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Purification and characterization of mouse glucose 6-phosphate dehydrogenase

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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 μm and 10 μm 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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Author notes

  1. James H. Yuan

    Present address: Department of Chemical Sciences, Old Dominion University, 23508, Norfolk, Virginia

  2. James M. Kramer

    Present address: Department of Human Genetics, University of Michigan, School of Medicine, 48109, Ann Arber, Michigan

Authors and Affiliations

  1. Laboratory of Environmental Mutagenesis, National Institute of Environmental Health Sciences, Research Triangle Park, 27709, North Carolina

    Chi-Yu Lee, James H. Yuan, David Mouer & James M. Kramer

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  1. Chi-Yu Lee
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  2. James H. Yuan
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  3. David Mouer
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  4. James M. Kramer
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Lee, CY., Yuan, J.H., Mouer, D. et al. Purification and characterization of mouse glucose 6-phosphate dehydrogenase. Mol Cell Biochem 24, 67–73 (1979). https://doi.org/10.1007/BF00314887

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