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 + or - 0.03 nmol product formed) measured using only 2 microL of crocodile serum, with maximum activity measured using 500 microL 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 + or - 1.3 microM and 75.5 + or - 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 + or - 0.24 nmol product formed) measured at 180 min. Crocodile serum DPP4 activity was temperature-dependent, with steadily increased activity from 5 to 40 degrees C.