Identification and characterization of dipeptidyl peptidase IV enzyme activity in the American crocodile ( Crocodylus acutus)

Abstract

Serum from the American crocodile was assayed for dipeptidyl peptidase IV (DPP4) activity. We measured the DPP4-mediated hydrolysis of Ala–Pro–AFC. The generation of AFC was dependent on the titer of serum, with significant DPP4 activity (0.20 ± 0.03 nmol product formed) measured using only 2 μL of crocodile serum, with maximum activity measured using 500 μL of serum. The hydrolysis of substrate was inhibited in a concentration-dependent manner by diprotin A, a specific inhibitor of DPP4 activity, indicating that this activity was due to the presence of DPP4. The crocodile serum DPP4 exhibited classical Michaelis–Menten kinetics, with K m and V max extrapolated, by double-reciprocal plot, to be 14.7 ± 1.3 μM and 75.5 ± 4.3 nmol/min, respectively. Crocodile DPP4 catalyzed the hydrolysis of Ala–Pro–AFC rapidly, with substantial activity measured within 5 min of the addition of substrate. After an initial rapid increase in activity, near maximal activity (7.43 ± 0.24 nmol product formed) measured at 180 min. Crocodile serum DPP4 activity was temperature-dependent, with steadily increased activity from 5 to 40 °C.