Hicks G R, Rayle D L, Jones A M, Lomax T L
Department of Botany and Plant Pathology, Oregon State University, Corvallis 97331-2902.
Proc Natl Acad Sci U S A. 1989 Jul;86(13):4948-52. doi: 10.1073/pnas.86.13.4948.
Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.
通过水相分配从西葫芦（南瓜属）下胚轴组织中分离出质膜囊泡，并使用膜特异性酶测定法评估其同质性。高度纯净（约95%）的质膜囊泡维持跨膜pH差异，并以类似于[3H]吲哚乙酸（IAA）积累的方式积累3-吲哚乙酸（IAA）的氚化叠氮类似物5-叠氮基-[7-3H]IAA（[3H]N3IAA）。[3H]N3IAA与膜囊泡的结合是可饱和的，并受到IAA和生长素类似物的竞争。在暴露于紫外光（15秒；300纳米）之前，通过向质膜囊泡中添加[3H]N3IAA对生长素结合蛋白进行光亲和标记，并通过随后的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（NaDodSO4/PAGE）和荧光自显影检测。当反应温度降至-196℃时，观察到40 kDa和42 kDa多肽的高特异性活性标记。Triton X-100（0.1%）增加了标记的特异性活性并降低了背景，这表明标记的多肽是内在膜蛋白。正如生长素受体所预期的那样，标记的多肽丰度较低。此外，向光亲和反应混合物中添加IAA和生长素类似物导致标记减少，其性质与它们对膜囊泡中放射性标记IAA积累的影响相似。总体而言，这些结果表明放射性标记的多肽是生长素受体。标记的共价性质应有助于受体的纯化和进一步表征。
