SEP-1 - a subtilisin-like serine endopeptidase from germinated seeds of Hordeum vulgare L. cv. Morex

Debora Fontanini, Berne L Jones
Planta 2002, 215 (6): 885-93
Proteolysis is crucial for all living cells. It regulates protein processing, intracellular protein levels and removes abnormal or damaged proteins from the cell, working as a cellular housekeeper. By means of proteolysis, cells can control the short-lived regulatory proteins that affect processes such as signal transduction and reception, transcription, division and cellular growth. Proteolysis also furnishes amino acids for the de novo synthesis of proteins. In germinating seeds, its main role is to degrade storage proteins into small peptides and amino acids that can be used by the embryo during autotrophic growth. We have isolated and purified a serine endopeptidase, one of the many proteolytic enzymes that occur in germinated barley seeds (green malt), using chromatofocusing and DEAE-, CM-, and size-exclusion chromatographies. The enzyme, named SEP-1, has a molecular weight of 70 kDa, as estimated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography. SEP-1 was detected and measured by its ability to digest gelatin in gels and to hydrolyze the synthetic substrate N-succinyl Ala-Ala-Pro-Leu p-nitroanilide. The hydrolysis of the synthetic substrate was optimal at pH 6.5 and 50 degrees C with a K(m) of 2.6 mM. The enzyme was inhibited by phenylmethylsulfonyl fluoride and p-amidinophenyl methanesulfonyl fluoride but not by any other class-specific inhibitor, suggesting it was a serine endopeptidase. Its amino acid sequence was similar to those of other plant subtilisin-like serine peptidases (EC 3.4.21), especially to the cucumisin-like group. SEP-1 was present in resting seeds, and its activity increased during germination in all of the malted barley tissues except for the endosperm, where it never occurred, suggesting that the enzyme is not likely involved in storage-protein degradation.

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