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 re sidues. A peptidase cleaving after Xaa-Pro- at the N terminus of peptides w as purified from green barley malt. It was identified as a serine-type dipe ptidyl peptidase (DPP), based on inhibitor studies, and the nature of the c leavage 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 op timum at 7.2. Substrate specificity was determined with a series of fluorog enic 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 sub strates were Xaa = lysine and arginine, while the poorest were Xaa = aspart ic acid, phenylalanine, and glutamic acid. The K-m values ranged from 0.071 to 8.9 mu M, compared with values of 9 to 130 mu M 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 th e germinating barley grain.