Journal of Cellular Biochemistry 97:1259–1266 (2006) 24,25-Dihydroxyvitamin D 3 Binds to Catalase Dennis Larsson, Deryk Anderson, Nathan M. Smith, and Ilka Nemere* Department of Nutrition and Food Sciences and the Center for Integrated Biosystems, Utah State University, Logan, Utah 84322 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 (20,000g, 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 ¼ 5.6  2.7 nM) and a B max ¼ 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. ß 2005 Wiley-Liss, Inc. Key words: vitamin D; rapid actions; chick intestine; phosphate uptake A growing body of evidence has indicated that the vitamin D metabolite 24,25-dihydroxyvita- min D 3 (24,25(OH) 2 D 3 ) has specific, physiologi- cally significant actions that are distinct from 1,25-dihydroxyvitamin D 3 [reviewed in Larsson and Nemere, 2001; Farach-Carson and Nemere, 2003]. In the perfused duodenal loop of chick, we have found that 24,25(OH) 2 D 3 inhibits the rapid 1,25(OH) 2 D 3 -mediated stimulation of phosphate transport [Nemere, 1996], as well as calcium transport [Nemere et al., 2002]. While the 24,25(OH) 2 D 3 binding protein is evident on the cell surface [Nemere et al., 1994; Larsson et al., 2001], a larger pool of binding activity was found intracellularly [Nemere et al., 2002]. In concert with the reports from the white Leghorn cockerel, putative receptor proteins for 24,25(OH) 2 D 3 in plasma membranes and evi- dence for physiological functions have been demonstrated in several other animal species and tissues [Seo et al., 1996; Kato et al., 1998; Pedrozo et al., 1999; Larsson et al., 2001, 2003; Boyan et al., 2002]. The putative receptors for 24,25(OH) 2 D 3 show allosteric binding to 24,25(OH) 2 D 3 , and mediate rapid membrane- initiated responses regulating calcium home- ostasis, cell differentiation and maturation, and trigger second messenger systems in ß 2005 Wiley-Liss, Inc. Grant sponsor: Swedish Foundation for International Cooperation in Research and Higher Education; Grant number: 99/820; Grant sponsor: USDA NRI-CSREES; Grant number: 2004-35206-14134; Grant sponsor: CURI. Dennis Larsson’s present address is School of Life Sciences, Biomedicine, University of Sko ¨vde, Ho ¨gskoleva ¨gen 1, Box 408, 541 28 Sko ¨vde, Sweden. Deryk Anderson’s present address is Kansas City Uni- versity of Medicine and Biosciences, 1750 Independence Ave., Kansas City, MO 64106-1453. *Correspondence to: Ilka Nemere, PhD, Department of Nutrition and Food Sciences, Utah State University, Logan, UT 84322-8700. E-mail: Nemere@cc.usu.edu Received 12 August 2005; Accepted 20 October 2005 DOI 10.1002/jcb.20717