Polypeptide components of oligomeric legumin-like thiamin-binding protein from buckwheat seeds characterized by partial amino acid sequencing and photoaffinity labeling.
Among thiamin-binding proteins that ubiquitously occur in plant seeds, that of common buckwheat became a model of extensive studies of the chemical mechanism of ligand-protein interaction. In this work, the polypeptide components of buckwheat seed thiamin-binding protein (BSTBP) are identified and characterized. We suggest that BSTBP is probably a fraction of major storage 13 S globulin (legumin), has an average molecular mass of 235 kDa and comprises hexamers of 57-kDa and 38-kDa subunits in variable combinations. Each subunit is a pair of disulfide-linked polypeptide chains, 36 kDa plus 24 kDa and two-times 22 kDa, respectively. The N-terminal sequences of 22-kDa and 24-kDa components show strict homology with those reported for "basic subunits" of buckwheat legumin. By photoaffinity labeling of BSTBP with 4-azido-2-nitrobenzoylthiamine, it is shown that the 36-kDa chain plays the major role in thiamin binding, but the other chains may also be variably involved. Putative thiamin-binding fragments are identified and sequenced.
Rapala-Kozik
Maria
M
Department of Analytical Biochemistry, Faculty of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland. rapala@mol.uj.edu.pl
Ostrowska
Katarzyna
K
Bednarczyk
Katarzyna
K
Dulinski
Robert
R
Kozik
Andrzej
A
D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D019612:Fagopyrum / Q000737:chemistry*; D015394:Molecular Structure; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis; D019920:Photoaffinity Labels / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012639:Seeds / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D013831:Thiamine / Q000031:analogs & derivatives* / Q000737:chemistry; D000097793:Legumins
D002352:Carrier Proteins; D010455:Peptides; D019920:Photoaffinity Labels; D010940:Plant Proteins; D013831:Thiamine; D000097793:Legumins; C026989:thiamine-binding protein; C031810:4-azido-2-nitrobenzoylthiamine