Summary
An immunochemical method has been developed for the quantitative determination of species-specific gene products, for instance a-galactosidase and N-acetyl-a-galactosaminidase, in man-rodent hybrid cells and in the parental cell lines. Antisera raised against the purified enzymes are covalently coupled to Sepharose 4B. The gene products are specifically removed from a cell lysate by incubating with the appropriate Sepharose-coupled antiserum. After centrifugation followed by washing of the precipitated Sepharose, the enzymic activity can be quantitatively measured on the Sepharose beads. With this technique it has been demonstrated that the ability of human N-acetyl-a-galactosaminidase (also known as a-galactosidase B) to hydrolyze a-galactosidic linkages is lost when the enzyme is expressed in man-Chinese hamster hybrid cells.
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References
Bergmeyer, H. U., Gawehn, K., Grassl, M.: In: Methoden der Enzymatischen Analyse (H. U. Bergmeyer, Ed.), Vol. 1, p. 457. Weinheim: Verlag Chemie 1970
Beutler, E., Kuhl, W.: Purification and properties of human α-galactosidases. J. Biol. Chem. 247, 7195–7200 (1972)
Beutler, E., Guinto, E., Kuhl, W.: Variability of α-galactosidase A and B in different tissue of man. Amer. J. Hum. Genet. 25, 42–46 (1973)
Champion, M. J., Shows, T. B.: Mannosidosis: Assignment of the lysosomal α-mannosidase B gene to chromsome 19 in man. Proc. Natl. Acad. Sci. USA 74, 2968–2972 (1977)
Chern, C. J.: Detection of active heteropolymeric β-glucuronidase in hybrids between mouse cells and human fibroblasts with β-glucuronidase deficiency. Proc. Natl. Acad. Sci. USA 74, 2948–2952 (1977)
Chern, C. J., Beutler, E., Kuhl, W., Gilbert, F., Mellman, W. J., Croce, C. M.: Characterization of heteropolymeric hexosaminidase A in man x mouse hybrid cells. Proc. Natl. Acad. Sci. USA 73, 3637–3640 (1976)
Cuatrecasas, P.: Protein purification by affinity chromatography. J. Biol. Chem. 245, 3059–3065 (1970)
Dean, K. J., Sung, S. S. J., Sweeley, C. C.: The identification of α-galactosidase B from human liver as an N-acetyl-α-galactosaminidase. Biochem. Biophys. Res. Commun. 77, 1411–1417 (1977)
Groot, P. G. de, Westerveld, A., Meera Khan, P., Tager, J. M.: Localization of a gene for human α-galactosidase B(=N-acetyl-α-D-galactosaminidase) on chromosome 22. Hum. Genet. 44, 305–312 (1978)
Grzeschik, K. H., Grzeschik, A. M., Banhof, S., Romeo, G., Siniscalco, M., Someren, H. van, Meera Khan, P., Westerveld, A., Bootsma, D.: X-linkage of human α-galactosidase. Nature New Biol. 240, 48–50 (1972)
Hamers, M. N., Westerveld, A., Meera Khan, P., Tager, J. M.: Characterization of α-galactosidase isoenzymes in normal and Fabry human-Chinese hamster somatic hybrids. Hum. Genet. 36, 289–297 (1977)
Held, K. R., Kahan, B., DeMars, R.: Adenine phosphoribosyltransferase and hypoxanthineguanine phosphoribosyltransferase immunoprecipitation reactions in human-mouse and human-Chinese hamster hybrids. Hum. Genet. 30, 23–34 (1975)
Hoeksema, H. L., Reuser, A. J. J., Hoogeveen, A., Westerveld, A., Braidman, I., Robinson, D.: Characterization of β-D-N-acetylhexosaminidase isoenzymes in man-Chinese hamster somatic cell hybrids. Am. J. Hum. Genet. 29, 14–23 (1977)
Kusiak, J. W., Quirk, J. M., Brady, R. O.: Purification and properties of the two major isoenzymes of α-galactosidase from human placenta. J. Biol. Chem. 253, 184–190 (1978)
Lalley, P. A., Rattazzi, M. C., Shows, T. B.: Human β-D-N-acetyl-hexasaminidases A and B: Expression and linkage relationship in somatic cell hybrids. Proc. Natl. Acad. Sci. USA 71, 1569–1573 (1974)
Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with the folinphenol reagent. J. Biol. Chem. 193, 265–275 (1951)
McKusick, V. A., Ruddle, F. H.: The status of the gene map of the human chromosomes. Science 196, 390–405 (1977)
Meera Khan, P., Westerveld, A., Würzer-Figurelli, E. M., Bootsma, D.: α-Galactosidase in man-Chinese hamster somatic cell hybrids. Human Gene Mapping 2, Rotterdam Conference 1974, Vol. XI, pp. 205–210. New York: The National Foundation March of Dimes (1975)
Neuwelt, E., Kohler, P. F., Austin, J.: Primary enzyme immunoassay. Studies of the mutant enzyme in metachromatic leukodystrophy (primary enzyme immunoassay of arylsulfatase A). Immunochemistry 10, 767–773 (1973)
Palser, H. R., McAlpine, P. J.: An immunochemical method for the detection of the expression of human gene loci in human-rodent somatic cell hybrids with special reference to the GPI locus. Biochem. Genet. 14, 661–670 (1976)
Rebourcet, R., Weil, D., Van Cong, N., Frézal, J.: α-Galactosidase: A dimeric enzyme dependent on a structural locus on the X-chromosome. Cytogenet. Cell Genet. 14, 406–408 (1975)
Schram, A. W., Hamers, M. N., Brouwer-Kelder, B., Donker-Koopman, W. E., Tager, J. M.: Enzymological properties and immunological characterization of α-galactosidase isoenzymes from normal and Fabry human liver. Biochim. Biophys. Acta 482, 125–137 (1977a)
Schram, A. W., Hamers, M. N., Tager, J. M.: The identity of α-galactosidase B from human liver. Biochim. Biophys. Acta 482, 138–144 (1977b)
Shimizu, N., Shimizu, Y., Kucherlapati, R. S., Ruddle, F. H.: Immunochemical detection of human enzymes in hybrid cells. Cell 7, 123–130 (1976)
Someren, H. van, Beijersbergen van Henegouwen, H.: Independent loss of human hexosaminidases A and B in man-Chinese hamster somatic cell hybrids. Humangenetik 18, 171–174 (1973)
Swallow, D. M., Solomon, E., Pajunen, L.: Immunochemical analysis of the N-acetylhexosaminidases in human-mouse hybrids made using a double selective system. Cytogenet. Cell Genet. 18, 136–148 (1977)
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 New Biol. 234, 20–24 (1971)
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de Groot, P.G., Hamers, M.N., Westerveld, A. et al. A new immunochemical method for the quantitative measurement of specific gene products in man-rodent somatic cell hybrids. Hum Genet 44, 295–304 (1978). https://doi.org/10.1007/BF00394294
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DOI: https://doi.org/10.1007/BF00394294