用[3H]4-叠氮基-2-硝基苯甲酰硫胺对酵母质膜中硫胺结合成分进行光亲和标记。

Photoaffinity labeling of thiamin-binding component in yeast plasma membrane with [3H]4-azido-2-nitrobenzoylthiamin.

作者信息

Nishimura H, Sempuku K, Kawasaki Y, Nosaka K, Iwashima A

机构信息

Department of Biochemistry, Kyoto Prefectural University of Medicine, Japan.

出版信息

FEBS Lett. 1989 Sep 11;255(1):154-8. doi: 10.1016/0014-5793(89)81080-4.

DOI:10.1016/0014-5793(89)81080-4

PMID:2676598

Abstract

When prepared from Saccharomyces cerevisiae through an acid precipitation at pH 5.0 for a crude particulate fraction obtained by mechanical agitation of yeast protoplasts with glass beads, the plasma membranes have more remarkable binding quantities of [14C]thiamin (Kd, 51 nM; Bmax, 263 pmol per mg of protein) compared with our previously prepared membranes [(1986) Experientia 42, 607-608]. Photoaffinity labeling of these yeast plasma membranes with [3H]4-azido-2-nitrobenzoylthiamin resulted in the covalent modification of a membrane component with an apparent molecular mass of 6-8 kDa. The extent of its labeling was markedly decreased by previous addition of thiamin. This result suggests that the small membrane component (6-8 kDa) takes part in the thiamin binding of thiamin carrier protein(s) in yeast plasma membranes.

摘要

当通过用玻璃珠机械搅拌酵母原生质体获得的粗颗粒部分在pH 5.0下进行酸沉淀从酿酒酵母制备时，与我们之前制备的膜相比，质膜具有更显著的[14C]硫胺素结合量（Kd，51 nM；Bmax，每毫克蛋白质263 pmol）[(1986) Experientia 42, 607 - 608]。用[3H]4-叠氮基-2-硝基苯甲酰硫胺对这些酵母质膜进行光亲和标记，导致表观分子量为6 - 8 kDa的膜成分发生共价修饰。预先添加硫胺素可显著降低其标记程度。该结果表明，小的膜成分（6 - 8 kDa）参与酵母质膜中硫胺素载体蛋白的硫胺素结合。