Purification and biochemical characterization of a novel secretory dipeptidyl peptidase IV from porcine serum

• Published in: Molecular and cellular biochemistry Vol. 471; no. 1-2; pp. 71 - 80
• Main Authors: Kumar, Divya, Hamse, Vivek K., Neema, K. N., Babu Shubha, Priya, Chetan, D. M., Shivananju, Nanjunda Swamy
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.08.2020; Springer; Springer Nature B.V
• Subjects: Animals; Anion exchange; Anion exchanging; Biochemistry; Biological products; Biomedical and Life Sciences; Cardiology; Dextran; Dipeptides - metabolism; Dipeptidyl Peptidase 4 - blood; Dipeptidyl Peptidase 4 - chemistry; Dipeptidyl Peptidase 4 - isolation & purification; Dipeptidyl-peptidase IV; Dipeptidyl-Peptidase IV Inhibitors - pharmacology; Enzyme Assays - methods; Enzymes; Filtration; Gel electrophoresis; Gel filtration; Glycine; Glycoproteins; Kinetics; Life Sciences; Medical Biochemistry; Molecular Weight; Oligopeptides - pharmacology; Oncology; Peptidase; Plant extracts; Purification; Sodium lauryl sulfate; Substrate Specificity; Substrates; Swine; Type 2 diabetes; Diprotin A; DPP-IV; GLP-1; Terminalia chebula; Momordica carantia; Azadirachta indica
• ISSN: 0300-8177; 1573-4919
• DOI: 10.1007/s11010-020-03766-y

Purification of DPP-IV enzyme from porcine serum, is presented in this study for the first time. The high molecular weight DPP-IV from porcine serum was fractioned using Sephadex G-75 gel filtration followed by DEAE Sephadex anion exchange and Sephadex G-100 gel filtration chromatography columns with a final yield of 11.25%. The SDS-PAGE of the purified sample showed a single band of molecular mass nearing 160 kDa. Distinct single band was observed after PAS staining confirmed it to be a glycoprotein. The purified enzyme showed an optimum pH and temperature of 8 and 37 °C, respectively. The enzyme effectively cleaved fluorogenic substrate Gly-Pro-AMC with Km and Vmax of 4.578 µM and 90.84 nmoles/min, respectively. Purified DPP-IV activity was inhibited by Diprotin A with an IC 50 value of 8.473 µM. Among the three plant extracts used to study DPP-IV inhibition, the aqueous hot extract of Terminalia chebula showed the highest inhibition of 87.19%, followed by the aqueous cold extract of Momordica carantia , ( 31.6%) and Azadirachta indica (34.16%) at the concentration of 25 µg.