U.S. Department of Agriculture, Agricultural Research Service, 40546, Lexington, KY, USA.
Planta. 1990 Jun;181(3):287-95. doi: 10.1007/BF00195878.
Photoaffinity labeling with [(32)P] 8-azidoadenosine 5'-triphosphate (8-N3ATP) was used to identify putative binding sites on tobacco (Nicotiana tabacum L. and N. rustica L.) leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase, EC 4.1.1.39). Incorporation of (32)P was observed in polypeptides corresponding to both RuBPCase subunits when desalted leaf and chloroplast extracts, and purified RuBPCase were irradiated with ultraviolet light in the presence of [(32)P] 8-N3ATP. (32)P-labeling was dependent upon ultraviolet irradiation and occurred with [(32)P] 8-N3ATP labeled in the α-position, indicating covalent incorporation of the photoprobe. Both [(32)P] 8-N3ATP and [(32)P] 8-N3GTP were incorporated to a similar extent into the 53-kilodalton (kDa) "large" subunit (LSu), but incorporation of [(32)P] 8-N3GTP into the 14-kDa "small" subunit (SSu) of RuBPCase was <5% of that measured with [(32)P] 8-N3ATP. Distinct binding sites for 8-N3ATP on the two subunits were indicated by different apparent K D values, 3 and 18 μM for the SSu and LSu, respectively, and differences in the response of photoaffinity labeling to Mg(2+), anions and enzyme activation. Active-site-directed compounds, including the non-gaseous substrate ribulose 1,5-bisphosphate, the reaction intermediate analog 2-carboxyarabinitol-1,5-bisphosphate and several phosphorylated effectors afforded protection to the LSu site against photoincorporation but provided almost no protection to the SSu. These results indicate that 8-N3ATP binds to the active-site region of the LSu and a distinct site on the SSu of RuBPCase. Experiments conducted with intact pea (Pisum sativum L.) and tobacco chloroplasts showed that the SSu was not photolabeled with [(32)P] 8-N3ATP in organello or in undesalted chloroplast lysates but was photolabeled when lysates were ultrafiltered or desalted. These results indicate that 8-N3ATP binds to a site on the SSu that has physiological significance.
用 [(32)P] 8-叠氮腺苷 5'-三磷酸（8-N3ATP）进行光亲和标记，以鉴定烟草（Nicotiana tabacum L. 和 N. rustica L.）叶核酮糖-1,5-二磷酸羧化酶/加氧酶（RuBPCase，EC 4.1.1.39）的假定结合位点。当用 [(32)P] 8-N3ATP 辐照时，在经脱盐的叶片和叶绿体提取物以及纯化的 RuBPCase 中，观察到与 RuBPCase 的两个亚基相对应的多肽掺入 [(32)P]。(32)P 标记依赖于紫外线照射，并发生在 α-位标记的 [(32)P] 8-N3ATP 上，表明光探针的共价掺入。[(32)P] 8-N3ATP 和 [(32)P] 8-N3GTP 均以相似的程度掺入 53 千道尔顿（kDa）“大”亚基（LSu），但 RuBPCase 的 14 kDa“小”亚基（SSu）掺入的 [(32)P] 8-N3GTP 仅为 [(32)P] 8-N3ATP 的 5%。两个亚基上 8-N3ATP 的不同结合位点表明，SSu 的表观 K D 值分别为 3 和 18 μM，以及对 Mg2+、阴离子和酶激活的光亲和标记反应的不同响应。活性位点导向化合物，包括非气态底物核酮糖 1,5-双磷酸、反应中间类似物 2-羧基阿拉伯糖醇-1,5-双磷酸和几种磷酸化效应物，为 LSu 位点提供了对光掺入的保护，但几乎没有为 SSu 提供保护。这些结果表明，8-N3ATP 结合到 RuBPCase 的 LSu 的活性位点区域和 SSu 的一个不同位点。在完整的豌豆（Pisum sativum L.）和烟草叶绿体中进行的实验表明，SSu 在细胞器或未经脱盐的叶绿体裂解物中不能用 [(32)P] 8-N3ATP 进行光标记，但在用超滤或脱盐处理的裂解物中可以进行光标记。这些结果表明，8-N3ATP 结合到 SSu 的一个具有生理意义的位点。
