THE MAJORITY OF STEM-CELL FACTOR EXISTS AS MONOMER UNDER PHYSIOLOGICAL CONDITIONS

Soluble Escherichia coli-derived recombinant human stem cell factor (r hSCF) forms a non-covalently associated dimer. We have determined a di mer association constant (K-a) of 2-4 x 10(8) M(-1), using sedimentati on equilibrium and size exclusion chromatography. SCF has been shown p reviously to be present at concentrations of approximately 3.3 ng/ml i n human serum, Based on the dimerization K-a, greater than 90% of the circulating SCF would be in the monomeric form. When I-125-rhSCF was a dded to human serum and the serum analyzed by size exclusion chromatog raphy, 72-49% of rhSCF was monomer when the total SCF concentration wa s in the range of 10-100 ng/ml, consistent with the K-a determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characteriz ed. The dimer K-a values, biophysical properties, and biological activ ities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mi togenic activity, while the activity of the Cys(165)-Cys(165) disulfid e-linked SCF(A165C) dimer was 10-fold higher than that of wild type rh SCF. The results suggest a correlation between dimerization affinity a nd biological activity, consistent with a model in which SCF dimerizat ion mediates dimerization of its receptor, Kit, and subsequent signal transduction.