Tobacco nectarin I. Purification and characterization as a germin-like, manganese superoxide dismutase implicated in the defense of floral reproductive tissues

Nectarin I, a protein that accumulates in the nectar ofNicotiana sp., was determined to contain superoxide dismutase activity by calorimetric and in-gel assays. This activity was found to be remarkably thermostable. Extended incubations at temperatures up to 90 °C did not diminish the superoxide dismutase activity of nectarin I. This attribute allowed nectarin I to be purified to homogeneity by heat denaturation of the other nectar proteins. By SDS-polyacrylamide gel electrophoresis, nectarin I appeared as a 29-kDa monomer. If the protein sample was not boiled prior to loading the gel, then nectarin I migrated as 165-kDa oligomeric protein. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the protomer subunit was found to be a 22.5-kDa protein. Purified nectarin I contained 0.5 atoms of manganese/monomer, and the superoxide dismutase activity of nectarin I was not inhibited by either H₂O₂ or NaCN. Following denaturation, the superoxide dismutase activity was restored after Mn²⁺ addition. Addition of Fe²⁺, Cu²⁺, Zn²⁺, and Cu²⁺/Zn²⁺ did not restore superoxide dismutase activity. The quaternary structure of the reconstituted enzyme was examined, and only tetrameric and pentameric aggregates were enzymatically active. The reconstituted enzyme was also shown to generate H₂O₂. Putative nectarin I homologues were found in the nectars of several other plant species.