The purification and characterization of glutathione S-transferase from the hepatopancreas of the blue crab, Callinectes sapidus |
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Authors: | W S Keeran R F Lee |
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Institution: | 1. Department of Physical, Earth and Environmental Sciences, University of Siena, via Mattioli, 4, 53100 Siena, Italy;2. CRTM Ente Parco Regionale della Maremma, Via Nizza, 24, 58010 Talamone, Gr, Italy;3. CRTM Legambiente, Sp.141 delle Saline, 71043 Manfredonia, FG, Italy;4. SUBMON, Rabassa 49, 08024 Barcelona, Spain;5. Department of Management and Quantitative Studies, University of Naples “Parthenope”, via generale Parisi 13, 80132 Naples, Italy |
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Abstract: | High glutathione S-transferase activity was found in the cytosol of F-cells from the hepatopancreas of the blue crab (Callinectes sapidus). Purification of glutathione S-transferase from hepatopancreas extracts by Sephadex G-200, DEAE-Sephacel, and chromatofocusing resulted in the isolation of two isozymes with isoelectric points of 5.9 and 5.7, as determined by analytical isoelectric focusing. Using 1-chloro-2,4-dinitrobenzene as the substrate the specific activities of the two purified isozymes were 222 and 182 mumol/min/mg, respectively. There was no evidence for basic transferase isozymes. In addition to 1-chloro-2,4-dinitrobenzene the purified glutathione S-transferase isozymes showed activity with p-nitrophenyl acetate, p-nitrobenzyl chloride, bromosulfophthalein, and benzopyrene oxide. Thus, both substitution and addition reactions associated with vertebrate glutathione S-transferase were found in the crab transferases. There was no when ethacrynic acid, methyl iodide, trans-4-phenyl-3-buten-2-one, 1,2-epoxy-(p-nitrophenoxy)propane, cumene hydroperoxide, and t-butyl hydroperoxide were used as substrates. The lack of peroxidase activity is of interest since this activity is commonly found in vertebrate transferase isozymes. The two transferases had a dimeric Mr of 40,800 with similar amino acid compositions and similar kinetic parameters (Vmax, Km, and pH maxima) with 1-chloro-2,4-dinitrobenzene as substrate. The two transferases could be distinguished by their isoelectric points, molecular mass of the monomers (22,300 for GST 1 and 22,300 and 22,400 for GST 2), and different inhibitor mechanisms with hematin and bromosulfophthalein. |
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