Abstract
Proteolytic enzymes hydrolyze cereal seed storage proteins into small peptides and amino acids, which are very important for seed germination and the malting process. A cysteine-class endopeptidase was purified from 4-d-germinated barley (Hordeum vulgare L. cv. Morex). Four purification steps were used, carboxymethyl cellulose cation-exchange chromatography, chromatofocusing, size-exclusion chromatography, and electroelution from a polyacrylamide gel. The endopeptidase was most active at pH 4.5. It's isoelectric point (pI) was 4.4, as determined by isoelectric focusing, and it's SDS-PAGE molecular size was 31 kDa. The enzyme specifically hydrolyzed peptide bonds when the S2 site contained relatively large hydrophobic amino acids. The N-terminal amino acid sequence residues (1–9) of the 31-kDa endopeptidase had high homology to those of the EP-A and EP-B cysteine proteinases reported previously. The 31-kDa endopeptidase had a hydrolytic specificity similar to that of the Morex green malt 30-kDa endopeptidase we characterized previously, and also reacted with the antibody raised against the purified 30-kDa proteinase, but the two had different mobilities on non-denaturing PAGE. The hydrolytic specificities of both 30- and 31-kDa endopeptidases are such that both would very quickly cleave hordein (barley storage) proteins to small glutamine- and proline-rich peptides that could be quickly degraded to amino acids by barley exopeptidases.
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Abbreviations
- CMC:
-
carboxymethyl cellulose
- E-64:
-
transepoxysuccinyl-l-leucylamido-(4-guanidino)butane
- EMI:
-
N-ethylmaleimide
- IEF:
-
isoelectricfocusing
- Phen:
-
1,10-phenanthroline
- PI:
-
isoelectric point
- PMSF:
-
phenylmethylsulfonyl fluoride
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We thank the American Malting Barley Association for partially funding this work. Germinated barley seeds were kindly prepared by Eddie D. Goplin. Special thanks to Laurie Marinac for her excellent technical assistance.
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Zhang, N., Jones, B.I. Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley. Planta 199, 565–572 (1996). https://doi.org/10.1007/BF00195188
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DOI: https://doi.org/10.1007/BF00195188