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  • Our previous work has demonstrated that two glutamate

    2019-12-03

    Our previous work has demonstrated that two glutamate residues (Glu-305 and Glu-331 in murine and human TPP II) in the active site are important for exopeptidase activity in mTPP II [17]. At least one of these, Glu-331, seems to form a salt bridge to the N-terminal amino group of the substrate, positioning it for cleavage at the third peptide bond from the N-terminus [17]. Recently, a study confirmed the importance of the homologous glutamate residues in dTPP II (Glu-312 and Glu-343) for the exopeptidase activity, and proposed a role for Glu-312 in hindering peptides from being cleaved endopeptidolytically [18]. Although quite low compared to the exopeptidase activity, the endopeptidase activity is nevertheless claimed to be as efficient as the proteasome at degrading at least some substrates [2]. The characteristics of the endopeptidase activity are still largely unknown. So far, only 10 cleavage sites in four different peptides have been reported [2], [3], [18]. The endopeptidase activity shows a preference for basic amino acids in the P1 position [2], [3], using the nomenclature of Schechter and Berger [19], in contrast to the exopeptidase activity, which has a preference for aliphatic and aromatic amino Pioglitazone australia residues in the P1 position [20], [21]. In addition, it has been claimed that the endopeptidase activity is dependent on a free N-terminus in the peptide substrate, even though it does not cleave at a specific position in the peptide [22].
    Materials and methods
    Results and discussion
    Acknowledgments
    Introduction Extensive proteolysis in ensiled legume forages usually reduces its protein quality for ruminants. During ensiling, most herbage proteins are degraded into oligopeptides, free amino acids (AA), ammonia and other forms of non-protein N (NPN; Ohshima and McDonald, 1978). Most true protein fractions within ensiled legumes are converted into NPN, and the concentration of AA is reduced extensively after ensiling (Guo et al., 2008). The rapid degradation of a high proportion of silage NPN in the rumen is often poorly synchronized with release of energy yielding substrates for protein synthesis by rumen microorganisms and, as a result, silage N is utilized less efficiently by rumen microbes than N from fresh or dried forages (Siddons et al., 1985, Givens and Rulquin, 2004). There is still no satisfactory or feasible means of inhibiting proteolysis in ensiling alfalfa because the most rapid proteolysis in ensiled alfalfa occurs within 24–48h of ensiling (Fairbairn et al., 1988, Guo et al., 2007). Although formic acid is one of the most effective additives in inhibiting proteolysis, cost and logistics of this chemical limit its utilization in practice. Proteolysis in ensiled forage results mainly from plant proteolytic enzymes (Ohshima and McDonald, 1978, McKersie, 1981, Heron et al., 1988). McKersie (1981) demonstrated the presence of at least three proteolytic enzymes in Lucerne (i.e., carboxypeptidase, aminopeptidase, acid proteinase), and each differed in its pH and temperature optima and sensitivity to inhibitors. Metallo, aspartic and cysteine endopeptidases were in perennial ryegrass (PRG; Wetherall et al., 1995, Nsereko et al., 1998) and specific inhibitors of these classes of peptidase decreased proteolysis in ensiled PRG (Nsereko and Rooke, 1999, Nsereko and Rooke, 2000). However, despite applying specific inhibitors to ensilage, substantial proteolysis still occurred indicating that the targeted Pioglitazone australia enzymes were not inhibited completely by spraying the inhibitor solution onto the surface of the forage, perhaps because most plant peptidases are located inside vacuoles and because other classes of peptidase may have been present. Pichard et al. (2006) reported that the main peptidases in extracts of six grass and legume species belonged to the serine class, whereas Nieri et al. (1998) described a metallopeptidase responsible for 90% of the proteolytic activity in senescent alfalfa leaves.