Vol.:(0123456789) 1 3 Molecular and Cellular Biochemistry https://doi.org/10.1007/s11010-020-03766-y Purifcation and biochemical characterization of a novel secretory dipeptidyl peptidase IV from porcine serum Divya Kumar 1  · Vivek K. Hamse 3  · K. N. Neema 1  · Priya Babu Shubha 2  · D. M. Chetan 4  · Nanjunda Swamy Shivananju 1 Received: 22 February 2020 / Accepted: 23 May 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020 Abstract Purifcation of DPP-IV enzyme from porcine serum, is presented in this study for the frst time. The high molecular weight DPP-IV from porcine serum was fractioned using Sephadex G-75 gel fltration followed by DEAE Sephadex anion exchange and Sephadex G-100 gel fltration chromatography columns with a fnal yield of 11.25%. The SDS-PAGE of the purifed sample showed a single band of molecular mass nearing 160 kDa. Distinct single band was observed after PAS staining confrmed it to be a glycoprotein. The purifed enzyme showed an optimum pH and temperature of 8 and 37 °C, respectively. The enzyme efectively cleaved fuorogenic substrate Gly-Pro-AMC with Km and Vmax of 4.578 µM and 90.84 nmoles/ min, respectively. Purifed 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. Keywords DPP-IV · GLP-1 · Diprotin A · Terminalia chebula · Momordica carantia · Azadirachta indica Abbreviations DPP-IV Dipeptidyl peptidase IV GLP-1 Glucagon-like peptide 1 Gly-Pro-AMC Glycyl prolyl 7-amino 4-methyl coumarin Introduction The earliest report of DPP-IV as a serine protease was described by Hopsu-Havu and Glenner in 1966 [1]. It was later named as dipeptidyl aminopeptidase IV (DAP-IV) or post-proline dipeptidyl peptidase IV by Yoshimoto et al. [2]. DPP-IV belongs to the Dipeptidyl peptidase subgroup, as its fourth member. Being a cell surface antigen, this enzyme is found to be widely distributed in several tissues like kid- neys, intestines, spleen, lungs and in serum in a soluble form [3, 4]. DPP-IV possesses numerous biological functions such as receptor and co-stimulatory activities, binding and reciprocation with various proteins, in addition, to assist in apoptosis [5]. It has a highly restricted substrate specifc- ity, thus it catalyses only the peptides that have proline or alanine residue towards the N-terminus at its catalytic site [6, 7]. GLP-1 is one such peptide which is secreted in the distal small intestine by the endocrine L cells [8, 9]. It is an insulinotropic hormone which stimulates insulin release by promoting the growth of β-cells and glycogenesis in liver and muscles [10]. It also plays an important role in down- regulating the gastric emptying and secretion of gastric acid in order to bring down the postprandial glucose levels [11]. Unfortunately, GLP-1 is rapidly inactivated by DPP-IV * Nanjunda Swamy Shivananju nanjundaswamy@sjce.ac.in; nanjuchem@gmail.com 1 Department of Biotechnology, Sri Jayachamarajendra College of Engineering, JSS Science and Technology University, JSS TI Campus, Mysuru, Karnataka 570006, India 2 Department of Studies in Chemistry, University of Mysore, Manasagangothri, Mysuru, Karnataka 570006, India 3 Faculty of Natural Sciences, Adichunchanagiri University, Bellur Cross, B.G. Nagara, Mandya, Karnataka, India 4 Department of Biotechnology, NMAM Institute of Technology, Nitte, Karkala Taluk, Udupi, Karnataka 574110, India