DIPEPTIDYL PEPTIDASE-IV FROM PORCINE SEMINAL PLASMA - PURIFICATION, CHARACTERIZATION, AND N-TERMINAL AMINO-ACID-SEQUENCE

Dipeptidyl peptidase IV (DPP IV) was purified to homogeneity from porc ine seminal plasma by polyacrylamide gel electrophoresis (PAGE). The m olecular weight of the purified enzyme was calculated to be approximat ely 290,000 on PAGE in the absence of sodium dodecyl sulfate (SDS) and 310,000 on Sephacryl S-300 HR column chromatography, and to be 115,00 0 and 105,000 on SDS-PAGE in the absence and presence of beta-mercapto ethanol. The enzyme is suggested to be composed of three identical sub units. The enzyme rapidly hydrolyzed the substrate Gly-Pro-MCA, and we akly the substrate Lys-Ala-MCA. It was strongly inhibited by diisoprop ylphosphofluoridate (DFP), and moderately by both phenylmethyl-sulfony l fluoride (PMSF) and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF ). It was also strongly inhibited by zinc ion. The amino acid sequence of the first 18 residues of the enzyme was -Ala-Ala-Ala-Asp-Ser-Arg-A rg-Thr-Tyr-Thr-Leu-Thr-. This sequence was highly homologous to the se quences in the rear of the transmembrane site of human and rat liver D PP IVs and mouse thymus DPP IV. The native DPP IV is suggested to be r eleased into the seminal plasma after the cleavage of the hydrophobic N-terminal domain by chymotrypsin-like or pepsin-like enzymes. Other p roperties of DPP IV including kinetic parameters, pH stability and hea t stability were characterized.