Abstract
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 x 10(8) M-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 Ka determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer Ka values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165 disulfide-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.
Highlights
Soluble Escherichia coli-derived recombinant human stem cell factor forms a non-covalently associated dimer
When 125I-recombinant human stem cell factor (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 Ka determination
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
Summary
(Received for publication, September 10, 1996, and in revised form, December 13, 1996). 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 ؋ 108 M؊1, using sedimentation equilibrium and size exclusion chromatography. The dimer Ka values, biophysical properties, and biological activities of these variants were studied. Valently associated dimers, as determined by sedimentation equilibrium and size exclusion chromatography at protein concentrations above 0.4 mg/ml [3]. We arrive at a value of 2– 4 ϫ 108 MϪ1 for the dimer association constant (Ka) of E. coli-derived rhSCF, based on several approaches including ultracentrifugation and size exclusion chromatography at low SCF concentrations. Characterization of dimer association constant Ka, biophysical properties, and biological activities of these variants give results consistent with the hypothesis that SCF dimerization is important for Kit dimer-. Models for SCF-induced Kit dimerization are discussed in light of the results
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