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ARS Home » Midwest Area » Madison, Wisconsin » Cereal Crops Research » Research » Publications at this Location » Publication #64095
Title: PURIFICATION AND PARTIAL CHARACTERIZATION OF A 31KD CYSTEINE ENDOPROTEINASEFROM GERMINATED BARLEY

Author
item ZHANG, NINGYAN - UNIV OF WISCONSIN
item Jones, Berne

Submitted to: Planta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/14/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: Enzymes that break down proteins during the germination of barley seeds are very important to the seed germination process and to commercial malt production, because the products released during protein break down are necessary for both germination and malting. In order for plant breeders to more efficiently develop improved malting barleys, we need to know what enzymes are present in barley and which can readily break down the proteins that are present. This paper reports how we purified and characterized a novel protein-degrading enzyme from germinated barley seeds. The enzyme was similar to, but different from, a second protein-destroying enzyme that we had studied previously. Both the previously studied enzyme and the one reported in this paper have characteristics that indicate they are probably very important to the seed germination process. They are therefore also very critical to the preparation of malt for brewing beer from barley. Now, plant breeders and other researchers will know that they need to retain, and possibly increase, the amounts of these two enzymes in any new barley varieties that they produce.

Technical Abstract: Proteolytic enzymes hydrolyze cereal storage proteins into small peptides and amino acids, which are very important for seed germination and malting process. A cysteine class endopeptidase was purified from four-day germinated barley (Hordeum vulgare L. cv. 'Morex'). Four purification steps were used, carboxymethyl cellulose (CMC) 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 kD. 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 kD endopeptidase had high homology to those of the EP-A and EP-B cysteine proteinases reported previously. The 31 kD endopeptidase had a hydrolytic specificity similar to that of the 30 kD endopeptidase, and also reacted with the antibody raised against the purified 30 kD proteinase, but the two had different mobilities on non-denaturing PAGE. The hydrolytic specificities of both 30 and 31 kD endopeptidases are such that both could 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.