Study of metallopeptidase isozymes from malted barley (Hordeum vulgare cv.Morex)

It has been reported that germinated barley contains peptidases that are se nsitive to metal-chelating agents; however, none of these enzymes have been isolated, nor have their roles in germinated barley been investigated. Ani on-exchange chromatography and chromatofocusing have been used to isolate a group of peptidases from barley (Hordeum vulgare cv. Morex) green malt tha t are sensitive to metal-chelating agents. Their activities were studied us ing one- and two-dimensional polyacrylamide gel electrophoresis. When analy zed on two-dimensional PAGE gels that contained gelatin as substrate, the e nzymes separated into three major and approximately six-minor activity spot s with acidic pl values. The enzymes were optimally active against the gela tin substrate at pH 8.9 and were completely inhibited by 1,10-phenanthrolin e 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 hydroly sis of gelatin substrate was also impaired by the presence of reducing agen ts. The metallopeptidases readily digested, in vitro, the barley prolamine D hordein, indicating that they may be involved in degrading storage protei ns during barley germination.