Biochemical studies on dwarf-8 (D_8) seedlings of Zea mays L.

从Dwarf-8(D8)及其正常等位基因暗培养的玉米幼苗中提取赤霉素结合蛋白(GBP)。经Scatchard图谱分析，正常幼苗的GBP中含有高亲和力的Kd组分，而D_8幼苗的GBP中未检测到高亲和力成分，但含有低亲和力的Kd组分，为13µM。正常幼苗的H-GA_4与GBP的结合被GA_1和GA_3抑制，而D_8幼苗的H-GA_4与GBP的结合被GA_1和GA_3弱抑制。，不活跃的GA对结合没有影响。当用Native-PAGE分离GBP时，D8幼苗的GBP中缺少正常幼苗GBP中存在的一个组分(GBP-3)。GBP-3是从正常幼苗中提纯的。经Sephacryl S-300凝胶过滤，测得其相对分子质量约为28万。它由一个分子量为70,000的亚基组成。GA_4的解离常数为0.5微米。根据这些结果，我们认为GBP-3可能是玉米D_8幼苗所缺乏的赤霉素受体的候选基因。为了阐明工作假说，我们正在使用一种针对GBP-3的抗体来研究GBP-3的cDNA克隆。

Gibberellin-binding proteins (GBPs) were extracted from dark-grown maize seedlings of dwarf-8 (D_8) and its normal allele. The proteins were partially purified by affinity chormatography on GA_3-linked EAH-Sepharose 4B.On the basis of Scatchard plot analysis, GBPs from normal seedlings contained a high affinity component of Kd for GA_4 of o.3 uM.While the high affinity component was not detected in GBPs from D_8 seedlings but they contained a low affinity component of Kd of 13uM.The binding of H-GA_4 to GBPs from normal seedlings was inhibited by GA_1 and GA_3, but that from D_8 seedlings was weakly inhibited by GA_1 and GA_3. GA_<13>, an inactive GA, had no effect on the binding. When GBPs were separated by native-PAGE, it was clarified that the GBPs from D_8 seedlings lacked one component (GBP-3) which existed in the GBPs from normal seedlings. The GBP-3 was purified from normal seedlings. The molecular weight was estimated to be about 280,000 by gel filtration on Sephacryl S-300. It consisted of one subunit with molecular weight of 70,000. The dissociation constant for GA_4 was 0.5 uM.From these results, we considered that GBP-3 might be a candidate of a gibberellin receptor lacked in D_8 seedlings of Zea mays. To clarify the working hypothesis, we are studying cDNA clining of GBP-3 using an antibody made against GBP-3.