scholarly journals Two cytochrome P-450 isoforms catalysing O-de-ethylation of ethoxycoumarin and ethoxyresorufin in higher plants

1990 ◽  
Vol 270 (3) ◽  
pp. 729-735 ◽  
Author(s):  
D Werck-Reichhart ◽  
B Gabriac ◽  
H Teutsch ◽  
F Durst
Keyword(s):  
Plant Species ◽  
Higher Plants ◽  
Cumene Hydroperoxide ◽  
Jerusalem Artichoke ◽  
Erod Activity ◽  
Plant Tissues ◽  
Chemical Inducers ◽  
Nadph Cytochrome ◽  
Cytochrome P 450 ◽  
Specific Inhibitors

The O-dealkylating activities of 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) have been fluorimetrically detected in microsomes prepared from manganese-induced Jerusalem artichoke tubers. Cytochrome P-450 dependence of the reactions was demonstrated by light-reversed CO inhibition, NADPH-dependence, NADH-NADPH synergism and by use of specific inhibitors: antibodies to NADPH-cytochrome P-450 reductase, mechanism-based inactivators and tetcyclasis. Apparent Km values of 161 microM for 7-ethoxycoumarin and 0.4 microM for 7-ethoxyresorufin were determined. O-De-ethylase activity was also detected in microsomes prepared from several other plant species, including wheat, maize, tulip, avocado and Vicia. ECOD and EROD were low or undetectable in uninduced plant tissues, and both activities were stimulated by wounding or by chemical inducers. Two distinct cytochrome P-450 isoforms are involved in ECOD and EROD activities since (1) they showed different distributions among plant species; (2) they showed contrasting inhibition and induction patterns; and (3) ECOD but not EROD activity was supported by cumene hydroperoxide.

scholarly journals Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants

1989 ◽  
Vol 259 (3) ◽  
pp. 847-853 ◽  
Author(s):  
I Benveniste ◽  
A Lesot ◽  
M P Hasenfratz ◽  
F Durst
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Higher Plants ◽  
Polyclonal Antibodies ◽  
Reductase Activity ◽  
Jerusalem Artichoke ◽  
Cross Reactivity ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Cytochrome P 450

Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.

scholarly journals Characterization of Saccharomyces cerevisiae CYP51 and a CYP51 fusion protein with NADPH cytochrome P-450 oxidoreductase expressed in Escherichia coli.

1997 ◽  
Vol 41 (4) ◽  
pp. 776-780 ◽  
Author(s):  
K Venkateswarlu ◽  
D E Kelly ◽  
S L Kelly
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Reductase Activity ◽  
Spectral Response ◽  
Cumene Hydroperoxide ◽  
Type Ii ◽  
Nadph Cytochrome ◽  
Binding Spectra ◽  
Cytochrome P 450 ◽  
Fus Protein

Saccharomyces cerevisiae CYP51, target of azole antifungal agents, and CYP51 fused with S. cerevisiae cytochrome P-450 oxidoreductase (FUS protein) were expressed in active forms in Escherichia coli by cloning into pET15b. The expression was monitored immunologically, catalytically, and by using reduced carbon monoxide difference and type II binding spectra. CYP51 and FUS enzymes were located in membranes and produced a Soret peak at 448 nm in the reduced CO difference spectrum. The cytochrome P-450 contents in the membrane fractions containing CYP51 and FUS proteins were 12.8 +/- 2.6 and 17.4 +/- 3.7 pmol/mg of protein, respectively. The NADPH cytochrome P-450 oxidoreductase (CPR) content was estimated to be 15.7 +/- 1.1 pmol/mg of protein in FUS membrane fractions. FUS protein catalyzed the demethylation of substrate at the 14alpha position, with a turnover number of 1.96 +/- 0.37 min(-1) in the presence of NADPH. No reductase activity was observed in membrane fractions containing CYP51, and therefore, CYP51 did not function catalytically in the presence of NADPH, but in the presence of an artificial electron donor, cumene hydroperoxide, activity was comparable to that of the FUS enzyme. Further support for a normal structure for the hemoproteins was obtained from type II binding spectra, in which the spectral response was saturated with an equimolar concentration of ketoconazole.

scholarly journals Histidine residues in rabbit liver microsomal cytochrome P-450 2B4 control electron transfer from NADPH-cytochrome P-450 reductase and cytochrome b5

1996 ◽  
Vol 318 (3) ◽  
pp. 857-862 ◽  
Author(s):  
Peter HLAVICA ◽  
Michael LEHNERER ◽  
Manfred EULITZ
Keyword(s):  
Electron Transfer ◽  
Cytochrome B5 ◽  
Cumene Hydroperoxide ◽  
Differential Susceptibility ◽  
Histidine Residues ◽  
Nadph Cytochrome ◽  
Fluorescence Measurements ◽  
Redox Partners ◽  
Nadh Cytochrome ◽  
Cytochrome P 450

Treatment of cytochrome P-450 2B4 (P-450 2B4) with diethylpyrocarbonate to introduce 10–11 equivalents of acylating agent per polypeptide chain resulted in the selective derivatization of histidine residues characterized by differential susceptibility toward the modifier. Second-derivative spectral analysis as well as fluorescence measurements disproved gross alterations in P-450 2B4 structure as a consequence of labelling. The modified haemoprotein retained its ability to bind hexobarbital and catalyse cumene hydroperoxide-sustained N-demethylation of the barbiturate. However, there was a steady attenuation of NAD(P)H-driven electron flux with increasing extent of P-450 2B4 carbethoxylation in reconstituted systems fortified with either NADPH-cytochrome P-450 reductase or NADH-cytochrome b5 reductase/cytochrome b5 as the redox partners, with 50% inhibition occurring when 6–7 histidines were blocked. Hampered P-450 2B4 reductase activities recovered to differing degrees upon treatment of the acylated mono-oxygenase with neutral hydroxylamine. Spectral data indicated that docking of the redox components to derivatized P-450 2B4 was not perturbed, so that disruption of the electron flows most likely resulted from some injury of the electron-transfer mechanisms.

scholarly journals Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction

1986 ◽  
Vol 235 (2) ◽  
pp. 365-373 ◽  
Author(s):  
I Benveniste ◽  
B Gabriac ◽  
F Durst
Keyword(s):  
Cytochrome C ◽  
Microsomal Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Jerusalem Artichoke ◽  
Higher Plant ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Fixed Concentration ◽  
Cytochrome P 450 ◽  
Nadph Cytochrome C Reductase

NADPH-cytochrome P-450 (cytochrome c) reductase (EC 1.6.2.4) was solubilized by detergent from microsomal fraction of wounded Jerusalem-artichoke (Helianthus tuberosus L.) tubers and purified to electrophoretic homogeneity. The purification was achieved by two anion-exchange columns and by affinity chromatography on 2′,5′-bisphosphoadenosine-Sepharose 4B. An Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. The purified enzyme exhibited typical flavoprotein redox spectra and contained equimolar quantities of FAD and FMN. The purified enzyme followed Michaelis-Menten kinetics with Km values of 20 microM for NADPH and 6.3 microM for cytochrome c. In contrast, with NADH as substrate this enzyme exhibited biphasic kinetics with Km values ranging from 46 microM to 54 mM. Substrate saturation curves as a function of NADPH at fixed concentration of cytochrome c are compatible with a sequential type of substrate-addition mechanism. The enzyme was able to reconstitute cinnamate 4-hydroxylase activity when associated with partially purified tuber cytochrome P-450 and dilauroyl phosphatidylcholine in the presence of NADPH. Rabbit antibodies directed against plant NADPH-cytochrome c reductase affected only weakly NADH-sustained reduction of cytochrome c, but inhibited strongly NADPH-cytochrome c reductase and NADPH- or NADH-dependent cinnamate hydroxylase activities from Jerusalem-artichoke microsomal fraction.

scholarly journals Haem synthesis during cytochrome P-450 induction in higher plants. 5-Aminolaevulinic acid synthesis through a five-carbon pathway in Helianthus tuberosus tuber tissues aged in the dark

1988 ◽  
Vol 249 (2) ◽  
pp. 473-480 ◽  
Author(s):  
D Werck-Reichhart ◽  
O T G Jones ◽  
F Durst
Keyword(s):  
Pyridoxal Phosphate ◽  
Higher Plants ◽  
Jerusalem Artichoke ◽  
Acid Synthesis ◽  
Chlorophyll Synthesis ◽  
Higher Plant ◽  
Aminolaevulinic Acid ◽  
C5 Pathway ◽  
Suicide Inhibitors ◽  
Cytochrome P 450

Chlorophyll and haem synthesis in illuminated Jerusalem artichoke tuber tissues were very efficiently inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). This inhibition seems to be due specifically to a blockade of the pathway for 5-aminolaevulinate biosynthesis which used glutamate as a substrate (the so-called C5 pathway) since we could not detect any inhibition of protein synthesis in the treated tissues and there was no effect of gabaculine on the glycine-dependent yeast 5-aminolaevulinate synthase used as a model. In dark-aged artichoke tissues, gabaculine also effectively blocked cytochrome P-450 induction, peroxidase activity and 5-aminolaevulinic acid synthesis, thus suggesting the involvement of a C5 pathway in cytoplasmic and microsomal haemoprotein synthesis in this higher plant. Allylglycine and (2-amino-ethyloxyvinyl)glycine, two olefinic glycine analogues which are potential suicide inhibitors of pyridoxal phosphate enzymes, were also demonstrated to be effective blockers of chlorophyll synthesis in artichoke tuber and Euglena cells exposed to light.

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