Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants.
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.
Benveniste
I
I
Laboratoire d'Enzymologie Cellulaire et Moléculaire, CNRS UA 1182, Université Louis-Pasteur, Strasbourg, France.
Lesot
A
A
Hasenfratz
M P
MP
Durst
F
F
D003429:Cross Reactions; D006368:Helianthus / Q000201:enzymology*; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000276:immunology*; D008861:Microsomes / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000276:immunology*; D010944:Plants / Q000201:enzymology
D007074:Immunoglobulin G; D009251:NADPH-Ferrihemoprotein Reductase