Purification and characterization of an esterase isozyme involved in hydrolysis of organophosphorus compounds from an insecticide resistant pest, Helicoverpa armigera (Lepidoptera: Noctüidae).
An esterase isozyme was purified from the insecticide resistant pest, Helicoverpa armigera collected from field crops. Purification involved ammonium sulfate precipitation, hydrophobic interaction and ion exchange chromatography followed by gel filtration chromatography. The purification was 212-fold with 1% yield of the enzyme. The optimum pH of the isozyme was found to be 10.5 and 8.5 for p-nitrophenyl phosphate and paraoxon, respectively. The enzyme was unstable at temperature >50 degrees C. The molecular mass determined by SDS-PAGE was 66 kDa. Cations such as Hg(+2), Ag(+2), Cd(+2) inhibited the activity while Zn(+2) stimulated it. Kinetic studies indicated that the enzyme had low K(m) values of 0.238 and 0.348 mM for p-nitrophenyl phosphate and paraoxon, respectively. The enzyme had broad substrate specificity with high K(m) values for ATP, ADP and beta-glycerophosphate. This enzyme was partially sequenced and identified as an alkaline phosphatase.
Srinivas
R
R
Department of Biochemistry, Gulbarga University, Gulbarga-585 106, India.
Jayalakshmi
S K
SK
Sreeramulu
K
K
Sherman
N E
NE
Rao
Jayasimha
J
D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D006863:Hydrogen-Ion Concentration; D007305:Insecticide Resistance / Q000502:physiology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000201:enzymology*; D009943:Organophosphorus Compounds / Q000378:metabolism; D044345:Phosphoric Triester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D007527:Isoenzymes; D009943:Organophosphorus Compounds; D000469:Alkaline Phosphatase; D044345:Phosphoric Triester Hydrolases