Summary
A simple and unique procedure was developed to purify phosphoglucose isomerase variants from the whole mouse body extracts and Drosophila homogenate. It involved the use of an 8-(6-aminohexyl)-amino-ATP-Sepharose column followed by a preparative isoelectric focusing. In each case, the enzyme in the homogenate was adsorbed by ionic interaction on the ATP-Sepharose column. Substantial purification was achieved by the affinity elution with the substrate-glucose-6-phosphate. Mouse and Drosophila phosphoglucose isomerase as well as the corresponding variants were shown to be dimers of similar molecular weight and to exhibit similar kinetic properties. The isoelectric points for the variants from DBA/2J and C57BL/6J mice were determined to be 8.4 and 8.7 respectively, while they were 6.8 and 6.3 respectively for Drosophila and 4/4 variants. Differential thermal stability was observed for the two mouse variants but not for the Drosophila ones. Amino acid composition analysis was performed for both mouse and Drosophila enzymes. Rabbit antisera for mouse (DBA/2J) and Drosophila (2/2) enzymes were raised. Within each species, complete immunological identity was observed between the variants. The antisera were used to characterize the null mutants of phosphoglucose isomerase identified in the mouse and Drosophila populations. By rocket immunoelectrophoresis, the null allele of the naturally occurring heterozygous null variant of Drosophila was shown to express no cross-reacting materials (CRM).
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References
Lee, C.-Y., Charles, D., Bronson, D., Griffin, M. and Bennett, L., 1979. Mol. Gen. Genetics (in press).
Mukai, T. and Cockerham, C. C., 1977. Proc. Nat. Acad. Sci. 74, 2514–2517.
Lee, C.-Y., Charles, D. and Bronson, D., 1979. J. Biol. Chem. 254, 6375–6381.
Lee, C.-Y. and Pegoraro, B., 1979. Biochem. Genetics 17, 631–644.
Racine, R., Langley, C. H., and Voelker, R. A., 1979. Genetics (submitted).
Scares, E. R., 1978 Mouse News Lett. 59, 11–12.
Voelker, R. A., Onishi, S. and Langley, C. H. 1979) Genetics (submitted)
DeLorenzo, R. J. and Ruddle, F. H. 1969. Biochem. Genetics 3, 151–162.
Carter, N. D. and Yosida, A. 1969. Biochem. Biophys. Acta 181, 12–19.
Kempe, T. D. Nakagawa, Y. and Noltmann, E. A. 1974. J. Biol. Chem. 249, 4617–4624.
Noltmann, E. A. 1966. In Methods in Enzymology, vol. 9 (Wood, W. A., ed.) pp. 557–565, Academic Press, New York and London.
Reithel, F. J. 1966. In Methods in Enzymology, vol. 9 (Wood, W. A., ed.) pp. 565–568, Academic Press, New York and London.
Muirhead, H. and Shaw, P. J. 1974. J. Mol. Biol. 89, 195–203.
Nosoh, Y. 1975. In Methods in Enzymology, vol. 41, Part B (Wood, W. A., ed.) pp. 353–387, Academic Press, New York and London.
Böhlen, P., Stein, S., Dairman, W. and Undefriend, S. 1973. Arch. Biochem. Biophys. 155, 213–220.
Laemmli, U. K. 1970. Nature 227, 680–685.
Lee, C.-Y., Lazarus, L. H., Kabakoff, D. S., Laver, M. B., Russell, P. J. and Kaplan, N. O. (1977) Arch. Biochem. Biophys. 178, 8–18.
Martin, R. G. and Ames, B. N. 1961. J. Biol. Chem. 236, 1372–1379.
Siegel, L. M. and Monty, K. 1966. Biochem. Biophys Acta 112, 346–362.
Vesterburg, O. and Svensson, H. 1966. Acad. Chem. Scand. 20, 820–834.
Ouchterlony, O., 1958. In Progress in Allergy vol. 5 (Kallos, P. T., ed.) pp. 1–78, S. Kager, Basel and New York.
Hirs, C. H. W., 1967. In Methods in Enzymology vol. 11 (Hirs, C. H. W., ed) pp. 197–199, Academic Press, New York and London.
Matsubara, H. and Sasaki, R. M., 1969. Biochem. Biophys. Res. Comm. 35, 175–181.
Lee, C.-Y. and Johansson, C.-J., 1977. Anal. Biochem. 77, 90–102.
Lee, C.-Y., Leigh-Brown, A., Langley, C. H. and Charles, D., 1978. J. Solid Phase Biochem. 2, 213–223.
Lee, C.-Y., Neisel, D. and Bewley, G., 1979. Biochem. Genet. (submitted).
Jackson, D. A., Symons, R. H. and Berg, P., 1972. Proc. Nat. Acad. Sci., 69, 2904–2909.
Morrow, J. F., Cohen, S. N., Chang, A. C. Y., Boyer, H. W., Goodman, H. M., and Helling, R. B., 1974. Proc. Nat. Acad. Sci. 71, 1743–1747.
Tanaka, T., Weisblem, B., Schons, M. and Inman, R. B., 1974. Biochem. 14, 2064–2072.
Glover, D. M., white, R. L., Finnegan, D. J., and Hogness, D. S., 1975. Cell 5, 149–157.
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Charles, D., Lee, CY. Biochemical characterization of phosphoglucose isomerase and genetic variants from mouse and Drosophila melanogaster . Mol Cell Biochem 29, 11–21 (1980). https://doi.org/10.1007/BF00230952
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DOI: https://doi.org/10.1007/BF00230952