Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley.
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.
Zhang
N
N
USDA/Agricultural Research Service, Cereal Crops Research Unit, Madison, WI 53705, USA.
Jones
B L
BL
D023181:Antimicrobial Cationic Peptides; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005983:Glutens; D001467:Hordeum / Q000201:enzymology*; D010940:Plant Proteins / Q000378:metabolism
D023181:Antimicrobial Cationic Peptides; D010940:Plant Proteins; D005983:Glutens; C016085:purothionin; D003546:Cysteine Endopeptidases