Photoinhibition in Chilling Stressed Leguminosae: Comparison of Vicia Faba and Pisum Sativum

The concentrations of photosynthetic pigments decreased in both chilling stressed species but the ratios of chlorophyll (Chl) a/b and total carotenoids (Car)/Chls were depressed only in faba bean. The contents of + carotene and lutein+lutein-5,6-epoxide remained unaffected in both species, but the de-epoxidation state involving the components of xanthophyll cycle increased in pea. Under chilling stress the photosynthetic electron transport associated with photosystem 2, PS2 (with and without the water oxidising complex) decreased in both plant species, the inhibition being higher in faba bean. The intrachloroplast quinone pool also decreased in both stressed species, yet an opposite trend was found for cytochrome b _559LP. Under stress an increasing peroxidation of thylakoid acyl lipids was detected in pea, but higher protein/Chl ratio was detected in faba bean. Thus the acceptor side of PS2 is mostly affected in both chilling stressed species, but faba bean is more sensitive.     

Regulation of calbindin-D9k expression by 1,25-dihydroxyvitamin D(3) and parathyroid hormone in mouse primary renal tubular cells

Membranes of the obligate methylotroph Methylobacillus flagellatus KT contained hemes B, O, and C and cytochromes b, o, and c both in batch and in continuous cultures. Neither heme A nor heme D was detected in the membranes. The cytochromes o and bb were the main components reversibly binding carbon monoxide (CO) in the terminal part of the respiratory chain. The alpha-region and especially the alpha-peaks at 568 and 573 nm and the alpha-troughs at 586 and 592 on the CO-difference spectra were diagnostic for the cytochromes o and bb, respectively. The cytochrome o content increased up to 1.8 times upon increasing the dilution rate of the culture from 0.15 to 0.55 h(-1) under methanol limitation. By contrast, the level of the CO-binding cytochrome bb was not affected by methanol concentration but its content increased up to 1.9 times when the level of oxygen decreased from 95 to 21 microM under the constant dilution rate (mu = 0.55 h(-1)). The maximum ratio between the cytochromes o and bb reached 2 during continuous cultivation under methanol-limited conditions (mu = 0.55 h(-1)), whereas the minimum ratio between them was about 0.7 during batch cultivation at stationary phase of growth. The synthesis of the CO-binding cytochrome bb but not of the cytochrome o in M. flagellatus KT was assumed to depend on the ambient redox potential of the medium. The cytochrome o synthesis was supposed to depend on the transmembrane gradient of protons (Delta(mu)H+).     

Novel cytochromes P450 applications arising from the directed-evolution of recombinant micro-organisms

AIMS: Directed (forced) evolution of cytochromes P450 (overall 2700 CYP isoforms in non-recombinant biota) is a method that has been investigated in yeasts (and other micro-organisms) by aerobically growing brewers' yeast Saccharomyces cerevisiae in very high glucose (20%) media. METHODS AND RESULTS: Mitochondrial repression subverts cytochrome oxidase biosynthesis into manifest cytochromes P450 accumulation in brewers' yeast. A similar phenomenon is observed with the acridine-induced petit mutant. Cytochromes P450 EC 1.14.14.1 (and mimics) display a range of redox iron-mediated bioconversions in food processing, with mixed function oxidase (O2:mono-oxygenase) intervention results. Unfortunately these enzymes generate reactive oxygen species (ROS) through redox electron recycling, whilst isoform CYP 1A1 can activate precarcinogens such as benzo(a)pyrene to the ultimate (proximate) carcinogen that binds to nuclear DNA. CONCLUSIONS: In conclusion, another 5000 CYP isoforms, for example, might be identified in micro-organisms and many more made to order through recombinant DNA technology and utilized both in vitro and in vivo for aimed bioconversions in industry and in the environment, as part of the impact of greener-approach supporting strategies to minimize global pollution.     

Cerebral Cortex Synaptic Heavy Mitochondria May Represent the Oldest Synaptic Mitochondrial Population: Biochemical Heterogeneity and Effects of L-Acetylcarnitine

The microheterogeneous nature of intrasynaptic mitochondria has been demonstrated and iswidely accepted. However, evidence is still lacking about the role played by the differentintrasynaptic mitochondrial subpopulations. The data obtained support the hypothesis thatheavy mitochondria could represent old mitochondrial populations: in fact, in addition tothe well known impairment of typical mitochondrial functions, they possess the highest levelsof hydroperoxides and their fatty acids pattern is completely modified. The qualitative andquantitative fatty acid modifications suffered by these organelles deeply altered theirprotein/lipid ratio, thus modifying their mode of action. The present work also collects a large bodyof evidence that a subchronic L-acetylcarnitine treatment in 28 days does not structurallyaffect both nonsynaptic and intrasynaptic mitochondria of normal rat in asteady-statemetabolic condition.     

Wild-type CYP102A1 as a biocatalyst: turnover of drugs usually metabolised by human liver enzymes

This work provides functional data showing that the bacterial CYP102A1 recognises compounds metabolised by human CYP3A4, CYP2E1 and CYP1A2 and is able to catalyse different reactions. Wild-type cytochrome CYP102A1 from Bacillus megaterium is a catalytically self-sufficient enzyme, containing an NADPH-dependent reductase and a P450 haem domain fused in a single polypeptidie chain. An NADPH-dependent method (Tsotsou et al. in Biosens. Bioelectron. 17:119–131, 2002) together with spectroscopic assays were applied to investigate the catalytic activity of CYP102A1 towards 19 xenobiotics, including 17 commercial drugs. These molecules were chosen to represent typical substrates of the five main families of drug-metabolising human cytochromes P450. Liquid chromatography–mass spectrometry analysis showed that CYP102A1 catalyses the hydroxylation of chlorzoxazone, aniline and p-nitrophenol, as well as the N-dealkylation of propranolol and the dehydrogenation of nifedipine. These drugs are typical substrates of human CYP2E1 and CYP3A4. The K _M values calculated for these compounds were in the millimolar range: 1.21 ± 0.07 mM for chlorzoxazone, 2.52 ± 0.08 mM for aniline, 0.81 ± 0.04 mM for propranolol. The values of v _max for chlorzoxazone and propranolol were 46.0 ± 9.0 and 7.6 ± 3.4 nmol min⁻¹ nmol⁻¹, respectively. These values are higher then those measured for the human enzymes. The v _max value for aniline was 9.4 ± 1.3 nmol min⁻¹ nmol⁻¹, comparable to that calculated for human cytochromes P450. The functional data were found to be in line with the sequence alignments, showing that the identity percentage of CYP102A1 with CYP3A4 and CYP2E1 is higher than that found for CYP1A2, CYP2C9 and CYP2D6 families.     

Liver microsomal cytochromes P450‐dependent alkoxyphenoxazone O‐dealkylation in vitro by alligator and rat: Activities,inhibition, substrate preference,and discrimination factors

Six substituted alkoxyphenoxazones (resorufins) and four inhibitors of P450‐dependent mixed‐function oxygenases (MFO) were used to probe the breadth and extent of P450 metabolism induced by pretreatment with five xenobiotic chemicals in liver microsomes of the American alligator, Alligator mississippiensis. Phenobarbital (PB), 3‐methylcholanthrene (3MC), and PB–3MC co‐pretreatment elicited major induction of alligator MFO activity measured by alkoxyresorufin O‐dealkylation (AROD). The induced levels of activities observed with appropriate substrate, 7‐ethoxy, 7‐methoxy, 2‐phenylbenzyloxy, 7‐pentoxy, or 7‐benzyloxyresorufin (EROD, MROD, PBROD, PROD and BROD, respectively), were 10 to 100 times lower in alligator as compared to rat. The exception was a higher level of isopropoxyresorufin O‐dealkylation (IPROD) in alligator. The induction regimes used in alligator and rat revealed marked differences in substrate preference, discrimination factors (DF) for various inducible P450 isoforms. EROD, a classic indicator of CYP1A activity in rat, had a low DF in alligator. MROD was the best discriminator in alligator of CYP1A‐type induction. In contrast to rats, pretreatment of alligators with Aroclor 1254, 2,2′,4,4′ tetrachlorobiphenyl, and clofibrate caused minor alterations in AROD relative to untreated controls. The inhibitors, α‐napthaflavone, 1‐ethynylpyrene, SKF 525A, and 9‐ethynylphenanthrene, inhibited AROD activity of the expected P450 isoform. For example, 10 μM α‐napthaflavone inhibited liver microsomal EROD catalyzed by 3MC‐inducible isoforms from alligator by 90% and from rat by 97%. Similarly, 10 μM SKF 525A inhibited PROD catalyzed by PB‐inducible isoforms by 63% and 79% in alligator and rat liver microsomes, respectively. To the best of our knowledge, the present studies are the first to show PB induction of P450 activities typical of the mammalian CYP2 family and their inhibition with classical inhibitors in alligator liver. While our data indicate metabolism of P450 substrates with preferences to certain isoforms, it remains to be established which isoforms exert catalytic function in alligator and whether these are homologues or orthologues of mammalian isoforms. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 17–27, 1999     

Redox chemistry of low-pH forms of tetrahemic cytochrome c3

Desulfovibrio vulgaris Hildenborough cytochrome c₃ contains four hemes in a low-spin state with bis-histidinyl coordination. High-spin forms of cytochrome c₃ can be generated by protonation of the axial ligands in order to probe spin equilibrium (low-spin/high-spin). The spin alterations occurring at acid pH, the associated changes in redox potentials, as well as the reactivity towards external ligands were followed by the conjunction of square wave voltammetry and UV–visible, CD, NMR and EPR spectroscopies. These processes may be used for modelling the action of enzymes that use spin equilibrium to promote enzyme activity and reactivity towards small molecules.