title: Purification and characterization of barley dipeptidyl peptidase IV.
abstract: Barley (Hordeum vulgare L.) storage proteins, which have a high content of proline (Pro) and glutamine, are cleaved by cysteine endoproteases to yield peptides with a Pro next to the N-terminal and/or C-terminal amino acid residues. A peptidase cleaving after Xaa-Pro- at the N terminus of peptides was purified from green barley malt. It was identified as a serine-type dipeptidyl peptidase (DPP), based on inhibitor studies, and the nature of the cleavage product. It is a monomeric glycoprotein with an apparent molecular mass of 105 kD (85 kD after deglycosylation), with a pI of 3.55 and a pH optimum at 7.2. Substrate specificity was determined with a series of fluorogenic peptide substrates with the general formula Xaa-Pro-AMC, where Xaa is an unspecified amino acid and AMC is 7-amino-4-methylcoumarin. The best substrates were Xaa = lysine and arginine, while the poorest were Xaa = aspartic acid, phenylalanine, and glutamic acid. The K(m) values ranged from 0.071 to 8.9 microM, compared with values of 9 to 130 microM reported for mammalian DPP IVs. We discuss the possible role of DPP IV in the degradation of small Pro-containing peptides transported from the endosperm to the embryo of the germinating barley grain.
authors.affiliation: Carlsberg Research Laboratory, Carlsberg Laboratory, Gamle Carlsbergvej 10, DK-2500 Valby, Denmark.
authors.forename: A
authors.initials: A
authors.lastname: Davy
authors.forename: K K
authors.initials: KK
authors.lastname: Thomsen
authors.forename: M A
authors.initials: MA
authors.lastname: Juliano
authors.forename: L C
authors.initials: LC
authors.lastname: Alves
authors.forename: I
authors.initials: I
authors.lastname: Svendsen
authors.forename: D J
authors.initials: DJ
authors.lastname: Simpson
mesh_terms: D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D003374:Coumarins / Q000378:metabolism; D018819:Dipeptidyl Peptidase 4 / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D001467:Hordeum / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D010455:Peptides; D018819:Dipeptidyl Peptidase 4; C028743:7-amino-4-methylcoumarin