Purification and characterization of a CM-glycinin digesting protease from soybean seeds.
A glycinin-digesting protease was purified from 4-h-imbibed soybean seeds to apparent homogeneity by a combination of DEAE-cellulose, gel filtration and Shodex IEC QA-824 chromatography. The purified soybean protease showed a single band on SDS-PAGE and degraded the carboxymethylated glycinin molecule at neutral or alkaline pH. The glycinin-digesting protease has an apparent molecular mass of 98 kilodaltons as estimated by SDS-PAGE under both reduced and non-reduced conditions. A peptidic substrate for the soybean protease was detected and purified from a tryptic digest of glycinin A5A4B3 complex; it corresponded to Ala(375)-Lys(381) of the B3 subunit. It was found that the protease could hydrolyze the peptide bond between Tyr(378) and Asn(389) of that peptide. The soybean protease was significantly inhibited by PMSF, indicating the endopeptidase is a serine protease. Moreover, the glycinin-digesting activity is also susceptible to EDTA and the inactivated enzyme could be restored by the addition of MnCl2, suggesting that Mn2+ is an important factor for the endopeptidase activity. To our knowledge, this is the first report of purification and characterization of a glycinin-digesting protease from soybean seeds.
Akhtaruzzaman
M
M
Department of Agricultural Science, Faculty of Agriculture, Okayama University, Japan.
Kimura
Y
Y
Takagi
S
S
D000409:Alanine / Q000032:analysis; D000595:Amino Acid Sequence; D001216:Asparagine / Q000032:analysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005916:Globulins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008239:Lysine / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014674:Plant Proteins, Dietary / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology*; D030262:Soybean Proteins; D013025:Glycine max / Q000201:enzymology*; D014443:Tyrosine / Q000032:analysis
D005916:Globulins; D014674:Plant Proteins, Dietary; D030262:Soybean Proteins; D014443:Tyrosine; D001216:Asparagine; C011474:glycinin; D010450:Endopeptidases; D008239:Lysine; D000409:Alanine