, Volume 95, Issue 1, pp 37–42 | Cite as

Antigenic and catalytic disparity in the distribution of cytochrome P-450-dependent 25-hydroxyvitamin D3-1α- and 24-hydroxylases

  • K. Takezawa
  • B. Moorthy
  • M. L. Mandel
  • J. C. Garancis
  • J. G. Ghazarian


Chick 25-hydroxyvitamin D3-1α-hydroxylase, a cytochrome P-450 monooxygenase with a molecular weight of 57 kDa, can be isolated as described by Mandel et al. (1990b). Under normal physiological circumstances, it occurs exclusively in kidney mitochondria. An isozyme of the 1α-hydroxylase, known as the 24-hydroxylase, which uses the same substrate to yield an isomeric product, is also a cytochrome P-450 monooxygenase, has a molecular weight of 55 kDa, and likewise occurs in kidney mitochondria. The amino-terminal sequences of the first 10 residues of the two isozymes are 100% homologous. Monoclonal antibodies of the IgM class raised against the 1α-hydroxylase, which quantitatively discriminate against other P-450 cytochromes of mitochondrial or microsomal origin, recognize and interact with the 24-hydroxylase as an antigen. In the present study we show that the intestine, which is the only non-renal tissue with demonstrable 24-hydroxylase activity, gives a positive peroxidase-antiperoxidase immunohistochemical reaction using the monoclonal antibodies against the 1α-hydroxylase. The reactions revealed that the antigen in the kidney is restricted to the cortical proximal tubular cells while in the intestine, the antigen is localized in the enterocytes of the villi. In kidney medullary or intestinal crypt cells, or in liver, heart and lung tissues where 1α-hydroxylase or 24-hydroxylase activity could not be detected using cell or tissue homogenates, the immunohistochemical reactions were also negative. Since it has been reported that chick embryonic intestine possesses 1α-hydroxylase activity that is absent in the mature intestine, our results would suggest that the mature intestinal 24-hydroxylase represents a modified 1α-hydroxylase as a consequence of developmentally imposed requirements regulating calcium homeostatic activity in this tissue. The difference in the molecular weights of the two enzymes would indicate either genomic processing prior to the translation of their respective mRNAs, or a post-translational processing of the larger 1α-hydroxylase to the smaller 24-hydroxylase.


Crypt Cell Proximal Tubular Cell Immunohistochemical Reaction Intestinal Crypt Kidney Mitochondrion 
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  1. Bikle DD (1980) Studies of the chick renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase. Biochim Biophys Acta 615:208–222Google Scholar
  2. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem 37:911–917Google Scholar
  3. Burgos-Trinidad M, Brown AJ, DeLuca HF (1986) Solubilization and reconstitution of chick renal mitochondrial 25-hydroxyvitamin D3-24-hydroxylase. Biochemistry 25:2692–2696Google Scholar
  4. Burgos-Trinidad M, Ismail R, Prahl J, De Luca HF (1990) Monoclonal antibodies to the chick renal mitochondrial 1,25-dihydroxyvitamin D3-24-hydroxylase. FASEB J 4:A645 abstract 2201Google Scholar
  5. DeLuca HF (1988) The vitamin D story: a collaborative effort of basic science and clinical medicine. FASEB J 2:224–236Google Scholar
  6. Dutta C, Henry HL (1989) Generation of antibodies against renal mitochondrial cytochrome P-450. J Bone Min Res 4:S220 abstract 408Google Scholar
  7. Frolik CA, DeLuca HF (1971) 1,25-Dihydroxycholecalciferol: the metabolite of vitamin D responsible for increased intestinal calcium transport. Arch Biochem Biophys 147:143–147Google Scholar
  8. Garabedian M, Liberherr M, N'Guyen TM, DuBois MB, Balsan S (1978) The in vitro production and activity of 24,25-dihydroxycholecalciferol in cartilage and calvarium. Clin Orthop Rel Res 135:241–247Google Scholar
  9. Garancis CJ, Miller L, Tomita TJ, Tieu TM, Clowry L (1983) Immunoperoxidase localization of estrogen receptors in human breast carcinoma. Cancer Detect Prev 6:235–239Google Scholar
  10. Ghazarian JG, Jefcoate CR, Knutson JC, Orme-Johnson WH, De Luca HF (1974) Mitochondrial cytochrome P-450: a component of chick kidney 25-hydroxycholecalciferol-1α-hydroxylase. J Biol Chem 249:3026–3033Google Scholar
  11. Ghazarian JG, DeLuca HF (1980) Determination of vitamin D and its metabolites. Handbuch der inneren medizin band 1A. Springer, Berlin Heidelberg New York, pp 635–655Google Scholar
  12. Gray RW, Ghazarian JG (1989) Solubilization and reconstitution of kidney 25-hydroxyvitamin D3 1α- and 24-hydroxylases from vitamin D-replete pigs. Biochem J 259:561–568Google Scholar
  13. Henry HL, Norman AW (1974) Studies on calciferol metabolism: renal 25-hydroxyvitamin D3-1-hydroxylase; involvement of cytochrome P-450 and other properties. J Biol Chem 249:7529–7535Google Scholar
  14. Kawashima H, Kurokawa K (1986) Metabolism and sites of action of vitamin D in the kidney. Kidney Int 29:98–107Google Scholar
  15. Kumamoto T, Ito A, Omura T (1986) Characterization of a mitochondrial matrix protease catalyzing the processing of adrenodoxin precursor. J Biochem 100:247–254Google Scholar
  16. Kumar R, Schnoes HK, DeLuca HF (1978) Rat intestinal 25-hydroxyvitamin D3- and 1α,25-dihydroxyvitamin D3-24-hydroxylase. J Biol Chem 253:3804–3809Google Scholar
  17. Luben RA, Mohler MA (1980) In vitro immunization as an adjunct to the production of hybridomas producing antibodies against the lymphokine osteoclast activating factor. Mol Immunol 17:635–639Google Scholar
  18. Mandel ML, Moorthy B, Swartz SJ, Garancis JC, Ghazarian JG (1990) Monoclonal antibodies to chick renal calcium regulating hemeproteins. J Clin Lab Immunol (in press)Google Scholar
  19. Mandel ML, Moorthy B, Ghazarian JG (1990a) Reciprocal post-translational regulation of renal 1α and 24-hydroxylases of 25-hydroxyvitamin D3 by phoshorylation of ferredoxin: mRNA-directed cell-free synthesis and immunoisolation of ferredoxin. Biochem J 266:385–392Google Scholar
  20. Mandel ML, Swartz SJ, Ghazarian JG (1990b) Avian kidney mitochondrial hemeprotein P-450: isolation, characterization and NADPH-ferredoxin reductase-dependent activity. Biochim Biophys Acta 1034:239–246Google Scholar
  21. Mason DY, Farrell C, Taylor CR (1975) The detection of intracellular antigens in human leucocytes by immunoperoxidase staining. Br J Haematol 31:361–369Google Scholar
  22. Matocha MF, Waterman MR (1985) Synthesis and processing of mitochondrial steroid hydroxylases. In vivo maturation of the precursor forms of cytochrome P-450scc, cytochrome P-45011b, and adrenodoxin. J Biol Chem 260:12259–12265Google Scholar
  23. Norman A (1987) Studies on the vitamin D endocrine system in the avian. J Nutr 117:797–807Google Scholar
  24. Pedersen JI, Ghazarian JG, Orme-Johnson NR, DeLuca HF (1976) Isolation of chick renal mitochondrial ferredoxin active in the 25-hydroxyvitamin D3-1α-hydroxylase system. J Biol Chem 251:3933–3941Google Scholar
  25. Pedersen JI, Shobaki HH, Holmberg I, Bergseth S, Bjorkhem I (1983) 25-Hydroxyvitamin D3-24-hydroxylase in rat kidney mitochondria. J Biol Chem 258:742–746Google Scholar
  26. Postlind H (1990) Separation of the cytochromes P-450 in pig kidney mitochondria catalyzing 1α-24- and 26-hydroxylations of 25-hydroxyvitamin D3. Biochem Biophys Res Commun 168:261–266Google Scholar
  27. Puzas JE, Turner RT, Forte M, Kenny AD, Baylink DJ (1980) Metabolism of 25(OH)D3 by chick chorioallantoic cells in culture. Gen Comp Endocrinol 42:116–122Google Scholar
  28. Puzas JE, Turner RT, Howard GA, Baylink DJ (1983) Cells isolated from embryonic intestine synthesize 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in culture. Endocrinology 112:378–380Google Scholar
  29. Reinhardt TA, Horst RL, Orf JW, Hollis BW (1984) A microassay for 1,25-dihydroxyvitamin D not requiring high performance liquid chromatography: application to clinical studies. J Clin Endocrinol Metab 58:91–98Google Scholar
  30. Tanaka Y, Halloran B, Schnoes HK, DeLuca HF (1979) In vitro production of 1,25-dihydroxyvitamin D3 by rat placental tissue. Proc Natl Acad Sci USA 76:5033–5035Google Scholar
  31. Turner RT, Puzas JE, Forte MD, Lester GE, Gray TK, Howard GA, Baylink DJ (1980) In vitro synthesis of 1α,25-dihydroxycholecalciferol by isolated calvarial cells. Proc Natl Acad Sci USA 77:5720–5724Google Scholar
  32. Vieth R, Fraser D (1979) Kinetic behavior of 25-hydroxyvitamin D-1-hydroxylase and 24-hydroxylase in rat kidney mitochondria. J Biol Chem 254:12455–12460Google Scholar
  33. Warner M (1982) Catalytic activity of partially purified renal 25-hydroxyvitamin D hydroxylases from vitamin D-deficient and vitamin D-replete rats. J Biol Chem 257:12995–13000Google Scholar
  34. Waterman MR, Simpson ER (1985) Regulation of the biosynthesis of cytochromes P-450 involved in steroid hormone synthesis. Mol Cell Endocrinol 39:81–89Google Scholar
  35. Weisman Y, Harell A, Edelstein S, David M, Spirer Z, Golander A (1979) 1α,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in vitro synthesis by human decidua and placenta. Nature 281:317–319Google Scholar
  36. Whitsett JA, Ho M, Tsang RC, Norman EJ, Adams KG (1981) Synthesis of 1,25-dihydroxyvitamin D3 by human placenta in vitro. J Clin Endocrinol Metab 53:484–488Google Scholar
  37. Yanda DM, Ghazarian JG (1981) Vitamin D and 25-hydroxyvitamin D in rainbow trout (Salmo gairdneri): cytochrome P-450 and biotransformations of the vitamins. Comp Biochem Physiol 69B:183–188Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • K. Takezawa
    • 1
    • 3
  • B. Moorthy
    • 1
  • M. L. Mandel
    • 1
  • J. C. Garancis
    • 2
  • J. G. Ghazarian
    • 1
  1. 1.Department of BiochemistryMedical College of WisconsinMilwaukeeeUSA
  2. 2.Department of PathologyMedical College of WisconsinMilwaukeeeUSA
  3. 3.Department of Internal MedicineToho University School of MedicineTokyoJapan

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