Abstract
Diaphorase-1 and diaphorase-2 were isolated from twoDrosophila species,D. virilis andD. melanogaster, and purified by gel filtration, affinity chromatography, immunoaffinity chromatography, and ion-exchange chromatography. The molecular weights of both enzymes were the same in each species. The molecular weight of diaphorase-1 was the same under both denaturating and nondenaturating conditions, close to 60,000, indicating a monomeric structure. Sodium dodecyl sulfate (SDS) electrophoresis of the purified diaphorase-2 revealed the presence of a single protein band of 55,000 Da, while the molecular weight of the native enzyme was found to be 67,000. The two diaphorases were further characterized by their pH optima, isoelectric points, and kinetic parameters, and antibodies were raised in rabbits against the purified enzymes fromD. virilis. The antibodies showed no cross-reactions but recognized the corresponding diaphorases inD. melanogaster andD. novamexicana as well asD. virilis. The data obtained confirmed the hypothesis of an independent genetic control of diaphorase-1 and diaphorase-2 inDrosophila.
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References
Bellstedt, D. U., Human, P. A., Rowland, G. F., and Van der Merwe, K. J. (1987). Acid-treated, naked bacteria as immune carriers for protein antigens.J. Immunol. Methods 98249.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72248.
Chrambach, A., and Rodbard, D. (1971). Polyacrilamide gel electrophoresis.Science 172440.
Dunkov, B. C. (1988). Ph.D. thesis, Sofia University, Sofia, Bulgaria.
Dunkov, B. C., Georgieva, T. G., and Ralchev, K. H. (1991). Diaphorase activity associated withDrosophila xanthine dehydrogenase.Dros. Inform. Serv. (in press).
Engvall, E., and Perlmann, P. (1972). Enzyme-linked immunosorbent assay, ELISA. III. Quantitation of specific antibodies by enzyme-labelled anti-immunoglobulin in antigencoated tubes.J. Immunol. 109129.
Ernster, L., Danielson, L., and Ljunggren, M. (1962). DT diaphorase. I. Purification from the soluble fraction of rat-liver cytoplasm, and properties.Biochim. Biophys. Acta 58471.
Ferguson, K. A. (1964). Starch-gel electrophoresis—application to the classification of pituitary proteins and polypeptides.Metabolism 13985.
Georgieva, T. G., Dunkov, B. C., and Ralchev, K. H. (1991). Effects of inhibitors and activators on the activity of two diaphorases fromDrosophila virilis, Insect Biochem. 21145.
Harano, T., and Chino, H. (1971). A new diaphorase fromBombyx silkworm eggs-cytochrome c reductase activity mediated with xanthommatin.Arch. Biochem. Biophys. 146467.
Hawkes, R., Niday, E., and Gordon, J. (1982). A dot-immunoblotting assay for monoclonal and other antibodies.Anal. Biochem. 119142.
Hedrick, J. L., and Smith, A. J. (1968). Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis.Arch. Biochem. Biophys. 126155.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of bacteriophage T4.Nature 227680.
Ralchev, K. H., Dunkov, B. C., Doichinov, A., and Simeonovska, M. (1987). Electrophoretic spectrum of diaphorases inDrosophila, Genet. Breed. 20196.
Ralchev, K. H., and Harisanova, N. T. (1985). A convenient method for mass production and harvesting of synchronousDrosophila embryos.Dros. Inform. Serv. 61196.
Sedmak, J. J., and Grossberg, S. E. (1977). A rapid, sensitive, and versatile assay for protein using coomassie brilliant blue G250.Anal. Biochem. 79544.
Thomas, N., Jones, C. N., and Thomas, P. L. (1988). Low volume processing of protein blots in rolling drums.Anal. Biochem. 170393.
Yoshimura, H., Hokama, Y., Wada, I., Koga, N., and Yoshihara, S. (1987). Comparison of DT-diaphorases purified from the liver cytosol of untreated, and 3,4,5,3′,4′-pentachlorobiphenyl- and 3-methylcholanthrene-treated rats.J. Biochem. 1021547.
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Ralchev, K.H., Petkov, P.M. & Dunkov, B.H. Purification and characterization of diaphorases from someDrosophila species. Biochem Genet 30, 305–315 (1992). https://doi.org/10.1007/PL00020474
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DOI: https://doi.org/10.1007/PL00020474