Summary
A simple, fast and direct staining method for the detection of hypoxanthineguanine phosphoribosyltransferase is described. It is based on the conversion of inosine monophosphate to hypoxanthine, which is then enzymatically oxidized. This oxidation is coupled to the reduction of a tetrazolium salt to blue formazan. The electrophoretic pattern of HGPRT in man-Chinese hamster hybrid cells shows a three-banded pattern, suggesting a dimeric structure for this enzyme.
Zusammenfassung
Eine einfache, rasche und direkte Färbemethode für die Darstellung der Hypoxanthin-Guanin-Phosphoribosyltransferase wird beschrieben. Sie beruht auf der Umwandlung von Inosin-Monophosphat in Hypoxanthin, das dann enzymatisch oxydiert wird. Diese Oxydation ist mit einer Reduktion eines Tetrazoliumsalzes zu blauem Formazan gekoppelt. Die Elektrophorese von HGPRT in Mensch-Chinesischer Hamster-Hybridzellen zeigt ein Muster mit drei Banden, das eine dimere Struktur dieses Enzyms nahelegt.
This is a preview of subscription content, log in via an institution to check access.
References
Adams, A., Anderson, J. M., Nicol, A. D., Harkness, R. A.: The development of hypoxanthine/guanine phosphoribosyltransferase activity in man. Biochem. J. 125, 36P (1971)
Arnold, W. J., Kelley, W. N.: Human hypoxanthine-guanine phosphoribosyltransferase: Purification and subunit structure. J. biol. Chem. 246, 7398–7404 (1971)
Bakay, B., Nyhan, W. L.: Electrophoretic properties of hypoxanthine-guanine phosphoribosyl transferase in erythrocytes of subjects with Lesch-Nyhan syndrome. Biochem. Genet. 6, 139–146 (1972)
Der Kaloustian, V. M., Byrne, R., Young, W. J., Childs, B.: An electrophoretic method for detecting hypoxanthine-guanine phosphoribosyl transferase variants. Biochem. Genet. 3, 299–302 (1969)
Edwards, Y. H., Hopkinson, D. A., Harris, H.: Inherited variants of human nucleoside phosphorylase. Ann. hum. Genet. 34, 395–408 (1971)
Littlefield, J. W.: Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709–710 (1964)
Meera Khan, P.: Enzyme electrophoresis on cellulose acetate gel: Zymogram patterns in man-mouse and man-Chinese hamster somatic cell hybrids. Arch. Biochem. Biophys. 145, 470–483 (1971)
Murray, A. W., Friedrichs, B.: Inhibition of 5′-nucleotidase from Ehrlich ascites-tumour cells by nucleoside triphosphates. Biochem. J. 111, 83–89 (1969)
Newcombe, D. S., Willard, J. M.: A spectrophotometric assay for hypoxanthine-guanine phosphoribosyltransferase. Analyt. Biochem. 43, 454–459 (1971)
Ropers, H. H.: Simplified methods for the measurement and electrophoretic demonstration of hypoxanthine-guanine phosphoribosyl transferase. Humangenetik 17, 69–74 (1972)
Seegmiller, J. E., Rosenbloom, F. M., Kelley, W. N.: Enzyme defect associated with a sexlinked human neurological disorder and excessive purine synthesis. Science 155, 1682–1684 (1967)
Shin, S., Meera Khan, P., Cook, P. R.: Characterization of hypoxanthine-guanine phosphoribosyl transferase in man-mouse somatic cell hybrids by an improved electrophoretic method. Biochem. Genet. 5, 91–99 (1971)
Spencer, N., Hopkinson, D. A., Harris, H.: Adenosine deaminase polymorphism in man. Ann. hum. Genet. 32, 9–14 (1968)
Westerveld, A., Visser, R. P. L. S., Meera Khan, P., Bootsma, D.: Loss of human genetic markers in man-Chinese hamster somatic cell hybrids. Nature (Lond.) New Biol. 234, 20–24 (1971)
Author information
Authors and Affiliations
Additional information
This work was supported in part by the Netherlands Foundation for Medical Research.
Rights and permissions
About this article
Cite this article
van Someren, H., van Henegouwen, H.B. & Khan, P.M. Rapid detection of hypoxanthine-guanine phosphoribosyltransferase on cellogel. Humangenetik 22, 75–80 (1974). https://doi.org/10.1007/BF00338138
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00338138