Effect of zinc and calcium ions on the rat kidney membrane-bound form of dipeptidyl peptidase IV

摘要

Dipeptidyl peptidase IV (DPP-IV) is an ectopeptidase with many roles, and a target of therapies for different pathologies. Zinc and calcium produce mixed inhibition of porcine DPP-IV activity. To investigate whether these results may be generalized to mammalian DPP-IV orthologues, we purified the intact membrane-bound form from rat kidney. Rat DPP-IV hydrolysed Gly-Pro-e?‘?-nitroanilide with an average Vmax of 0.86?±0.01 e???mol mina€“1mLa€“1 and KM of 76?±6 e???M. The enzyme was inhibited by the DPP-IV family inhibitor L-threo-Ile-thiazolidide (Ki=64.0?±0.53 nM), competitively inhibited by bacitracin (Ki=0.16?±0.01 mM) and bestatin (Ki=0.23?±0.02 mM), and irreversibly inhibited by TLCK (IC50 value of 1.20?±0.11 mM). The enzyme was also inhibited by divalent ions like Zn2+ and Ca2+, for which a mixed inhibition mechanism was observed (Ki values of the competitive component: 0.15?±0.01 mM and 50.0?±1.05 mM, respectively). According to bioinformatic tools, Ca2+ ions preferentially bound to the e???-propeller domain of the rat and human enzymes, while Zn2+ ions to the e???-e??? hydrolase domain; the binding sites were essentially the same that were previously reported for the porcine DPP-IV. These data suggest that the cationic susceptibility of mammalian DPP-IV orthologues involves conserved mechanisms.