Partial characterization of phosphotriesterase activity from the earthworm, Eisenia andrei
Introduction
Organophosphate-degrading enzymes have been intensively investigated in microorganisms, since they can be potentially utilized to detoxify environmental pollutants such as industrial wastes and pesticides (Caldwell 1991a, Caldwell 1991b; Omburo et al., 1993; Benning et al., 1994). Phosphotriesterase (PTE) is an enzyme that is able to hydrolyze organophosphate triesters. PTE was first detected in the soil microorganisms, Pseudomonas diminuta and Flavobacterium sp., which is capable of hydrolyzing paraoxon and parathion at a high catalytic rate (Munnecke and Hsieh, 1974). PTE is a protein encoded by a plasmid-borne gene, known as opd (organophosphate degradation gene) (Serdar and Gibson, 1985; Harper et al., 1988). It has been also reported that PTE from P. diminuta is a monomeric protein having an apparent molecular weight of 39 kDa and its activity is inhibited by dithiothreitol and dependent on zinc ion (Dumas et al., 1989). In addition, this enzyme appeared to be able to hydrolyze commonly used insecticides and associated metabolites such as parathion, diazinon, cyanophos and paraoxon. The enzymatic hydrolysis of anticholinesterase organophosphorous compounds has been identified in various organisms including bacteria (Attaway et al., 1987), protozoa (Landis et al., 1987), clam (Anderson et al., 1988), squid (Hoskin and Roush, 1982) and human (Mueller et al., 1983; Ortigoza-Ferado et al., 1984). The PTE system from P. diminuta has been intensively investigated.
In soil macroinvertebrates such as earthworms, PTE system was found, to effectively hydrolyze organophosphorous pesticides applied to soil (Stenersen, 1979). It is likely that in earthworm, this enzyme is involved in the bioactivation of selected organophosphates by producing metabolites which are more toxic than the parent compound. Earthworms play an important role in the disposition of soil xenobiotics. Among enzyme systems responsible for xenobiotic metabolism, the characteristics and significance of phosphomonoesterase and phosphodiesterase activities in the earthworm have been investigated (Park 1990, Park 1992). These enzymes were shown to bioactivate pesticides containing p-nitrophenyl moiety such as p-nitrophenylphosphate, bis(p-nitrophenyl)phosphate, parathion and paraoxon by releasing p-nitrophenol as metabolite (Park et al., 1993).
In the earthworm, it has been reported that paraoxon hydrolase, a PTE, was able to hydrolyze paraoxon to produce diethylphosphate and p-nitrophenol in vivo and in vitro. This activity appeared to be associated with the microsomal fraction and was not inhibited by EDTA and 4-(chloromercuri)benzoic acid and incompletely inhibited by aldicarb (Stenersen, 1979). However, the histological distribution and fundamental biochemical properties have not been determined, which would be essential to better understand the physiological and ecotoxicological significance of PTE system in this animal species. The present study reports the selected biochemical properties of an earthworm phosphotriesterase which differs significantly from the bacterial phosphotriesterases.
Section snippets
Experimental animals
Sexually mature Eisenia andrei were reared by the method of Park 1992, Park 1996. Each plastic container containing about 60 worms was placed in a darkened incubator at 23±1°C and powdered cow dung was daily supplied as food material. Before use, worms were placed on filter paper moistened with earthworm saline (Storey, 1989) for at least 24 h to induce purging of the intestine. To minimize phosphotriesterase activity derived from gut microflora, approximately 0.5 ml of earthworm saline was very
Results and discussion
As shown in Fig. 1, all tissues tested exhibited PTE activity. However, over 90% of PTE activity appeared to be associated with gut tissues such as chloragogue and epithelial tissues. The highest level PTE activity was found in the epithelial tissue. Although it is not clear whether PTE of the earthworm is released into the lumen, its distribution suggests that organophosphotriesters ingested with soil would be more effectively hydrolyzed by epithelial PTE, since they are usually hydrophobic
Acknowledgements
This paper was supported by NON DIRECTED RESEARCH FUND, Korea Research Foundation, 1996.
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