24,25-dihydroxyvitamin D3 binds to catalase

There is increasing evidence that the vitamin D metabolite, 24,25-dihydroxyvitamn D-3 (24,25(OH)(2)D-3) has endocrine actions. In the crrent work, we report that an endogenous binding protein for 24,25(OH)(2)D-3 is catalase, based on sequence analysis of the isolated protein. An antibody (Ab 365) generated against equivalent protein recognized bovine catalase and a 64 kDa band in subcellular fractions of chick intestine. A commercially available anti-catalase antibody reduced specific [H-3]24,25(OH)(2)D-3 binding in subcellular fractions of chick intestine by greater than 65%, relative to the same fractions treated with art unrelated antibody (Ab 099). The same commercially available anti-catalase was able to block the inhibitory actions of 24,25(OH)(2)D-3 Oil P-32 uptake in isolated intestinal epithelial cell suspensions. We subsequently characterized binding of steroid to commercially available catalase, and found that between 0 and 5 nM of enzyme added to subcellular fraction P-2 (20,000g, 10-min post-nuclear pellet) resulted in a linear increase in the amount of [H-3]24,25(OH)(2)D-3 specifically bound. Additional studies indicated that 25(OH)D-3 was an effective competitor for binding, whereas 1,25(OH)(2)D-3 only poorly displaced [H-3]24,25(OH)(2)D-3. Saturation analyses with added catalase yielded a physiologically relevant affinity constant (K-D=5.6 +/- 2.7 nM) and a B-max = 209 +/- 34 fmols/mg protein, comparable to previous studies using purified basal lateral membranes or vesicular fractions. Moreover, in a study on subcellular fractions isolated from chickens of varying ages, we found that in females, both specific [H-3]24,25(OH)(2)D-3 binding and catalase activity increased from 7-to58-week-old birds,whereas in males, elevated levelsof both parameters were expressed in preparations of 7- and 58-week-old birds. The data suggest that signal transduction may occur through modulation of hydrogen peroxide production.