Hydrolysis of various bioactive peptides by goat brain dipeptidylpeptidase-III homologue Suman Dhanda * , Jasbir Singh and Hari Singh Department of Biochemistry, Kurukshetra University, Kurukshetra, Haryana, India DPP-III from goat brain was purified to apparent electrophoretic homogeneity which showed several characteristics similar to other reported DPP-IIIs although it possesses dissimilar molecular weight and different inhibition behavior. Thin layer chromatographic studies with goat brain DPP-III revealed that it hydrolyses Leu-enkephalin (Tyr–Gly–Gly–Phe–Leu) at the Gly–Gly bond producing Tyr–Gly and Gly–Phe–Leu with no further degradation of liberated tripeptide. (Ala) 4 is hydrolyzed to dialanine whereas trialanine is not cleaved. ACTH, angiotensin II and III were also hydrolyzed whereas angiotensin I was not. It was concluded that the enzyme requires at least a tetrapeptide to act and that it removes a dipeptidyl moiety from the NH 2 -terminus of the studied peptides. Goat brain DPP-III may be involved in the metabolism of very important bioactive peptides such as enkephalins and angiotensins. Copyright # 2007 John Wiley & Sons, Ltd. key words — DPP; enkephalin; angiotensin; thin layer chromatography; goat brain abbreviations — ACTH, adrenocorticotropic hormone; DPP, dipeptidyl peptidase; DTE, dithioerythritol; DTT, dithiothreitol; PMSF, phenylmethanesulphonyl fluoride; Arg–Arg–4mbNA, arginyl– arginyl–4-methoxy-b-naphthylamide; b-ME, b-mercaptoethanol; TLC, thin layer chromatography. INTRODUCTION Several peptides possessing neurotransmitter proper- ties have been reported in brain, 1 which are acted upon by a variety of peptidases. The physiological role of particular peptidases in the metabolism and regulation of neuropeptide disposition is unclear. Specific enzyme systems involved in hydrolyzing enkephalins (Leu- and Met-enkephalins), which deprive them of their biological activities have been purified and characterized. 2 Enkephalins are degraded after admin- istration in vivo and their analgesic activity does not last. 3 Both these peptides are hydrolyzed by brain enzymes designated as aminopeptidases 4–7 and enke- phalinases. 8,9 Aminopeptidases have been purified and investigated from animal brains 10,11 and human serum. 12 They hydrolyze at a Tyr–Gly bond of enkephalins. One of these enzymes is dipeptidylpeptidase (DPP), which is known to hydrolyze the Gly–Gly bond of enkephalins and was first purified from bovine pituitary by Ellis and Nuenke 13 and later on from several sources. Recently, we purified a DPP-III from goat brain in homogenous state, 14 that is, another candidate for involvement in enkephalin and angio- tensin degradation. The enzyme exhibits affinity for enkephalins in the micromolar range. 15 In the present study, we have confirmed the enkephalin hydrolysis using thin layer chromatography (TLC) with removal of a dipeptide (Tyr–Gly) moiety from the NH 2 -terminus. Furthermore, we also observed the degradation of angiotensins, adrenocorticotropic hormone (ACTH), and other peptides. Goat brain DPP-III may play an important role in the metabolism of specific peptides in addition to general peptide degradation. MATERIALS AND METHODS Materials All the chemicals used for the experiments were of highest possible quality. Gastrin tetrapeptide amide cell biochemistry and function Cell Biochem Funct 2008; 26: 339–345. Published online 7 December 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/cbf.1448 * Correspondence to: Dr S. Dhanda, Department of Biochemistry, Kurukshetra University, Kurukshetra-136119, Haryana, India. Tel: 0091-1744-238196-2752. E-mails: dhandasuman@rediffmail.com; jaid22in@rediffmail.com Copyright # 2007 John Wiley & Sons, Ltd. Received 25 June 2007 Revised 20 August 2007 Accepted 29 August 2007