Human and rat dipeptidyl peptidase III: Biochemical and mass spectrometricarguments for similarities and differences

Dipeptidyl peptidase (DPP tit) was purified from rat and human erythrocytes using an identical procedure. Electrophoretic analyses revealed the same m olecular size and pi for both enzymes. The molecular mass of the human enzy me, measured by matrix-assisted laser desorption/ionization MS, was 82500 /- 60 Da, Its tryptic peptide mass profile was determined using the same te chnique, and the amino acid sequence of two internal peptides was obtained by tandem MS and Edman degradation. A search of databases revealed a high s imilarity between the human eryrthrocyte and rat liver DPP III: 21 matches out of 34 detected peptides were found, covering 40% of the total sequence. Matched peptides included the peptide harboring the characteristic HELLGH sequence motif, and a stretch of 19 identical amino acids, containing Glu, a putative ligand of active site zinc. Both enzymes preferred Arg-Arg-2-naphthylamide, and were activated by micro molar Co2+, differing in their pH optima and k(cat)/K-m, Zn2+ ions, sulfhyd ryl reagents, andaminopeptidase inhibitors, especially probestin, inhibited the rat DPP III more potently, The two enzymes showed the highest affinity for angiotensin III (K-i < 1 <mu>M) and a preference for a hydrophobic res idue at the P-1' site, However, significant differences in the binding cons tants for several peptides indicated non-identity in the active site topogr aphy of human and rat erythrocyte DPP III.