Study of metallopeptidase isozymes from malted barley (Hordeum vulgare cv. Morex).
It has been reported that germinated barley contains peptidases that are sensitive to metal-chelating agents; however, none of these enzymes have been isolated, nor have their roles in germinated barley been investigated. Anion-exchange chromatography and chromatofocusing have been used to isolate a group of peptidases from barley (Hordeum vulgare cv. Morex) green malt that are sensitive to metal-chelating agents. Their activities were studied using one- and two-dimensional polyacrylamide gel electrophoresis. When analyzed on two-dimensional PAGE gels that contained gelatin as substrate, the enzymes separated into three major and approximately six minor activity spots with acidic pI values. The enzymes were optimally active against the gelatin substrate at pH 8.0 and were completely inhibited by 1,10-phenanthroline and EDTA, indicating that they belonged to the metallopeptidase class (EC 3.4.24.x). After the enzymes were inhibited with EDTA, the activities were recovered in the presence of low concentrations of metal ions. The hydrolysis of gelatin substrate was also impaired by the presence of reducing agents. The metallopeptidases readily digested, in vitro, the barley prolamine D hordein, indicating that they may be involved in degrading storage proteins during barley germination.
Fontanini
D
D
Department of Agronomy, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA. dfontani@facstaff.wisc.edu
Jones
B L
BL
D000838:Anions; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005780:Gelatin / Q000378:metabolism; D005983:Glutens; D001467:Hordeum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D007527:Isoenzymes / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D008666:Metalloendopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D010940:Plant Proteins / Q000378:metabolism
D000838:Anions; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010618:Phenanthrolines; D010940:Plant Proteins; D005983:Glutens; D005780:Gelatin; D004492:Edetic Acid; D008666:Metalloendopeptidases; C025205:1,10-phenanthroline