Plant Physiol, February 2000, Vol. 122, pp. 425-432

Purification and Characterization of Barley Dipeptidyl Peptidase IV¹

Carlsberg Research Laboratory (A.D.), Department of Physiology (K.K.T., D.J.S.), and Department of Chemistry (I.S.), Carlsberg Laboratory, Gamle Carlsbergvej 10, DK-2500 Valby, Denmark; and Department of Biophysics, Escola Paulista de Medicina, Rua Tres de Maio 100, Sao Paulo 04044-020, Brazil (M.A.J., L.C.A.).

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 µM, compared with values of 9 to 130 µM 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.