Organophosphate-detoxicating enzymes inE. coli. Gelfiltration and isoelectric focusing of DFPase, paraoxonase and unspecific phosphohydrolases

Zech, Ronald; Wigand, K. D.

doi:10.1007/bf01990678

Cited by 34 publications

(7 citation statements)

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“…Our previous report suggested that rat liver paraoxonase requires to be solubilized prior to purification [26]. Loss of activity was observed during the various steps of purification in spite of maintenance of calcium and Triton X-100 throughout the whole process, as previously reported for serum A-esterases [23,[33][34][35][36]. The loss of activity during purification could explain the low purification indexes obtained in previous reports of attempts to purify A-esterases.…”

Section: Discussionmentioning

confidence: 76%

Purification and characterization of paraoxon hydrolase from rat liver

Rodrigo

Gil

Hernández

et al. 1997

Biochemical Journal

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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.

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Section: Discussionmentioning

confidence: 76%

Purification and characterization of paraoxon hydrolase from rat liver

Rodrigo

Gil

Hernández

et al. 1997

Biochemical Journal

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“…We studied microbial utilization of the model OPC diisopropyl fluorophosphate (DFP) (used in ophthalmic medications and in the treatment of myasthenia gravis) and its breakdown product, diisopropyl phosphate (DIPP), in Escherichia coli K-12. It has been reported previously that E. coli K-12 possesses hydrolytic enzymes that detoxify OPCs, including DFP, soman, and paraoxon (16,17,54). Crude extracts of E. coli K-12 strain JA221 are capable of hydrolyzing DFP to DIPP and hydrofluoric acid (unpublished data).…”

mentioning

confidence: 81%

phnE and glpT Genes Enhance Utilization of Organophosphates in Escherichia coli K-12

Elashvili

DeFrank

Culotta

1998

Appl Environ Microbiol

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Wild-type Escherichia coli K-12 strain JA221 grows poorly on low concentrations (≤1 mM) of diisopropyl fluorophosphate and its hydrolysis product, diisopropyl phosphate (DIPP), as sole phosphorus sources. Spontaneous organophosphate utilization (OPU) mutants were isolated that efficiently utilized these alternate sources of phosphate. A genomic library was constructed from one such OPU mutant, and two genes were isolated that conferred the OPU phenotype to strain JA221 upon transformation. These genes were identified asphnE and glpT. The original OPU mutation represented phnE gene activation and corresponded to the same 8-bp unit deletion from the cryptic wild-type E. coliK-12 phnE gene that has been shown previously to result inphnE activation. In comparison, sequence analysis revealed that the observed OPU phenotype conferred by the glpT gene was not the result of a mutation. PCR clones of glpT from both the mutant and the wild type were found to confer the OPU phenotype to JA221 when they were present on the high-copy-number pUC19 plasmid but not when they were present on the low-copy-number pWSK29 plasmid. This suggests that the OPU phenotype associated with theglpT gene is the result of amplification and overproduction of the glpT gene product. Both the active phnEand multicopy glpT genes facilitated effective metabolism of low concentrations of DIPP, whereas only the active phnEgene could confer the ability to break down a chromogenic substrate, 5-bromo-4-chloro-3-indoxyl phosphate-p-toluidine (X-Pi). This result indicates that in E. coli, X-Pi is transported exclusively by the Phn system, whereas DIPP (or its metabolite) may be transported by both Phn and Glp systems.

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“…Kinetics. The Km0 and V000r for free parathion hydrolysis activity were determined to be 18 ,uM and 3 ,umol of parathion hydrolyzed/liter per min, respectively (Fig. 2).…”

Section: Downloaded Frommentioning

confidence: 94%