The present study describes the partial purification and characterization of dolicholbinding component(s). [³H]dolichol binding was assessed by a newly developed binding assay of fast flow liquid chromatography on DEAE-Toyopearl 650 M (1 ml). The bin...
The present study describes the partial purification and characterization of dolicholbinding component(s). [³H]dolichol binding was assessed by a newly developed binding assay of fast flow liquid chromatography on DEAE-Toyopearl 650 M (1 ml). The binding component(s) for dolichol in rat liver cytosol was purified about 60-fold by heat treatment (80℃, 5 min) and DEAE-Toyopearl 650 M column chromatography. [³H]dolichol complex of the partially purified component(s) behaved as an anionic entity on DEAE-Toyopearl and the binding was abolished by Pronase proteolysis. Scatchard analysis revealed that the partially purified binding component(s) possessed a high affinity (K_d=1.25 × 10^(-7) M) and low capacity (242 pmol/㎎ protein) binding site, and also a low affinity and high capacity site(s). By competitive binding assay, the partially purified binding component(s) showed an affinity with dolichol and its derivatives, but not with palmitic acid, oleic acid and retinol. Though not dolichol congeners, squalene, ubiquinone and cholesterol were partially competitive. No lipid binding (carrier) proteins so far described accounted for the dolichol binding activity in our preparation, and the total binding capacity estimated herein comparable to the amounts of dolichol in rat liver cytosol.