25-Hydroxyvitamin D3-1 alpha-hydroxylase in porcine hepatic tissue: subcellular localization to both mitochondria and microsomes.
In vitro studies were performed to assess the ability of hepatic homogenates, mitochondria, and microsomes to 1 alpha-hydroxylate 25-hydroxyvitamin D3 [25(OH)D3]. Addition of 25(OH)D3 to either hepatic mitochondria or microsomes caused a concentration-dependent increase in the production of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Hepatic homogenates also produced purported 1,25(OH)2D3, although at a much reduced efficiency as compared with hepatic mitochondria or microsomes. Purported 1,25(OH)2D3 synthesized by hepatic mitochondria or microsomes was identified by its mobility on several high-performance liquid chromatographic systems and, ultimately, by its ability to interact with the bovine thymus 1,25(OH)2D3 receptor protein. Production of 1,25(OH)2D3 by hepatic mitochondria and microsomes was dependent on time of incubation, protein content, and pH of incubation medium, and it required an adequate source of reducing equivalents. Generation of 1,25(OH)2D3 by these organelles could be totally blocked by the cytochrome P-450 inhibitor ketoconazole. The microsomal 1 alpha-hydroxylase could not be saturated even at the highest concentration (240 microM) of 25(OH)D3 used. The mitochondrial 1 alpha-hydroxylase, however, displayed saturation at approximately 40 microM 25(OH)D3. Eadie-Hofstee reciprocal plot analysis of the hepatic mitochondrial 1 alpha-hydroxylase gave a Km of 17 microM 25(OH)D3 and a Vmax of 481 pg of 1,25(OH)2D3 per min per mg of protein. Because of its inability to achieve substrate saturation, meaningful kinetic parameters could not be calculated for the hepatic microsomal 1 alpha-hydroxylase. These data demonstrate the liver to be an even more dynamic organ than was previously believed with respect to vitamin D metabolism in that the liver has the potential to produce 1,25(OH)2D3 in situ by at least two separate mechanisms.
Hollis
B W
BW
Department of Pediatrics, School of Medicine, Medical University of South Carolina, Charleston 29425.
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D007523:Isocitrates / Q000494:pharmacology; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013552:Swine; D014461:Ultracentrifugation
D007523:Isocitrates; D009249:NADP; C034219:isocitric acid; D013250:Steroid Hydroxylases; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D007654:Ketoconazole