caa a22 4500 477101739 CHVBK 20180405111543.0 cr unu---uuuuu 170330e19960801xx s 000 0 eng 10.1007/BF00195188 doi (NATIONALLICENCE)springer-10.1007/BF00195188 Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley [Elektronische Daten] [Ningyan Zhang, Berne Jones] 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. Springer-Verlag, 1996 Endopeptidase purification nationallicence Germination nationallicence Hordeum nationallicence Malting nationallicence Protein hydrolysis nationallicence Storage protein nationallicence CMC : carboxymethyl cellulose nationallicence E-64 : transepoxysuccinyl-l-leucylamido-(4-guanidino)butane nationallicence EMI : N-ethylmaleimide nationallicence IEF : isoelectricfocusing nationallicence Phen : 1,10-phenanthroline nationallicence PI : isoelectric point nationallicence PMSF : phenylmethylsulfonyl fluoride nationallicence Zhang Ningyan Cereal Crops Research Unit, USDA/Agricultural Research Service, 501 N. Walnut St., 53705, Madison, WI, USA aut Jones Berne Department of Agronomy, University of Wisconsin-Madison, USA aut Planta Springer-Verlag 199/4(1996-08-01), 565-572 0032-0935 199:4<565 1996 199 425 https://doi.org/10.1007/BF00195188 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00195188 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Zhang Ningyan Cereal Crops Research Unit, USDA/Agricultural Research Service, 501 N. Walnut St., 53705, Madison, WI, USA aut NATIONALLICENCE 700 1- Jones Berne Department of Agronomy, University of Wisconsin-Madison, USA aut NATIONALLICENCE 773 0- Planta Springer-Verlag 199/4(1996-08-01), 565-572 0032-0935 199:4<565 1996 199 425 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer