Abstract
Phosphotriesterase (PTE) activities in mammalian serum are typically found in the lipoprotein fraction. This PTE requires Ca++ for activity and is consequently inactivated by ethylenediaminetetraacetic acid (EDTA). There is also a little known PTE in mammal serum that is resistant to EDTA inactivation. In this work, the PTE activities for the substrates O-hexylO-2,5-dichlorophenyl phosphoramidate (HDCP) andO,O′-diethyl p-nitrophenyl phosphate were purified from rabbit serum by ultracentrifugation, molecular exclusion, and anion exchange chromotography. Rabbit serum produced two PTE activities. One was sensitive and the other was resistant to EDTA inhibition. The EDTA-resistant HDCP hydrolyzing activity and paraoxonase activities of rabbit serum were purified to homogeneity. These activities copurified and were associated to albumin. This EDTA-resistant activity exhibited no stereoselectivity in the hydrolysis of HDCP. The EDTA-sensitive activity was isolated in the lipoprotein fraction and stereoselectively hydrolyzed theS-HDCP over the R-HDCP. Other differences between the EDTA-sensitive paraoxonase and HDCP hydrolyzing activity were discovered in response top-nitrophenylbutkyrate, 5,5-dithio-bis(2-nitrobenzoic acid), caprylic acid, sodium ions, and ammonium ions. This work demonstrates the existence of two well differentiated PTE activities in rabbit serum. One is sensitive to EDTA, stereoselective, and found in the lipoprotein fraction, and the other is resistant to EDTA inhibition and nonstereospecific.
Footnotes
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Send reprint requests to: Professor E. Vilanova, Unidad de Toxicologı́a y Seguridad Quı́miCa Instituto de Bioingenierı́a, Universidad Miguel Hernández, Campus de San Juan. E-03550 San Juan de Alicante, Alicante, Spain. E-mail:evilanova{at}umh.es
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This study was financed by DGICT SAF96/0168. M.A.S. and I.S. received a grant from the Spanish Ministry of Education and Science and from the Xunta de Galicia, respectively.
- Abbreviations used are::
- DCP
- 2,5-dichlorophenol
- EDTA
- ethylenediaminetetraacetic acid
- HDCP
- O-hexyl,O-2,5-dichlorophenyl phosphoramidate
- HDCPase
- HDCP hydrolyzing activity
- L fraction
- lipoprotein fraction
- paraoxon
- O,O′-diethyl p-nitrophenyl phosphate
- paraoxonase
- paraoxon hydrolyzing activity
- p-NP
- p-nitrophenol
- PTE
- phosphotriesterase
- S fraction
- soluble fraction
- SS fraction
- S fraction from a second centrifugation
- Received March 12, 1998.
- Accepted August 20, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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