Identification of paraoxonase 3 in rat liver microsomes: purification and biochemical properties

Abstract

Three paraoxonase genes (PON1, PON2 and PON3) have been described so far in mammals. Although considerable information is available regarding PON1, little is known about PON2 and PON3. PON3 has been isolated recently from rabbit serum [Draganov, Stetson, Watson, Billecke and La Du (2000) J. Biol. Chem. 275, 33435-33442] and liver [Ozols (1999) Biochem. J. 338, 265-275]. In the present study, we have identified the presence of PON3 in rat liver microsomes and a method for the purification to homogeneity is presented. PON3 has been purified 177-fold to apparent homogeneity with a final specific activity of 461 units/mg using a method consisting of seven steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA, two DEAE-cellulose steps and a final affinity chromatography on concanavalin A-Sepharose. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent molecular mass of 43 kDa. The isolated protein was identified by nanoelectrospray MS. Internal amino acid sequences of several peptides were determined and compared with those of human, rabbit and mouse PON3, showing a high similarity. Some biochemical properties of PON3 were also studied, including optimum pH, K(m) and heat and pH stability.

Effects of Some Antibiotics on Paraoxonase from Human Serum in Vitro and from Mouse Serum and Liver in Vivo

“…16) Different protocols are available for PON enzyme purification from serum and liver using three, four and seven steps. 14,20,21) We previously reported a purification strategy designed for the human PON1 enzyme consisting of two-step procedures resulting in a shorter and more straightforward approach in contrast to other purification procedures. 34,35) The gel for hydrophobic interaction chromatography was synthesized using Sepharose 4B, L-tyrosine and 1-napthylamine.…”

“…1, a single band, 43 kDa, was obtained, which corresponds to the results of previous studies. [20][21][22] [S] in the absence (᭜, controls) and in the presence of (, 0.0101 mg/ml), (᭡, 0.0135 mg/ml), (, 0.190 mg/ml), (᭡, 0.381 mg/ml) and (, 0.0476 mg/ml), (᭡, 0.0952 mg/ml) for sodium ampicillin ciprofloxacin and clindamycin phosphate, respectively. The inset shows the Dixon plot for K i determination.…”

The Past and Present of Paraoxonase Enzyme: Its Role in the Cardiovascular System and Some Diseases

“…After that, Rodrigo et al (36) purified it from rat liver microsomes and examined its biochemical properties. It was found that by hydrolyzing lipid peroxides, PON1 contributed to this effect of HDL (37), and that it could also limit the activity of platelet activating factor (PAF) (38).…”

Modulation of paraoxonase 1 and 3 expression after moderate exercise training in the rat

“…However, mouse HDL may not be a good acceptor for both human and mouse PON3; therefore, PON3 remains cellbound in mouse ( 22 ). Microsomal localization for PON3 has been demonstrated in rat liver ( 23 ).…”

“…PON1 is the most widely studied member of the family of mammalian enzymes; plasma PON1 has been extensively explored in relation to cardiovascular diseases, whereas few data are present on the hepatic enzyme. PON1 activity has been observed in rat (2 3, 43 ) hepatic microsomes; a portion of this enzyme is secreted into the circulation ( 23 ), and another portion is stored in the liver ( 43 ), where it seems to provide hepatic protection against oxidative stress ( 44 ). The presence and the physiological role of PON3 have been recently addressed ( 12 ); it was thought that the enzyme is absent in rat plasma, in analogy with the mouse, where PON3 remains cell-bound ( 22 ).…”