| Autor: |
Hsu, Yueh-Rong, Wu, Gay-May, Mendiaz, Elizabeth A., Syed, Rashid, Wypych, Jette, Toso, Robert, Mann, Michael B., Boone, Thomas C., Narhi, Linda O., Lu, Hsieng S., Langley, Keith E. |
| Zdroj: |
Journal of Biological Chemistry; March 1997, Vol. 272 Issue: 10 p6406-6415, 10p |
| Abstrakt: |
Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (Ka) of 2-4 × 108M−1, using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization Ka, greater than 90% of the circulating SCF would be in the monomeric form. When 125I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10-100 ng/ml, consistent with the Kadetermination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer Kavalues, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction. |
| Databáze: |
Supplemental Index |
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