Dipeptidyl peptidase II from porcine seminal plasma: Purification, characterization, and its homology to granzymes, cytotoxic cell proteinases (CCP 1-4)

Dipeptidyl peptidase II (DPP LI) was purified to homogeneity from porcine seminal plasma by polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was calculated to be approx. 185 000 and 200 000 on Superdex 200 column chromatography and non-denatured PAGE, respectively, and to be 58 000 and 61 000 on SDS-PAGE in the absence and presence of P-mercaptoethanol (P-ME), respectively. These findings suggested that the enzyme is composed of three identical subunits. The enzyme rapidly hydrolyzed the substrates Lys-Ala-MCA and Gly-Pro-MCA at acidic pH. The K-m and V-max values of DPP II at optimal pH (pH 6.0) were 1330 mu M and 2.9 mu mol/mg per min for Gly-Pro-MCA, and 360 mu M and 1.43 mu mol/mg per min for Lys-Ala-MCA, respectively, It was strongly inhibited by diisopropylphosphofluoride (DFP), and moderately by 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). These findings suggest that DPP II is a serine peptidase. Furthermore, the enzyme activity was also strongly inhibited by copper ions. The amino-acid sequence of the first 41 residues of the enzyme was determined as Ala(1)-Ser-Pro-Pro-Glu-Pro-Gly-Phe-Arg-Glu(10)-Val-Tyr-Phe-Glu-Gln-Leu-Leu-Asp-His-Phe(20)-Asn-Phe-Glu-Arg-Phe-Gly-Lys-Lys-Thr-Phe(30)-Arg-Gln-Arg-Phe-Leu-Val-Ser-Asp-Lys-Phe(40)-Trp. This sequence showed homology (11.6-30.2%) to the N-terminal amino-acid sequences of cytotoxic cell proteinases (CCP 1-4), granzymes. Other properties of DPP II including pH optimum, pH stability, and heat stability were characterized.