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 mere allowed to imbibe mater in darkness, the ac tivity expressed per seedling increased notably after d 1, reached a maximu m 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 se edlings, and could be separated by hydrophobic column chromatography. WEP-1 was further purified and identified as a 31-kDa polypeptide that was immun oreactive to antiserum raised against REP-1, a major rice cysteine endopept idase. Experiments with proteinase inhibitors revealed that WEP-1 and WEP-2 are cysteine and serine endopeptidases, respectively. The two enzymes diff ered in substrate specificity, pH dependence, and the ability to digest maj or wheat seed proteins. Determination of its amino-terminal amino acid sequ ence indicated the similarity of WEP-1 to other cereal cysteine endopeptida ses which are involved in the digestion of seed storage proteins, The expre ssion of WEP-1 in de-embryonated seeds was induced in the presence of gibbe rellic acid and its effect was eliminated by abscisic acid. In addition to WEP-1 and WEP-2, a legumain-like asparaginyl endopeptidase was identified i n the extract of seedlings on hydrophobic chromatography. The asparaginyl e ndopeptidase may function in the early step of mobilization of wheat storag e proteins in germinated seeds.