Photoaffinity labeling of thiamin-binding component in yeast plasma membrane with [3H]4-azido-2-nitrobenzoylthiamin.
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.
Nishimura
H
H
Department of Biochemistry, Kyoto Prefectural University of Medicine, Japan.
Sempuku
K
K
Kawasaki
Y
Y
Nosaka
K
K
Iwashima
A
A
D000135:Acid Phosphatase; D000345:Affinity Labels; D001665:Binding Sites; D002352:Carrier Proteins / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis; D004586:Electrophoresis / Q000379:methods; D010777:Photochemistry; D012441:Saccharomyces cerevisiae / Q000032:analysis*; D029701:Saccharomyces cerevisiae Proteins; D013831:Thiamine / Q000031:analogs & derivatives*
D000345:Affinity Labels; D002352:Carrier Proteins; D029701:Saccharomyces cerevisiae Proteins; C026989:thiamine-binding protein; C031810:4-azido-2-nitrobenzoylthiamine; D000135:Acid Phosphatase; C488913:PHO3 protein, S cerevisiae; C488916:PHO5 protein, S cerevisiae; D013831:Thiamine