolling in rice was investigated. Twenty-four hours (h) irradiation with white or blue light increased endogenous brassinosteroid levels, especially those of typhasterol and castasterone, in aerial tissues of rice seedlings. There was an accompanying up-regulation of transcript levels of CYP85A1/OsDWARF, encoding an enzyme catalyzing C-6 oxidation, after 6h under either white or blue light. These effects were not observed in seedlings placed under far-red or red light regimes. It was concluded that blue light up-regulates the levels of several cytochrome P450 enzymes including CYP85A1, thereby promoting the synthesis of castasterone, a biologically active brassinosteroid in rice. Based on these findings, it is considered that blue light-mediated rice leaf bending and unrolling are consequences of the enhanced biosynthesis of endogenous castasterone. In contrast to aerial tissues, brassinosteroid synthesis in roots appeared to be negatively regulated by white, blue and red light but positively controlled by far-red light.

Effect of high-dose total body irradiation on ACTH, corticosterone, and catecholamines in the rat.

Total body irradiation (TBI) or partial body irradiation is a distinct risk of accidental, wartime, or terrorist events. Total body irradiation is also used as conditioning therapy before hematopoietic stem cell transplantation. This therapy can result in injury to multiple tissues and might result in death as a result of multiorgan failure. The hypothalamic-pituitary-adrenal (HPA) axis could play a causative role in those injuries, in addition to being activated under conditions of stress. In a rat model of TBI, we have established that radiation nephropathy is a significant lethal complication, which is caused by hypertension and uremia. The current study assessed HPA axis function in rats undergoing TBI. Using a head-shielded model of TBI, we found an enhanced response to corticotropin-releasing hormone (CRH) in vitro in pituitaries from irradiated compared with nonirradiated rats at both 8 and 70 days after 10-Gy single fraction TBI. At 70, but not 8 days, plasma adrenocorticotrophic hormone (ACTH) and corticosterone levels were increased significantly in irradiated compared with nonirradiated rats. Plasma aldosterone was not affected by TBI at either time point, whereas plasma renin activity was decreased in irradiated rats at 8 days. Basal and stimulated adrenal steroid synthesis in vitro was not affected by TBI. In addition, plasma epinephrine was decreased at 70 days after TBI. The hypothalamic expression of CRH messenger RNA (mRNA) and hippocampal expression of glucocorticoid receptor mRNA were unchanged by irradiation. We conclude that the hypertension of radiation nephropathy is not aldosterone or catecholamine-dependent but that there is an abscopal activation of the HPA axis after 10 Gy TBI. This activation was attributable at least partially to enhanced pituitary ACTH production.