Identification and possible roles of three types of endopeptidase from germinated wheat seeds.
Little or no endopeptidase activity was detected in extracts of dry mature wheat seeds, but when they were allowed to imbibe water in darkness, the activity expressed per seedling increased notably after d 1, reached a maximum on d 3 and then decreased. Two major endopeptidases, named WEP-1 and WEP-2, were present in the 50-70% saturated ammonium sulfate fraction of d-3 seedlings, and could be separated by hydrophobic column chromatography. WEP-1 was further purified and identified as a 31-kDa polypeptide that was immunoreactive to antiserum raised against REP-1, a major rice cysteine endopeptidase. Experiments with proteinase inhibitors revealed that WEP-1 and WEP-2 are cysteine and serine endopeptidases, respectively. The two enzymes differed in substrate specificity, pH dependence, and the ability to digest major wheat seed proteins. Determination of its amino-terminal amino acid sequence indicated the similarity of WEP-1 to other cereal cysteine endopeptidases which are involved in the digestion of seed storage proteins. The expression of WEP-1 in de-embryonated seeds was induced in the presence of gibberellic acid and its effect was eliminated by abscisic acid. In addition to WEP-1 and WEP-2, a legumain-like asparaginyl endopeptidase was identified in the extract of seedlings on hydrophobic chromatography. The asparaginyl endopeptidase may function in the early step of mobilization of wheat storage proteins in germinated seeds.
Sutoh
K
K
Department of Biological Sciences, Tokyo Metropolitan University, Minami-ohsawa, Hachioji, Tokyo, 192-0397, Japan.
Kato
H
H
Minamikawa
T
T
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010450:Endopeptidases / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018525:Germination; D008970:Molecular Weight; D010940:Plant Proteins; D010944:Plants / Q000201:enzymology; D012697:Serine Endopeptidases; D014908:Triticum / Q000201:enzymology* / Q000235:genetics; D000099240:Asparaginyl Endopeptidase
D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D010940:Plant Proteins; D012697:Serine Endopeptidases; D000099240:Asparaginyl Endopeptidase