Feldwisch J, Zettl R, Campos N, Palme K
Max-Planck-Institut für Züchtungsforschung, Köln, Germany.
Biochem J. 1995 Feb 1;305 ( Pt 3)(Pt 3):853-7. doi: 10.1042/bj3050853.
A 23 kDa protein (p23) was identified in microsomal extracts from maize coleoptiles by photoaffinity labelling with 5-azido-[7-3H]indol-3-ylacetic acid ([3H]N3IAA). Labelling of p23 was blocked by unlabelled IAA, N3IAA, indol-3-ylbutyric acid and indol-3-yl-lactate. In addition, labelling was efficiently decreased by tryptophan, as well as by the scavenger p-aminobenzoic acid. Labelling was, however, not affected by synthetic auxins such as 1-naphthylacetic acid or 2,4-dichlorophenoxyacetic acid. Competition data suggest that the label was probably bound via the indole ring, and hence labelling was not specific for auxins. The 23 kDa protein was solubilized from crude microsomes by extraction with Triton X-100 and purified to homogeneity by ion-exchange, size-exclusion and reversed-phase chromatography. After electroblotting, the amino acid sequences of the p23 N-terminus as well as the several tryptic peptides were obtained. Database comparisons revealed sequence identity with a maize manganese superoxide dismutase. We conclude that photoaffinity labelling of p23 was pseudo-affinity, and therefore the binding site for IAA is not specific.
通过用5-叠氮基-[7-³H]吲哚-3-乙酸（[³H]N₃IAA）进行光亲和标记，在玉米胚芽鞘的微粒体提取物中鉴定出一种23 kDa的蛋白质（p23）。未标记的吲哚乙酸（IAA）、N₃IAA、吲哚-3-丁酸和吲哚-3-乳酸可阻断p23的标记。此外，色氨酸以及清除剂对氨基苯甲酸可有效降低标记。然而，标记不受合成生长素如1-萘乙酸或2,4-二氯苯氧乙酸的影响。竞争数据表明，标记可能是通过吲哚环结合的，因此标记对生长素不具有特异性。通过用Triton X-100提取，从粗微粒体中溶解出23 kDa的蛋白质，并通过离子交换、尺寸排阻和反相色谱法纯化至同质。电印迹后，获得了p23 N端以及几个胰蛋白酶肽段的氨基酸序列。数据库比较显示与玉米锰超氧化物歧化酶具有序列同一性。我们得出结论，p23的光亲和标记是假亲和性的，因此IAA的结合位点不具有特异性。
