title: Identification and purification of metalloprotease from dry grass pea (Lathyrus sativus L.) seeds.
abstract: Proteolytic enzymes play a central role in the biochemical mechanism of germination. The present study reported the presence of Zn(2+)-dependent endoproteases in the dry seeds of grass pea (Lathyrus sativus L.) with maximum caseinolytic activity observed at pH 8.0. Studies with class-specific inhibitors (specific for cysteine, serine, aspartate, and metalloproteases) on crude extract identified the inhibitory effect of 1,10-phenanthroline. This inhibitory effect was overcome by addition of Zn(2+), not with Fe, Ca, Cu, Mg, or Co and indicates that the protease is Zn(2+) dependent. This metalloprotease was further characterized by attempting gelatin-PAGE zymography and observed three distinct zones of proteolytic activity with higher mobility. The protease fraction consisted of three isoforms as evidenced by the appearance of three different bands on gelatin-PAGE zymogram. We also purified these proteases to 110-fold by a three-step procedure comprising crude extract from dry seeds, (NH(4))(2)SO(4) fractionation, and casein-alginate affinity chromatography. The molecular mass of isoforms of metalloproteases is 25, 18, and 14 kDa.
authors.affiliation: Department of Biotechnology, Sri Krishnadevaraya University, Andhra Pradesh, India. vrkrishna70@rediffmail.com
authors.forename: Vadde
authors.initials: V
authors.lastname: Ramakrishna
authors.forename: Sake
authors.initials: S
authors.lastname: Rajasekhar
authors.forename: Lokireddy Sudarsana
authors.initials: LS
authors.lastname: Reddy
mesh_terms: D003067:Coenzymes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D027807:Lathyrus / Q000201:enzymology*; D045726:Metalloproteases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D012639:Seeds / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism
chemical_list: D003067:Coenzymes; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D045726:Metalloproteases; D015032:Zinc