Determination of disulfide array and subunit structure of taste-modifying protein, miraculin.
The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity.
Igeta
H
H
Department of Chemistry, Faculty of Education, Yokohama National University, Japan.
Tamura
Y
Y
Nakaya
K
K
Nakamura
Y
Y
Kurihara
Y
Y
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003553:Cystine; D004220:Disulfides / Q000032:analysis; D004228:Dithionitrobenzoic Acid; D006023:Glycoproteins / Q000737:chemistry*; D008027:Light; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D012542:Scattering, Radiation; D013549:Sweetening Agents
D004220:Disulfides; D006023:Glycoproteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D013549:Sweetening Agents; C008533:miraculin protein, Synsepalum dulcificum; D003553:Cystine; D004228:Dithionitrobenzoic Acid