title: Purification and immunocharacterization of a plant cytochrome P450: the cinnamic acid 4-hydroxylase.
abstract: Cinnamic acid 4-hydroxylase (CA4H) was purified from microsomes of manganese-induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. The three-step purification procedure involved solubilization and phase partitioning in Triton X-114, followed by chromatography on DEAE-Trisacryl and hydroxylapatite columns. Purification was monitored using carbon monoxide and type I substrate binding properties of the enzyme. The protein, purified to electrophoretic homogeneity, showed an Mr of about 57,000 on SDS-PAGE. Polyclonal antibodies raised against this protein selectively reacted with a 57-kDa polypeptide on Western blots of induced Jerusalem artichoke microsomes. The antibody selectively and strongly inhibited CA4H activity from several plant species.
authors.affiliation: Laboratoire d'Enzymologie Cellulaire et Moléculaire, Institut de Botanique, Université Louis Pasteur, Strasbourg, France.
authors.forename: B
authors.initials: B
authors.lastname: Gabriac
authors.forename: D
authors.initials: D
authors.lastname: Werck-Reichhart
authors.forename: H
authors.initials: H
authors.lastname: Teutsch
authors.forename: F
authors.initials: F
authors.lastname: Durst
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000918:Antibody Specificity; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D007120:Immunochemistry; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D050564:Trans-Cinnamate 4-Monooxygenase
chemical_list: D000596:Amino Acids; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050564:Trans-Cinnamate 4-Monooxygenase