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

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 ( K d, 51 nM; B max, 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.