Purification and partial characterization of the glutathione S-transferase of rat erythrocytes |
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| Institution: | 1. School of International Trade and Economics, University of International Business and Economics, Beijing 100029, PR China;2. Department of Decision Sciences, NUS Business School, National University of Singapore, Singapore 119245, Singapore;1. Department of Chemical and Biological Engineering, Colorado School of Mines, Golden, Colorado, USA;2. Department of Chemistry, Colorado School of Mines, Golden, Colorado, USA;3. Department of Anesthesiology, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, USA;4. Departments of Bioengineering and Pediatrics, Hemophilia and Thrombosis Center, University of Colorado Denver, Anschutz Medical Campus, Aurora, Colorado, USA |
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| Abstract: | The single glutathione S-transferase (EC 2.5.1.18) present in rat erythrocytes was purified to apparent homogeneity by affinity chromatography on glutathione-Sepharose and hydroxyapatite chromatography. Approx. 1.86 mg enzyme is found in 100 ml packed erythrocytes and accounts for about 0.01% of total soluble protein. The native enzyme (Mr 48 000) displays a pI of 5.9 and appears to possess a homodimeric structure with a subunit of Mr 23 500. Enzyme activities with ethacrynic acid and cumene hydroperoxide were 24 and 3%, respectively, of that with 1-chloro-2,4-dinitrobenzene. The Km values for 1-chloro-2,4-dinitrobenzene and glutathione were 1.0 and 0.142 mM, respectively. The concentrations of certain compounds required to produce 50% inhibition (I50) were as follows: 12 μM bromosulphophthalein, 34 μM S-hexylglutathione, 339 μM oxidized glutathione and 1.5 mM cholate. Bromosulphophthalein was a noncompetitive inhibitor with respect to 1-chloro-2,4-dinitrobenzene (Ki = 8 μM) and glutathione (Kis = 4 μM; Kii = 11.5 μM) while S-hexylglutathione was competitive with glutathione (Ki = 5 μM). |
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