title: The partial purification and characterization of a gibberellin C-20 hydroxylase from immature Pisum sativum L. seeds.
abstract: A gibberellin (GA) C-20 hydroxylase that catalyses the conversion of GA53 to GA44 was purified from developing pea embryos by ammonium-sulfate precipitation, gel filtration and anion-exchange column chromatography. The purification was about 270-fold and 15% of the enzymic activity was recovered. The relative molecular mass was 44000 by Sephadex G-200 gel filtration. The apparent Michaelis constant was 0.7 μM and the isoelectric point was 5.6-5.9. The enzymic activity was optimal at pH 7.0 2-Oxoglutarate and ascorbate were required for activity. Low concentrations of Fe(2+) stimulated the reaction, but externally added Fe(2+) was not essential, even in the most purified preparation. Catalase and bovine serum albumin also stimulated. Dithiothreitol preserved the activity during purification but was not needed during incubation. In fact, the simultaneous presence of dithiothreitol and Fe(2+) in the incubation mixture was inhibitory to the purified enzyme. The cofactor requirements are typical for those of 2-oxoglutarate-dependent dioxygenases.When the incubation time was long enough, GA53 was converted to both GA44 and GA19. The proportions of these two products remained constant throughout the purification, but this does not necessarily mean that their formations is catalysed by a single enzyme. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis showed that the final preparation contained several proteins. Although the most prominent protein band was located within the range expected for the enzyme on the grounds of its molecular weight, this band did not represent the enzyme, since it separated from the GA C-20 hydroxylase activity on ultrathin-layer isoeletric focusing.
authors.affiliation: Pflanzenphysiologisches Institut der Universität, Untere Karspüle 2, D-3400, Göttingen, Federal Republic of Germany.
authors.forename: T
authors.initials: T
authors.lastname: Lange
authors.forename: J E
authors.initials: JE
authors.lastname: Graebe