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Influence of transformation by Rous sarcoma virus on the amount, phosphorylation and enzyme kinetic properties of enolase.
Author(s) -
Eigenbrodt E.,
Fister P.,
Rübsamen H.,
Friis R.R.
Publication year - 1983
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1983.tb01625.x
Subject(s) - enolase , rous sarcoma virus , biology , microbiology and biotechnology , dephosphorylation , phosphorylation , biochemistry , transformation (genetics) , enzyme , tyrosine , isoelectric focusing , phosphatase , immunohistochemistry , immunology , gene
Using chicken embryo fibroblasts infected with the NY68 transformation‐defective temperature‐sensitive mutant of Rous sarcoma virus, the phosphorylation and enzyme kinetic properties of enolase have been studied before, and at different stages after, the onset of transformation. A method for purification of enolase was developed, which minimized dephosphorylation. Two enolase (EC 4.2.1.11) isoenzymes were separated by isoelectric focussing revealing that it was the gammagamma form (pI 5.2‐6.7) which had become phosphorylated at tyrosine residues after transformation. The phosphorylation of enolase in tyrosine occurred slowly after shift to the permissive temperature, rising from undetectable levels in phenotypically normal cells, to < 10% of the total phosphoamino acid after 3 h, and reaching 30‐50% of the total phosphoamino acid by 16 h. Interestingly, the fraction of phosphorylated enolase molecules declined during transformation from 8% in normal cells to 5% by 16 h after temperature shift, due to a 3‐ to 5‐fold increase in the total amount of enolase present in the transformed cultures. Although transformation had no apparent effect on the K0.5 of enolase (26 +/‐ 4 microM for 2‐phosphoglycerate), its specific activity was reduced by about one third.