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 porcine seminal plasma by polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was calculated to be approximately 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 115,000 and 105,000 on SDS-PAGE in the absence and presence of beta-mercaptoethanol. The enzyme is suggested to be composed of three identical subunits. The enzyme rapidly hydrolyzed the substrate Gly-Pro-MCA, and weakly the substrate Lys-Ala-MCA. It was strongly inhibited by diisopropylphosphofluoridate (DFP), and moderately by both phenylmethyl-sulfonyl 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 Asn-Lys-Gly-Thr-Asp-Asp-Ala-Ala-Ala-Asp-Ser-Arg-Arg- Thr-Tyr-Thr-Leu-Thr-. This sequence was highly homologous to the sequences in the rear of the transmembrane site of human and rat liver DPP IVs and mouse thymus DPP IV. The native DPP IV is suggested to be released into the seminal plasma after the cleavage of the hydrophobic N-terminal domain by chymotrypsin-like or pepsin-like enzymes. Other properties of DPP IV including kinetic parameters, pH stability and heat stability were characterized.
Ohkubo
I
I
Department of Medical Biochemistry, Shiga University of Medical Science, Ohtsu.
Huang
K
K
Ochiai
Y
Y
Takagaki
M
M
Kani
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002413:Cations, Divalent; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry* / Q000302:isolation & purification*; D004795:Enzyme Stability; D007531:Isoflurophate; D008297:Male; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D012661:Semen / Q000201:enzymology*; D017385:Sequence Homology; D013379:Substrate Specificity; D013552:Swine; D015032:Zinc
D002413:Cations, Divalent; D011480:Protease Inhibitors; D007531:Isoflurophate; D018819:Dipeptidyl Peptidase 4; D015032:Zinc