DIPEPTIDYL-PEPTIDASE-IV-BETA - FURTHER CHARACTERIZATION AND COMPARISON TO DIPEPTIDYL-PEPTIDASE-IV ACTIVITY OF CD26

Dipeptidyl peptidase IV-beta (DPP IV-beta) is a novel protein which sh ows a peptidase activity similar to the T-cell-activation antigen CD26 . To further characterize this DPP IV-beta and confirm its cell surfac e expression, we have developed a purification strategy using the CD26 (-) cell line C8166. The purification process includes biotinylation o f cell surface proteins before preparation of cell extracts and proces sing by gel-filtration, ion-exchange and lectin chromatographies. Cons istent with the molecular mass of DPP IV-beta estimated by gel-filtrat ion chromatography, the final purified fraction, manifesting a typical DPP IV activity, showed a major biotinylated 75-80-kDa band in SDS/PA GE, thus suggesting the monomeric nature of this enzyme. Kinetic param eters of DPP IV-beta and the sensitivity to a new family of irreversib le DPP IV inhibitors, were studied in comparison to CD26. Both enzymes followed a Michaelis kinetics with different K-m values for Gly-Pro-N H-Np (NH-Np, para-nitroanilide) hydrolysis (0.28 +/- 0.05 mM and 0.12 +/- 0.02 mM). More significant differences were observed in the sensit ivity to inhibitors, which exerted a much higher activity on CD26 than on DPP IV-beta. These differences permitted us to study DPP IV-beta e xpression in CD26-expressing cells, showing the expression of this new enzyme in all lymphoid cells rested, and a rapid enhancement in phyto hemagglutinin-stimulated or protein-A-stimulated peripheral blood mono nuclear cells. Our results indicate that, although DPP IV-beta and CD2 6 are coexpressed and manifest a typical DPP IV activity, there are di stinct features in their catalytic activities that may confer to each enzyme a complementary role in peptide processing.