Carter C, Thornburg R W
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
J Biol Chem. 2000 Nov 24;275(47):36726-33. doi: 10.1074/jbc.M006461200.
Nectarin I, a protein that accumulates in the nectar of Nicotiana sp. , was determined to contain superoxide dismutase activity by colorimetric and in-gel assays. This activity was found to be remarkably thermostable. Extended incubations at temperatures up to 90 degrees 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 H(2)O(2) or NaCN. Following denaturation, the superoxide dismutase activity was restored after Mn(2+) addition. Addition of Fe(2+), Cu(2+), Zn(2+), and Cu(2+)/Zn(2+) 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 H(2)O(2). Putative nectarin I homologues were found in the nectars of several other plant species.
花蜜素I是一种在烟草属植物花蜜中积累的蛋白质，通过比色法和凝胶内测定法确定其具有超氧化物歧化酶活性。发现这种活性具有显著的热稳定性。在高达90摄氏度的温度下长时间孵育不会降低花蜜素I的超氧化物歧化酶活性。这一特性使得通过其他花蜜蛋白的热变性将花蜜素I纯化至同质。通过SDS-聚丙烯酰胺凝胶电泳，花蜜素I表现为29 kDa的单体。如果在加载凝胶之前蛋白质样品未煮沸，那么花蜜素I以165 kDa的寡聚蛋白形式迁移。通过基质辅助激光解吸/电离飞行时间质谱法，发现原体亚基是一种22.5 kDa的蛋白质。纯化的花蜜素I每个单体含有0.5个锰原子，花蜜素I的超氧化物歧化酶活性不受H₂O₂或NaCN的抑制。变性后，添加Mn²⁺后超氧化物歧化酶活性得以恢复。添加Fe²⁺、Cu²⁺、Zn²⁺和Cu²⁺/Zn²⁺不能恢复超氧化物歧化酶活性。对重组酶的四级结构进行了研究，只有四聚体和五聚体聚集体具有酶活性。重组酶还显示会产生H₂O₂。在其他几种植物物种的花蜜中发现了假定的花蜜素I同源物。
