Zamir L O, Ginsburg R
J Bacteriol. 1979 Jun;138(3):684-90. doi: 10.1128/jb.138.3.684-690.1979.
A simple technique was developed for the detection of intermediary metabolites of Aspergillus versicolor that are putative precursors of aflatoxin. Minicolony populations were allowed to metabolize [1,2-14C]acetate over various time intervals. The biosynthetic reactions were quenched by quick-freezing the minicolonies, the cells were disrupted, and the metabolites were extracted into acetone. Small silica thin-layer chromatographic plates were then used to separate any radioactive metabolites present. Elution in two or three different directions was often necessary. Radioautography of the thin-layer chromatography plates provided a sensitive assay for the appearance of the various intermediates in a timing pattern which implicated the sequence of formation. Transient intermediates were distinguished from dead-end metabolites by the rapid formation and disappearance of the former. At least five unknown precursors of versicolorin A, a dead-end metabolite, were recognized. The kinetic pulse-labeling technique should be generally applicable to other fungal species whenever the entrapment of intermediary metabolites in the mycelium poses and technical problem.
开发了一种简单的技术来检测杂色曲霉的中间代谢产物，这些代谢产物被认为是黄曲霉毒素的前体。让小菌落群体在不同的时间间隔内代谢[1,2-¹⁴C]乙酸盐。通过快速冷冻小菌落来终止生物合成反应，破碎细胞，然后将代谢产物萃取到丙酮中。接着使用小型硅胶薄层层析板分离存在的任何放射性代谢产物。通常需要在两个或三个不同方向上进行洗脱。薄层层析板的放射自显影提供了一种灵敏的检测方法，用于检测各种中间体以时间模式出现的情况，这暗示了形成顺序。通过前者的快速形成和消失将瞬时中间体与终产物代谢物区分开来。至少识别出了作为终产物代谢物的杂色曲菌素A的五种未知前体。只要菌丝体中中间代谢产物的截留存在技术问题，动力学脉冲标记技术通常就适用于其他真菌物种。
