利用单克隆抗体对番茄 1-氨基环丙烷-1-羧酸合成酶的调控进行研究。

Studies on the regulation of 1-aminocyclopropane-1-carboxylate synthase in tomato using monoclonal antibodies.

机构信息

MSU-DOE Plant Research Laboratory, Michigan State University, 48824-1312, East Lansing, MI, USA.

出版信息

Planta. 1988 Mar;173(3):385-90. doi: 10.1007/BF00401025.

Abstract

A partially purified preparation of 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) from tomato (Lycopersicon esculentum (Mill.) fruit tissue was used to generate monoclonal antibodies (MAb) specific for the two different MAbs yielded a 50-kDa polypeptide as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. An enzyme-linked immunosorbent assay (ELISA) capable of detecting <1 ng of antigen was developed. The ELISA system was used to demonstrate that two of the MAbs recognized different epitopes on the ACC-synthase protein. Wound-induced increases in ACC-synthase activity in tomato fruit tissue were correlated with changes in ELISA-detectable protein. In-vivo labeling of wounded tissue with [(35)S]methionine followed by extraction and immunopurification in the presence of various protease inhibitors yielded one major radioactive band of 50 kDa molecular mass. Pulse labeling with [(35)S]methionine at various times after wounding indicated that the wound-induced increase in ACC-synthase activity involved de-novo synthesis of a rapidly turning over 50-kDa polypeptide.

摘要

采用从番茄（Lycopersicon esculentum (Mill.) 果实组织中部分纯化的 1-氨基环丙烷-1-羧酸（ACC）合酶（EC 4.4.1.14）制备物，生成针对该酶的两种不同单克隆抗体（MAb）。两种单抗均产生 50 kDa 的多肤条带，这一点通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳得到证实。建立了一种可检测<1ng 抗原的酶联免疫吸附测定（ELISA）系统。该 ELISA 系统用于证明两种单抗识别 ACC 合酶蛋白上的不同表位。番茄果实组织中伤诱导的 ACC 合酶活性增加与 ELISA 可检测到的蛋白变化相关。用 [(35)S]甲硫氨酸对受伤组织进行体内标记，然后在存在各种蛋白酶抑制剂的情况下进行提取和免疫纯化，得到一条主要的 50 kDa 相对分子质量的放射性带。伤后不同时间进行 [(35)S]甲硫氨酸脉冲标记表明，伤诱导的 ACC 合酶活性增加涉及快速周转的 50 kDa 多肽的从头合成。