Purification and Partial Characterization of a Dipeptidase from Barley

Abstract

A peptidase hydrolyzing the dipeptide Ala-Gly optimally at pH 8 to 9 was pwified about 3500-fold from gerninated grains of baley (ffordeum vulgare L.). According to disc electrophoresis in the presence of sodium dodecyl sulfate, the preparation was about 90% pure.The enzyme preparation hydrolyzed all of the 15 neutnl dipeptides tested, but showed no activity on Lys-Gly or Asp-Ala. Three tripeptides and four synthetic aminopeptidase substrates were hydrolyzed at less than 0.1% of the rate for Ala-Gly; inhibition and inactivation tests indicated that these reactions were due to some contaminating aminopeptidase(s). The results suggest that the purified enzyme is a specific dipeptidase. Km values were determined for six dipeptides at pH 8.8; they varied from 0.3 to 15.8 mm. The enzyme was strongly inhibited both by metal chelators and by sulfhydryl reagents. The elution volumes of the enzyme in gel chromatography on Sephadex G-150 and Ultrogel AcA 22 corresponded to molecular weight 130,000 and 175,000, respectively. The migration rate in sodium dodecyl sulfate disc electropboresis indicated a subunit molecular weight of 50,000.The barley enzyme is remarkably similar to severl mmmalan and microbial dipeptidases.In 1929, Linderstrom-Lang and Sato (9) demonstrated that germinated barley contains high activities of two peptidases, one hydrolyzing Leu-Gly-Gly and Leu-Gly at pH 8.6, the other acting on Ala-Gly at pH 7.8. In a previous paper, we reported the purification and characterization of the former enzyme (22). This barley peptidase appeared remarkably similar to the mammalian and bacterial leucine aminopeptidases both in chemical and in enzymic properties.This paper contains a corresponding report for the Ala-Glysplitting barley peptidase. The results indicate that the enzyme is a specific dipeptidase, which is clearly similar to several mammalian and microbial dipeptidases.MATERIALS AND METHODS Plant Material. Pirkka "high enzyme" malt was obtained from Lahden Polttimo Oy, Lahti, Finland. Pirkka is a Finnish 6-row cultivar of barley (Hordeum vulgare L.).Reagents. The substrates were obtained from the following sources:

Contribution of exopeptidases to formation of nonprotein nitrogen during ensiling of alfalfa

“…The concentrations of inhibitors required for inhibiting activities of dipeptidase, dipeptidyl-peptidase, and tripeptidyl-peptidase in the alfalfa extracts were all greater than observed in previous reports (Sopanen, 1976;Davy et al, 2000;Book et al, 2005). The differences between inhibitor concentrations required to inhibit exopeptidases in our study relative to previous reports might be explained on the basis of the different forages used as fermentation substrates.…”

“…Previous studies suggested that carboxypeptidase and acid proteinase might be the principal enzymes hydrolyzing the proteins of ensiled alfalfa forage (McKersie and Buchanan-Smith, 1982;Guo et al, 2007). Studies characterizing exopeptidases in other plants have been well documented (Sopanen, 1976;Davy et al, 2000;Yang et al, 2004;Book et al, 2005;Kumada et al, 2007), whereas few studies have been conducted to characterize these exopeptidases in alfalfa. The present study indicates that 5 exopeptidases (aminopeptidase, carboxypeptidase, dipeptidase, dipeptidyl-peptidase, and tripeptidyl peptidase) existed in alfalfa.…”

“…The residual carboxypeptidase activity was determined using 2 mM N-carbobenzoxy-l-Phe-l-alanine as substrate in 100 mM Na-acetate, pH 5.0 (Feller et al, 1977). The residual dipeptidase activity was determined using 12.5 mM Ala-Gly in 25 mM Tris-HCl buffer, pH 8.8 (Sopanen, 1976). The residual dipeptidyl-peptidase activity was measured using 100 μM Lys-Pro-AMC in 50 mM 3-(Nmorpholino)-propane sulfonic acid (pH 7.2), and 2 mM β-mercaptoethanol as substrate (Davy et al, 2000).…”

Modification of the Methods for the Extraction of Carboxypeptidase and Proteinase Activities from Sorghum Malts

“…Some antioxidants such as cysteine, 2-mercaptoethanol (2-ME), dithiothreitol (DTT), dithioerythreitol (DTE), glutathione, thioglycollate and a host of others 4,12,24,41 are used as phenol-complexing agents or oxidase inhibitors. Several reports favour the use of varying concentrations of cysteine 8,9,29 and 2-mercaptoethanol 5,6,12,44 for the extraction of proteolytic enzymes from barley malts.…”

Purification and Properties of Malt Carboxypeptidases Attacking Hordein

“…These enzymes are also reported to be affected by di-isopropyl fluorophosphate (DIFP). [13][14][15][16][17] In view of the extreme toxicity, and consequent unavailability of this reagent, phenyl methyl sulphonyl fluoride (PMSF) which also inhibits active serinc residues, was used as a substitute. At a final concentration of 2mM, this reagent caused 34% inhibition of carboxypeptidase activity with N-CBZ-Phe-Ala.…”