title: Association of diamine oxidase and S-adenosylhomocysteine hydrolase in Nicotiana tabacum extracts.
abstract: The oxidative deamination of methylated putrescine by a diamine oxidase activity (DAO) is an important step in the biosynthesis of nicotine in tobacco and tropane alkaloids in several Solanaceous plants. A polyclonal rabbit antiserum was previously developed to a purported purified DAO enzyme from Nicotiana tabacum. The antiserum bound to a single 53 kDa protein and immunoprecipitated 80% of DAO activity from tobacco root extracts. In an effort to obtain DAO cDNAs, this antiserum was used to screen a tobacco cDNA expression library and three distinct immunoreactive cDNA clones were isolated. These cDNAs encoded predicted proteins that were either identical or nearly identical to predicted S-adenosylhomocysteine hydrolase (SAHH) from two Nicotiana species. Thus, the rabbit antiserum was not specific to DAO, even though it immunodepleted the majority of DAO activity from root extracts. Alternative hypotheses to explain the DAO immunodepletion results (such as poisoning of DAO activity or that SAHH is a bifunctional enzyme) were tested and ruled out. Therefore, we hypothesize that SAHH associates with DAO as part of a larger multienzyme complex that may function in planta as a nicotine metabolic channel.
authors.affiliation: Plant Pathology, Physiology, and Weed Science Deptartment, Fralin Biotechnology Center, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0346, USA.
authors.forename: William G
authors.initials: WG
authors.lastname: Heim
authors.forename: John G
authors.initials: JG
authors.lastname: Jelesko
mesh_terms: D043383:Adenosylhomocysteinase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006631:Amine Oxidase (Copper-Containing) / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive / Q000276:immunology; D007106:Immune Sera / Q000276:immunology; D010802:Phylogeny; D010936:Plant Extracts / Q000276:immunology / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics; D011485:Protein Binding / Q000276:immunology; D011817:Rabbits; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
chemical_list: D007106:Immune Sera; D010936:Plant Extracts; D010940:Plant Proteins; D006631:Amine Oxidase (Copper-Containing); D043383:Adenosylhomocysteinase