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PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES
Citation:
Hoehamer, C. F., C S. Mazur, AND N L. Wolfe. PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 53(1):90-97, (2005).
Impact/Purpose:
This task is divided into four major research areas: (1) Development of computational tools and databases for screening-level modeling of the environmental fate of organic chemicals; (2) Metabolism of xenobiotics: Enhancing the development of a metabolic simulator; (3) Metabonomics: The use of advanced analytical tools to identify toxicity pathways; and (4) Software infrastructure to support development and application of transformation/metabolic simulators.
For many chemicals, multiple transformation/metabolic pathways can exist. Consequently, transformation/metabolic simulators must utilize transformation rate data for prioritization of competing pathways. The prioritization process thus requires the integration of reliable rate data. When this data is absent, it is necessary to generate a database with metabolic and transformation rate constants based on: (1) experimentally measured values, including those requiring the use of advanced analytical techniques for measuring metabolic rate constants in vivo and in vitro; (2) rate constants derived from SPARC and mechanistic-based QSAR models; and (3) data mined from the literature and Program Office CBI. A long-term goal of this project is to build this database. This information will be used to enhance the predictive capabilities of the transformation/metabolic simulators. As indicated previously, exposure genomics, which provide early signs of chemical exposure based on changes in gene expression, will be used to guide chemical fate and metabolism studies. The incorporation of exposure genomics into fate studies will provide information concerning (1) the minimal concentrations at which biological events occur; and (2) the identification of biologically relevant chemicals(s) in mixtures.
The capability of categorizing chemicals and their metabolites based on toxicity pathway is imperative to the success of the CompTox Research Program. Metabonomics, which is the multi-parametric measurement of metabolites in living systems due to physiological stimuli and/or genetic modification, provides such a capability. The application of metabonomics to toxicity testing involves the elucidation of changes in metabolic patterns associated with chemical toxicity based on the measurement of component profiles in biofluids, and enables the generation of spectral profiles for a wide range of endogenous metabolites. Metabolic profiles can provide a measure of the real outcome of potential changes as the result of xenobiotic exposure.
Description:
An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase resolved a single protein band that migrated to approximately 60-kD. Nondenaturing SDS-PAGE electrophoresis revealed a single protein band around 120-kD after staining with Coomassie Brilliant blue. Quantitative gel filtration chromatography estimated a native molecular mass of this enzyme to be 120-kD. Thus, this acid phosphatase likely functions as a homodimer, consisting of two similar 60-kD subunits. An electrophoretic technique using the flourogenic substrate 4-methylumbelliferylphosphate enabled visualization of an acid phosphatase activity that corresponded to the protein band at 120-kD on a non-denaturing PAGE gel. It was determined that the acid phosphatase had a pH optimum of 6.0 at 25 degrees C. The enzyme activity appeared to be stable over a broad range of temperatures (10-40 degrees C) and in the presence of the metals Zn+2 , Mn+2 , and Mg+2 as well as the chelating agents EDTA and EGTA. It was shown that this acid phosphatase could hydrolyze a variety of physiological organophosphate compounds including, beta-glycerophosphate, phosphoserine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monphosphate (AMP) and pyrophosphate. Furthermore, analysis using capillary electrophoresis demonstrated this hydrolytic enzyme could transform a wide array of organophosphate pesticides including S-2-ethylthioethyl O, O-dimethyl phosphorothioate (demeton-S-methyl); S-1, 2-bis(ethoxycarbonyl)ethyl O, O-dimethyl phosphorodithioate (Malathion); O, O-dimethyl O-4-nitrophenyl (paraoxon); O, O, O, O-tetraethyl dithiopyrophosphate (sulfatep); O-2-chloro-4-nitrophenyl O, O-dimethyl phosphorothioate (dicapthon); and 2, 2-dichlorovinyl dimethyl phosphate (dichlorvos).