Abstract
Adenine uptake in cultured Chinese hamster fibroblasts showed biphasic saturation kinetics. The transport system was highly specific for adenine and was competitively inhibited by adenosine. Utilizing mutant clones of Chinese hamster fibroblasts that have either reduced or negligible adenine phosphoribosyltransferase (APRT) activity, we found that (1) adenine was not accumulated against a concentration gradient in the absence of APRT activity and (2) after rapid initial uptake equal to that of the parent the rates of adenine accumulation found for the mutants correlated strongly with their residual APRT activities. Furthermore, using either artificially depressed phosphoribosylpyrophosphate pool size and APRT activities or the mutants with decreased APRT activity, we found that adenine transport was independent of phosphorylation by APRT. These studies suggest that adenine is transported as the free base by facilitated diffusion and is subsequently phosphorylated by APRT.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alford, B. L., and Barnes, E. M. (1976). Hypoxanthine transport by cultured Chinese hamster lung fibroblasts. J. Biol. Chem. 2514823.
Berlin, R. D. (1970). Specificities of transport systems and enzymes. Science 1681539.
Hawkins, R. A., and Berlin, R. D. (1969). Purine transport in polymorphonuclear leukocytes. Biochim. Biophys. Acta 173324.
Hochstadt-Ozer, J., and Stadtman, E. R. (1971a). The regulation of purine utilization in bacteria. II. Adenine phosphoribosyl transferase in isolated membrane preparations and its role in transport of adenine across the membrane. J. Biol. Chem. 2465304.
Hochstadt-Ozer, J., and Stadtman, E. R. (1971b). The regulation of purine utilization in bacteria. IV. Roles of membrane localized and pericytoplasmic enzymes in the mechanism of purine nucleoside transport across isolated Escherichia coli membranes. J. Biol. Chem. 2472419.
Housset, P., and Nagy, M. (1977). Study of the role of purine phosphoribosyltransferase in the uptake of adenine and guanine by Schizosaccharomyces pombe cells. Eur. J. Biochem. 7399.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193265.
Quinlan, D. C., and Hochstadt, J. (1974). Uptake of hypoxanthine and inosine by purified membrane vesicles from BALB/C 3T3 cells. Fed. Proc. 331359.
Randerath, K., and Randerath, K. (1967). Thin layer separation methods for nucleic acid derivative. In Grossman and Moldave (eds.), Methods in Enzymology, Vol. XIIA, Academic Press, New York, pp. 323–347.
Rappaport, H., and DeMars, R. (1973). Diaminopurine resistant mutants of cultured Diploid Human fibroblasts. Genetics 75335.
Reem, G. H. (1975). Phosphoribosylphosphate overproduction. A metabolic abnormality in the Lesch-Nyhan syndrome. Science 1901098.
Taylor, M. W., Pipkorn, J. H., Tokito, M. K., and Pozzatti, R. O. (1977). Purine mutants of mammalian cell lines. III. Control of purine biosynthesis in adenine phosphoribosyl transferase mutants of CHO cells. Somat. Cell Genet. 3195–206.
Tsao, T., and Marzluff, G. A. (1976). Genetic and metabolic regulation of purine base transport in Neurospora crassa. Mol. Gen. Genet. 149347.
Zylka, J. M., and Plagemann, P. G. W. (1975). Purine and pyrimidine transport by cultured Novikoff cells: Specificities and mechanisms of transport and relationship to phosphoribosylation. J. Biol. Chem. 2505756.
Author information
Authors and Affiliations
Additional information
This research was supported by a grant from the USPHS, GM 18924.
Rights and permissions
About this article
Cite this article
Witney, F.R., Taylor, M.W. Role of adenine phosphoribosyltransferase in adenine uptake in wild-type and APRT− mutants of CHO. Biochem Genet 16, 917–926 (1978). https://doi.org/10.1007/BF00483743
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00483743