5-hydroxytryptophan and soluble pigment formation in Claviceps sp. PRL 1980

5-Hydroxytryptophan (156 mg/l) was identified in 15-day-old cultures of Claviceps sp. PRL 1980. [side-chain 3-¹⁴C] dl-Tryptophan and [side-chain 3-¹⁴C] 5-hydroxytryptophan were incorporated into the brown pigment in cultures of the same fungus, 6% and 24%, respectively.     

The roles of cytochrome b5 in a reconstituted N-demethylase system containing cytochrome P-450.

Compound 102804 isolated from $\text{Bacillus cereus}$ has been found to be a potent inhibitor of the N⁵-methyltetrahydrofolate-homocysteine transmethylase isolated from $\text{Escherichia coli}$ B. This inhibition was noted when 102804 was added to the enzyme reaction mixture after the reaction started or concurrently with the preparation of the mixture. Chemically inactivated 102804 has no activity as an inhibitor of this enzyme system.     

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 $\text{b}$₅ in the $\text{p}$-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 $\text{b}$₅. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome $\text{b}$₅ reductase, and Triton X-100 in addition to cytochrome $\text{b}$₅. The omission of cytochrome $\text{b}$₅ from the complete system entirely abolished the activity. These results clearly show that cytochrome $\text{b}$₅ is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome $\text{b}$₅ in such a way that the second electron is transferred from cytochrome $\text{b}$₅ and thus exhibits the demethylase activity.     

Formation of clavicipitic acid in cell-free systems of Claviceps sp.

4-Dimethylallyltryptophan-[3-¹⁴C] was converted to clavicipitic acid in cell-free extracts from Claviceps sp. SD 58 and Claviceps purpurea PRL 1980. Activity was concentrated in the microsomal fraction. Oxygen was required but there was no cofactor requirement. p-(Hydroxymercuri)benzoic acid strongly inhibited the conversion. Addition of diethyldithiocarbamate increased conversion 2·5 ×. Conversion was favored at high pH. Clavicipitic acid [¹⁴C] added to cultures of Claviceps sp. SD 58 was not significantly incorporated into elymoclavine.     

Purification and some properties of cytochrome P-450 specific for steroid 17 alpha-hydroxylation and C17-C20 bond cleavage from guinea pig adrenal microsomes

The energy requirements for mitochondrial protein synthesis were investigated in isolated rat liver mitochondria. Controlled changes in coupling efficiency were obtained by titration with FCCP in the presence of various substrates. No relationship was observed between the efficiency of oxidative phosphorylation and the inhibition of protein synthesis. With succinate-ADP as the substrate the ADP:O ratio was decreased by 70–80% with no effect on protein synthesis. In contrast, with acetate-ADP as substrate, a 10–20% reduction in the ADP:O ratio gave complete inhibition of protein synthesis. The data suggest that the rate of ATP production is more important for maintenance of protein synthesis than the efficiency of coupling $\text{per se}$. Thus, certain substrates can support maximal rates of protein synthesis even in relatively poorly coupled mitochondria. Analysis of mitochondrial translation products formed in the presence of increasing FCCP concentrations also showed that decreased efficiency of oxidative phosphorylation had no influence on the nature of the products.     

Purification and characterization of liver cytochrome P-446 isolated from protein energy malnourished rats

A liver cytochrome P-450 isozyme has been purified to homogeneity from protein-energy malnourished rats induced with -naphthoflavone (-NF). The purification steps included chromatography on DEAE-Sephadex-A-25, DEAE-cellulose (DE-53), hydroxylapatite (HA) and carboxymethyl-sephadex (CM) columns. The reduced carbon monoxide difference and absolute spectra showed a Soret peak at 446.5 nm. The wavelength maxima for the oxidized and reduced spectra were at 416 and 408 nm, respectively. Cytochrome P-446 appears to have a predominantly low spin ferric iron, migrates as a single band of molecular weight 56000 in sodium dodecyl sulfate polyacrylamide gels and has a specific content of 14 nmol/mg of protein. P-446 oxidized various substrates at different rates in a reconstituted system with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine. In this system turnover rates for benzo[]pyrene, testosterone and benzphetamine oxidation were: 81.10; 1.85 and 1.42 nmoles product/min/nmol P-446 respectively. While NH₂ terminal amino acid sequence analysis of 18 of the first 20 residues suggests that the cytochrome P-446 isolated from malnourished rats is identical with form c, the catalytic activities suggest that this isozyme may be a more effective or efficient catalyst for some substrates.Abbreviations -NF -napthoflavone - SDS-PAGE Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis - 3-MC 3-Methyl Cholanthrene - PEG Poly Ethylene Glycol - DTT Dithiothreitol - PMSF Phenyl Methyl Sulfonylfluoride - EDTA disodium ethylenediaminetetraacetate - NADPH reduced nicotinamide adenine dinucleotide phosphate - P-450 cytochrome P450, PB-1, PB-4, PB-5 and P-450 isozymes purified from phenobarbital induced rat liver - HPLC High Pressure Liquid Chromatography - B[]P benzo[]pyrene - CM Carboxymethyl Sephadex - PTH-amino acid phenylthiohydantoin amino acid, Cytochrome P-450 EC 1.14.14.1, NADPH Cytochrome P-450 (c) reductase ED 1.6.2.4     

Altered activation/detoxication enzymology following neonatal diethylstilbestrol treatment

The effects of neonatal exposure to diethylstilbestrol (DES) on hepatic activation/detoxication enzyme levels in the adult rat were investigated. Neonatal exposure of male rats to DES (DES males) decreased the endogenous levels of UDP-glucuronyltransferase as compared to control males. Female rats exposed neonatally to DES (DES females) had higher endogenous epoxide hydrolase and glutathione transferase activity levels than control females. Adult animals treated neonatally with DES also had altered metabolic potential following exposure to 3-methylcholanthrene and phenobarbital. The DES males treated in adulthood with 3-methylcholanthrene had higher benzo(a)pyrene hydroxylase activities and lower UDP-glucuronyltransferase activity levels than did control males treated in adulthood with 3-methylcholanthrene. The DES males exposed in adulthood to phenobarbital had reduced cytochrome P-450 and glutathione transferase activity levels as compared with respective controls. The DES females treated in adulthood with 3-methylcholanthrene had lower benzo(a)pyrene hydroxylase and epoxide hydrolase activity levels than control females receiving 3-methylcholanthrene. The DES females challenged in adulthood with phenobarbital also had decreased benzo(a)pyrene hydroxylase, epoxide hydrolase, UDP- glucuronyltransferase, and glutathione transferase activity levels as compared with respective controls. Our results demonstrated that neonatal exposure to DES changed the endogenous levels of specific hepatic enzymes and altered the metabolic response of these adult animals to a carcinogen and a drug.     

Cytochrome P-450 inducer/inhibitor effects on cell cultures of Catharanthus roseus

Cytochrome P-450 inducers phenobarbitone and β-naphthoflavone and cytochrome P-450 inhibitor ketoconazole were examined for their effect on Catharanthus roseus. Treatment was during growth on medium M3 which supports alkaloid synthesis. The inhibitor ketoconazole was found to inhibit serpentine accumulation prior to an effect on growth, while the inducers inhibited growth and in the case of phenobarbitone increased serpentine accumulation. No direct evidence of induction or inhibition of plant cytochrome P-450 is shown and the results are discussed in relation to possible effects on geraniol hydroxylase and other plant cytochromes P-450.     

Irritant phorbol derivatives from four Jatropha species

Four Jatropha species used in folk medicine were screened for irritant constituents. By chromatographic and countercurrent distribution procedures, highly irritant factors were isolated from each species. They represent new polyunsaturated esters of the tigliane-type diterpenoids 16-hydroxyphorbol (J. podagrica, J. multifida) and 12-deoxy-16-hydroxyphorbol (J. curcas, J. gossypifolia).     

The Induction of Cytochrome P-450 and Ethanol Oxidation in Yarrowia lipolytica Cells

The study of the effect of different ethanol concentrations in the medium on the growth and activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. However, when the initial concentration of ethanol in the medium was 4 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.     

Induction of cytochrome P-450-dependent drug metabolism by juvenile hormone mimics in mammalian cell culture

The monooxygenase activity of fetal hepatocytes in culture shows a differential response toward juvenile hormone I and analogs. Juvenile hormone I, R-20458, and Methoprene increase the deethyiation of 7-ethoxyresorufin while not affecting or even inhibiting the N-demethylation of p-chloro-N-methylaniline. RO-203600, a 1,3-benzodioxole-containing analog, increases both the deethylase and the N-demethylase, whereas Hydroprene does not affect either activity. The inductive effect with juvenile hormone I is obtained with exposure periods of at least 30 min and is maximum when the concentration of the hormone is 14 μM in the medium. This amount results in the covalent binding to cellular macromolecules of 1.3 × 19^?18 moles/cell. The induction requires continuous protein synthesis but RNA synthesis only for a short initial period. It is concluded that juvenile hormone and mimics induce specific cytochrome P-450 species in fetal liver cells even if the culture conditions are not optimal. The toxicological implications of these results are briefly discussed.     

Induction of cytochrome P-450 isozymes by hexachlorobenzene in rats and aromatic hydrocarbon (Ah)—Responsive mice

Hexachlorobenzene (HCB) differs markedly from other chlorinated benzenes (CBs) as an inducer of cytochrome P-450 (P-450) isozymes as determined by radioimmunoassay and immunoblotting. At > 99% pure, HCB induced both the phenobarbital-inducible forms, cytochromes P-450b + e (70X), and the 3-methylcholanthrene-inducible forms, cytochromes P-450c (58X) and P-450d (8X), in rat liver microsomes. The concentration of P-450d was considerably greater than that of P-450c in HCB-induced rat liver. In contrast to HCB, all lower chlorinated benzenes tested were PB-type inducers. Hexachlorobenzene increased the amounts of translatable messenger RNAs (mRNAs) for P-450b, P-450c, and P-450d in rat liver polysomes, suggesting that it increases the synthesis of these proteins. Evidence that HCB interacted with the putative Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was equivocal. Western blots of liver microsomes from Ahresponsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice demonstrated that HCB produced a large increase in P₃-450 and a very small increase in P₁-450 in the responsive strain. The increase in P₁-450 was not observed after HCB administration to nonresponsive mice, but a small increase in P₃-450 was noted. These findings suggested that HCB may act through the Ah receptor. However, HCB was at best a very weak competitor for specific binding of [³H]-TCDD to the putative receptor in rat or mouse hepatic cytosol in vitro, producing decreases in binding of [³H]-TCDD only at very high concentrations (10^?6 to 10^?5 M).     

Flavonoids and tannins of Acacia species

The heartwoods of Acacia giraffae and A. galpinii were selected from South African Acacias as representative of those with abnormally high and minimal tannin contents respectively. A. galpinii contains amongst other analogues, the first natural (+)-2,3-trans-3,4-trans-teracacidin (7,8,4′-trihydroxy-flavan-3,4-diol and novel 3-O-methyl-, 7,8-di-O-methyl- and 7,8,4′-tri-O-methylflavonol analogues. (−)-2,3-cis-3,4-cis-Melacacidin (7,8,3′,4′-tetrahydroxyflavan-3,4-diol) is also present, but tannins are absent. By contrast, from the large excess of leueofisetinidin tannins which characterizes the wood of A. giraffae, only (+)-catechin, (+)-2,3-trans-3,4-trans-leucofisetinidin (7,3′,4′,trihydroxyflavan-3,4-diol and all-trans-(+)-leueofisetinidin-(+)-catechin could be isolated.     

Interactions of nitrogen heterocycles with cytochrome P-450 and monooxygenase activity

Three groups of isomeric nitrogen heterocycles, phenylpyridines, phenylimidazoles and pyridylimidazoles were studied in relation to the effect of steric factors on type II binding to cytochrome P-450 and inhibition of aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity in hepatic microsomes from phenobarbital(PB)- and β-naphthoflavone(βNF)-induced rats. Type II binding affinity was lower (higher K_s) in compounds with substituents on the carbon adjacent to the nitrogen undergoing ligand interaction than in those where steric hindrance near the nitrogen was minimal. Binding affinities of the compounds as measured by their K_s values, were quite similar in both PB- and βNF-induced microsomes. In PB-induced microsomes, type II binding affinity was generally reflected by the ability of the compounds to inhibit AHH activity. In contrast, most of the compounds evaluated were inactive as AHH inhibitors in βNF-induced microsomes.     

Floral anthocyanins of some malesian Hibiscus species

In 14 Malesian species of Hibiscus (sensu lato) the most common floral anthocyanin was cyanidin 3-sambubioside. Cyanidin 3-glucoside was found     

Enantioselectivity in the Rabbit Liver Microsomal Biotransformation of Styrene and Phenylpropenes

The rabbit liver microsomal P-450 catalyzed oxidation of styrene (1a) and isomeric phenylpropenes, trans-1-phenylpropene (1b), cis-1-phenylpropene (1c) and 3-phenylpropene (1d), was investigated and the enantioselectivity of the epoxidation of the olefinic double bond was determined by checking the enantiomeric excesses of the corresponding first formed epoxides (2). These enantiomeric excesses were always modest, ranging between 7% of (1S,2S)-(2b) and 22% of (1R,2R)-(2c). In the case of (1d) a nonenantioselective hydroxylation at the benzylic-allylic C(3) was also oberved. The ratio between this hydroxylation and olefin epoxidation of (Id) was 1:2.