Purification and characterization of mouse glucose 6-phosphate dehydrogenase |
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Authors: | Chi-Yu Lee James H. Yuan David Mouer James M. Kramer |
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Affiliation: | (1) Laboratory of Environmental Mutagenesis, National Institute of Environmental Health Sciences, Research Triangle Park, 27709 North Carolina;(2) Present address: Department of Chemical Sciences, Old Dominion University, 23508 Norfolk, Virginia;(3) Present address: Department of Human Genetics, University of Michigan, School of Medicine, 48109 Ann Arber, Michigan |
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Abstract: | Summary Glucose-6-phosphate dehydrogenase was purified to homogeneity from testes and kidneys of the inbred strain of mice (DBA/2J) by a simple two-step affinity column procedure. This involved the sequential application of 8-(6-aminohexyl)-amino-AMP-and -2, 5-ADP-Sepharose columns and biospecific elution with NADP+ in both steps. The molecular and biochemical properties of the purified enzyme were studied in detail. These include the molecular weight determination, amino acid composition, steady-state kinetics, inactivation by high temperature, urea and iodoacetate, and immunology. The purified enzyme from mouse kidneys or testes was shown to be a tetramer with a molecular weight of 220,000. The enzyme is highly specific for glucose-6-phosphate, exhibits almost no activity with NAD+ as a coenzyme and is little inhibited by AMP or ATP. Michaelis constants for glucose-6-phosphate and NADP+ were determined to be 50 m and 10 m respectively. NADPH is a competitive inhibitor of NADP+ and has a Ki of 18 µm. Rabbit antisera against glucose-6-phosphate dehydrogenase were raised. The antisera also cross-react with the same enzyme from human and guinea pig. |
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