Biochemical characterization and purification of a binding protein for 24,25-dihydroxyvitamin D3 from chick intestine

An earlier study revealed that 24R,25-dihydroxyvitamin D-3 (24R,23(OH)(2)D-3) inhibits the rapid actions of 1,25(OH)(2)D-3 on stimulation of calcium transport in perfused duodena, as well as activation of protein kinases C and A. In the present work, a specific binding protein (24,25-BP) has been identified and partially characterized. Percoll-gradient resolution of differential centrifugation fractions from mucosal homogenates revealed the highest levels of specific [H-3]24R,25(OH)(2)D-3 binding to be in lysosomes (approximately eight to tenfold greater than in basal lateral membrane fractions). Incubation of isolated enterocytes with 6.5 nM [H-3]24R,25(OH)(2)D-3 for 10 s also demonstrated targeting of the steroid to lysosomal fractions. Using freshly isolated lysosomal fractions, time course studies indicated maximal specific binding after a 2-h incubation on ice. Western analyses revealed that the serum transport protein, DBP (vitamin D binding protein), was absent from both lysosomal and basal lateral membrane fractions. Protein dependence studies demonstrated linear binding between 0.05 and 0.155 mg of lysosomal protein, Saturation analyses yielded K-d = 7.4 +/- 1.8 nM B-max = 142 +/- 16 fmol/mg protein for lysosomes, and K-d = 8.5 nM. B-max = 149 +/- 25 fmol/mg protein for basal lateral membranes. Hill analyses of lysosomal binding yielded a Hill coefficient of 0.57 +/- 0.11, indicative of negative cooperativity. Studies with lysosomal proteins revealed a 81% +/- 7% competition of 24S,25(OH)(2)D-3 with [H-3]24R,25(OH)(2)D-3 for binding (P>0.05, relative to competition with 24R,25(OH)(2)D-3), while 25(OH)D3 and 1,25(OH)(2)D-3 yielded 53% +/- 13% and 39% +/- 11% competition respectively (each, P<0.05, relative to competition with 24R,25(OH)(2)D-3). The apparent affinity of 24S,25(OH)(2)D-3 for 24,25-BP led to testing of the metabolites effectiveness in the perfused duodenal loop system, Vascular perfusion with 130 pM 1.25(OH)(2)D-3 stimulated Ca-45 transport to 2.5-fold above control levels after 40 min, while simultaneous perfusion with 6.5 nM 24S,25(OH)(2)D-3 and 130 pM 1,25(OH)(2)D-3 abolished the stimulatory activity completely. Purification of the 24,25-BP by chromatography revealed a single protein band upon SDS-PAGE and silver staining of 66 kDa. The combined results suggest that 24R,25(OH)(2)D-3 may mediate its hormonal activities through a specific binding protein.