Purification and characterization of paraoxon hydrolase from rat liver.
Paraoxonase (paraoxon hydrolase), an enzyme that hydrolyses paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), is located in mammals primarily in the serum and liver. Although considerable information is available regarding serum paraoxonase, little is known about the hepatic form of this enzyme. The present work represents the first study on the purification of rat liver paraoxonase. This enzyme has been purified 415-fold to apparent homogeneity with a final specific activity of 1370 units/mg using a protocol consisting of five steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA and anion exchange on Mono Q HR 5/5. The presence of Ca2+ and Triton X-100 in the buffers throughout the purification procedure was essential for maintaining enzyme activity. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent Mr of 45 000. N-terminal and internal amino acid sequences were determined and compared with those of paraoxonases from human and rabbit serum and mouse liver, showing a high similarity. The pH profile showed optimum activity at pH 8.5. The pH stability and heat inactivation of the enzyme were also studied. The Km for liver paraoxonase was 1.69 mM.
Rodrigo
L
L
Departamento de Medicina Legal y Toxicología, Facultad de Medicina, Universidad de Granada, Spain.
Gil
F
F
Hernandez
A F
AF
Marina
A
A
Vazquez
J
J
Pla
A
A
D000818:Animals; D043303:Aryldialkylphosphatase; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D010261:Paraoxon / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D004950:Esterases; D043303:Aryldialkylphosphatase; D010261:Paraoxon