Abstract
There is increasing evidence that the vitamin D metabolite, 24,25‐dihydroxyvitamin D~3~ (24,25(OH)~2~D~3~) has endocrine actions. In the current 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 [^3^H]24,25(OH)~2~D~3~ binding in subcellular fractions of chick intestine by greater than 65%, relative to the same fractions treated with an unrelated antibody (Ab 099). The same commercially available anti‐catalase was able to block the inhibitory actions of 24,25(OH)~2~D~3~ on ^32^P 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~ (20,000__g__, 10‐min post‐nuclear pellet) resulted in a linear increase in the amount of [^3^H]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 [^3^H]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 [^3^H]24,25(OH)~2~D~3~ binding and catalase activity increased from 7‐ to 58‐week‐old birds, whereas in males, elevated levels of 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. J. Cell. Biochem. 97: 1259–1266, 2006. © 2005 Wiley‐Liss, Inc.