Identification of two rat liver proteins with paraoxonase activity: biochemical evidence for the identity of paraoxonase and arylesterase

The existence of two or more enzyme forms with paraoxonase activity has bee n reported in sheep, rabbit, human and rat serum and recently in mouse and rat liver. In this study we describe the presence of two peaks with paraoxo nase activity (M-1 and M-2) after non-specific affinity chromatography of r at liver microsomes on Cibacron Blue 3GA. The first peak (M-1) was obtained during the washing of the column and coeluted with albumin. The second act ive peak (M-2) was eluted with 1 M NaCl. The characterization of each peak was determined by SDS/PAGE electrophoresis and Western-blotting. A comparis on of both active fractions on the basis of kinetic parameters, heat inacti vation and pH stability, calcium requirement and inhibition by EDTA and sev eral metals was performed. Our results support the fact that two proteins c apable of hydrolyzing paraoxon are present in rat liver microsomes. Further more, during the purification to homogeneity of rat liver paraoxonase we ha ve performed a study of its hydrolytic ability against three different subs trates: paraoxon, phenylacetate and phenyl thioacetate (Paraoxonase (PON), Arylesterase (ArE), Phenyl thioacetate esterase (PTase)). The elution profi le in different chromatographic steps, as well as the activity ratios from the crude extract throughout the purification process, heat inactivation an d effect of inhibitors were used as identity criteria for the three hydroly tic activities. Our results show evidence for the hydrolysis of paraoxon an d phenylacetate by the same protein from rat liver (paraoxonase). (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.