Activity staining and inhibition characterization of dipeptidylpeptidase- III enzyme from goat brain

Abstract

Dipeptidylpeptidase-III (DPP-III) from goat brain was purified and characterized using Arginyl-Arginyl-4-methoxy - naphthylamide (Arg-Arg-4mβNA) substrate. This enzyme retained its activity in native 10% polyacrylamide gel when stained using Arg-Arg-4mNA. The activity was significantly increased by 100 mM chloride. Studies for its inhibition with some peptides and chemical inhibitors revealed that Leu-Trp-Met-Arg-Phe-Ala was most potent inhibitor followed by Arg-Phe-Ala and Gly-Phe-Leu. All the studied chemical inhibitors caused 40-50% inhibition at 1 mM. Metal ions helped to regain activity of EDTA pretreated enzyme. ZnCl2 at 50 μM almost completely restored the enzyme activity. Further ZnCl2 and CoCl 2 exerted protective effects on EDTA pretreated enzyme for its susceptibility to DTNB inhibition. Therefore, DPP-III is a metalloprotease with the involvement of cysteine residues either located at the catalytic site or involved in regulation. © 2011 Pooja Attri et al.