Identification and characterization of dipeptidyl peptidase IV enzyme activity in the American crocodile (Crocodylus acutus)
2010
Merchant, Mark | Mead, Stephanie | McAdon, Charles | McFatter, Justin | Wasilewski, Joe
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±0.03nmol product formed) measured using only 2μL of crocodile serum, with maximum activity measured using 500μL 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±1.3μM and 75.5±4.3nmol/min, respectively. Crocodile DPP4 catalyzed the hydrolysis of Ala-Pro-AFC rapidly, with substantial activity measured within 5min of the addition of substrate. After an initial rapid increase in activity, near maximal activity (7.43±0.24nmol product formed) measured at 180min. Crocodile serum DPP4 activity was temperature-dependent, with steadily increased activity from 5 to 40°C.
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