Dipeptidyl peptidase II from porcine seminal plasma: purification, characterization, and its homology to granzymes, cytotoxic cell proteinases (CCP 1-4).
Dipeptidyl peptidase II (DPP II) 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 beta-mercaptoethanol (beta-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 Km and V(max) values of DPP II at optimal pH (pH 6.0) were 1330 microM and 2.9 mumol/mg per min for Gly-Pro-MCA, and 360 microM and 1.43 mumol/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 Ala1-Ser-Pro-Pro-Glu-Pro-Gly-Phe-Arg- Glu10-Val-Tyr-Phe-Glu-Gln-Leu-Leu-Asp-His-Phe20-Asn-Phe-Glu- Arg-Phe- Gly-Lys-Lys-Thr-Phe30-Arg-Gln-Arg-Phe-Leu-Val-Ser-Asp-Lys-Phe40 -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.
Huang
K
K
Department of Medical Biochemistry, Shiga University of Medical Science, Otsu, Japan.
Takagaki
M
M
Kani
K
K
Ohkubo
I
I
D000595:Amino Acid Sequence; D000818:Animals; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012661:Semen / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine
D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100151:dipeptidyl peptidase II