Purification of two rat hepatic proteins with A-esterase activity toward chlorpyrifos-oxon and paraoxon.
A-esterases are calcium-dependent hydrolases that can detoxify the active metabolites (oxons) of organophosphorus insecticides such as chlorpyrifos and parathion. A-esterases from rat liver have previously been shown to hydrolyze chlorpyrifos-oxon but not paraoxon at low substrate concentrations. Two A-esterases were extracted by ammonium sulfate fractionation from solubilized rat liver microsomes followed by gel filtration chromatography and preparative scale isoelectric focusing. The proteins displayed similar characteristics and were difficult to separate; both had similar high molecular mass and isoelectric point range and exhibited A-esterase activity toward high and low concentrations of chlorpyrifos-oxon and high concentrations of paraoxon. Sufficient amounts of the higher molecular mass protein were obtained for kinetic studies, which yielded a Km of 0.93 mM toward high concentrations of chlorpyrifos-oxon and a Vmax of 369 nmoles product formed/mg protein-min. The protein hydrolyzed phenyl acetate, chlorpyrifos-oxon and paraoxon, suggesting that arylesterase and A-esterase activities are attributable to the same liver protein(s). Assays of purified protein and kinetic studies of microsomes suggested that the activity toward high (320 microM) and low (</=10(-5) M) concentrations of chlorpyrifos-oxon are due to the same protein(s), and that the activity toward low concentrations of chlorpyrifos-oxon is attributable to both a higher affinity and a higher Vmax (but primarily the latter) for chlorpyrifos-oxon than for paraoxon, which is not detectably hydrolyzed at low concentrations. The higher A-esterase activity with chlorpyrifos-oxon than paraoxon may be a major determinant in the observed lower acute toxicity of chlorpyrifos than parathion.
Pond
A L
AL
Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi, USA.
Chambers
H W
HW
Coyne
C P
CP
Chambers
J E
JE
D000818:Animals; D004390:Chlorpyrifos / Q000031:analogs & derivatives* / Q000378:metabolism; D004950:Esterases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007306:Insecticides / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D010261:Paraoxon / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D007306:Insecticides; C009618:O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphate; D004950:Esterases; D004390:Chlorpyrifos; D010261:Paraoxon