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Dipeptidyl peptidase IV from porcine seminal plasma: purification, characterization, and N-terminal amino acid
1Department of Medical Biochemistry, Shiga University of Medical Science, Ohtsu.
Abstract:
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.
Insights
Dipeptidyl peptidase IV (DPP IV) from porcine seminal plasma was purified and characterized. Its structure, substrate specificity, and inhibition patterns were elucidated, revealing homology to other DPP IV enzymes.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Dipeptidyl peptidase IV (DPP IV) is an enzyme with known roles in various biological processes.
- Understanding DPP IV in different biological contexts, such as seminal plasma, is crucial for its comprehensive characterization.
Purpose of the Study:
- To purify and characterize Dipeptidyl peptidase IV (DPP IV) from porcine seminal plasma.
- To investigate the enzyme's molecular properties, substrate specificity, inhibition profile, and N-terminal amino acid sequence.
Main Methods:
- Purification of DPP IV using polyacrylamide gel electrophoresis (PAGE).
- Molecular weight determination via PAGE, SDS-PAGE, and Sephacryl S-300 HR chromatography.
- Enzyme activity assays with specific substrates (Gly-Pro-MCA, Lys-Ala-MCA).
- Inhibition studies using DFP, PMSF, AEBSF, and zinc ions.
- N-terminal amino acid sequencing.
Main Results:
- DPP IV was purified to homogeneity, with estimated molecular weights suggesting a trimeric structure.
- The enzyme exhibited rapid hydrolysis of Gly-Pro-MCA and weak hydrolysis of Lys-Ala-MCA.
- DPP IV was strongly inhibited by DFP and zinc ions, and moderately by PMSF and AEBSF.
- The N-terminal sequence showed high homology to mammalian DPP IV enzymes, suggesting a common origin or function.
- The native enzyme is proposed to be released into seminal plasma via proteolytic cleavage.
Conclusions:
- Porcine seminal plasma DPP IV shares structural and functional similarities with DPP IV from other mammalian sources.
- The characterization provides insights into the enzyme's potential role and processing in the male reproductive tract.
- Further studies can explore the specific functions and therapeutic implications of this seminal plasma enzyme.