Purification and characterization of barley dipeptidyl peptidase IV.
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.
Davy
A
A
Carlsberg Research Laboratory, Carlsberg Laboratory, Gamle Carlsbergvej 10, DK-2500 Valby, Denmark.
Thomsen
K K
KK
Juliano
M A
MA
Alves
L C
LC
Svendsen
I
I
Simpson
D J
DJ
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
D003374:Coumarins; D010455:Peptides; D018819:Dipeptidyl Peptidase 4; C028743:7-amino-4-methylcoumarin