Biochemical Genetics

, Volume 17, Issue 7–8, pp 621–630 | Cite as

Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

  • Vassilis I. Zannis
  • Lorraine J. Gudas
  • David W. MartinJr.
Article

Abstract

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.

Purine nucleoside phosphorylase has been purified at least a hundredfold from 35S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of 35S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.

Key words

purine nucleoside phosphorylase human fibroblasts two-dimensional gels 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Agarwal, K. C., Agarwal, R. P., Parks, R. E., Jr., and Baldini, M. G. (1973). Electrophoretic heterogeneity and physiochemical properties of human erythrocytic purine nucleoside phosphorylase. Fed. Proc. 32581.Google Scholar
  2. Edwards, Y. H., Hopkinson, D. A., and Harris, H. (1971). Inherited variants of human nucleoside phosphorylase. Ann. Hum. Genet. 34395.Google Scholar
  3. Giblett, E. R., Ammann, A. J., Sandman, R., Wara, D. W., and Diamond, L. K. (1975). Nucleoside phosphorylase deficiency in a child with severely defective T-cell immunity and normal B-cell immunity. Lancet 11010.Google Scholar
  4. Gudas, L. J., Zannis, V. I., Clift, S. M., Ammann, A. J., Staal, G. E. J., and Martin, D. W., Jr. (1978). Characterization of mutant subunits of human purine nucleoside phosphorylase. J. Biol. Chem. 2538916.Google Scholar
  5. Kim, B. K., Cha, S., and Parks, R. E., Jr. (1968). Purine nucleoside phosphorylase from human erythrocytes 1. Purification and properties. J. Biol. Chem. 2431763.Google Scholar
  6. Korn, E. D., and Buchanan, J. M. (1955). Biosynthesis of the purines VI. J. Biol. Chem. 217183.Google Scholar
  7. Snyder, F. F., and Henderson, J. F. (1973) Alternative pathways of deoxyadenosine and adenosine metabolism. J. Biol. Chem. 2485899.Google Scholar
  8. Tsuboi, K. K., and Hudson, P. B. (1957) Enzymes of the human erythrocyte II. Purine nucleoside phosphorylase: specific properties. J. Biol. Chem. 224889.Google Scholar
  9. Zannis, V., Doyle, D., and Martin, D. W., Jr. (1978). Purification and characterization of human erythrocyte purine nucleoside phosphorylase and its subunits. J. Biol. Chem. 253504.Google Scholar
  10. Zimmerman, T. P., Gersten, N., and Miech, R. P. (1970). Adenine and adenosine metabolism in liver. Proc. Am. Assoc. Cancer Res. 1187.Google Scholar
  11. Zimmerman, T. P., Gersten, N., Ross, A. F., and Miech, R. P. (1971). Adenine as substrate for purine nucleoside phosphorylase. Can. J. Biol. 491050.Google Scholar

Copyright information

© Plenum Publishing Corporation 1979

Authors and Affiliations

  • Vassilis I. Zannis
    • 1
  • Lorraine J. Gudas
    • 1
  • David W. MartinJr.
    • 1
  1. 1.Howard Hughes Medical Institute Laboratory and Division of Medical Genetics, Department of Medicine and Department of Biochemistry and BiophysicsUniversity of California, San FranciscoSan Francisco

Personalised recommendations