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β-Carotene-15,15′-dioxygenase (EC 1.13.11.21) isolation reaction mechanism and an improved assay procedure

Published online by Cambridge University Press:  09 March 2007

Jannine Devery  and
B. V. Milborrow
Jannine Devery
Affiliation:
School of Biochemistry, University of New South Wales, PO Box I, Kensington, NSW, 2033, Australia
B. V. Milborrow
Affiliation:
School of Biochemistry, University of New South Wales, PO Box I, Kensington, NSW, 2033, Australia
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Abstract

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βCarotene-15,15′-dioxygenase (EC 1.13.11.21; β-carotene dioxygenase) activity in extracts from guinea-pig intestinal mucosa was assayed by supplying [15,15′-14C2l- or [15,15′-3H2]β-carotene dissolved in Tween 80. Methods were developed to minimize the breakdown of labelled β-carotene and β-carotene cleavage products during the isolation procedure. Antioxidants and unlabelled carriers were added to extracting solvents and C18 Sep-Pak cartridges were used to isolate the remaining β-carotene and retinaldehyde, which was the only cleavage product detected. The labelled material produced by the enzyme was analysed by either normal-phase TLC or reversed-phase HPLC and characterized chemically as retinaldehyde. The lack of other labelled up-carotenals isolated in these experiments and the formation of between 1.5 and 2 mol retinaldehyde/mol β-carotene consumed confirm the central cleavage mechanism for the enzyme's action. More β-carotene dioxygenase activity was obtained from guinea-pig mucosa than from chicken or pig intestinal mucosa. The β-carotene dioxygenase was obtained as a soluble enzyme which was partially purified by gel filtration and ion-exchange chromatography to a specific activity of 0.6 nmol retinaldehyde formedlmg protein per h. The formation of a lipid-protein aggregate containing the β-carotene dioxygenase activity, which has been reported to be present in the exclusion volume of Sephadex columns, was avoided if the mucosal serapings were homogenized in buffer at a proportion of 1:4 (w/v).

Keywords

β-Carotene dioxygenaseRetinaldehydeVitamin A
Type
Isolation and mechanism of B-carotene dioxygenase
Information
British Journal of Nutrition , Volume 72 , Issue 3 , September 1994 , pp. 397 - 414
Copyright
Copyright © The Nutrition Society 1994

References

REFERENCES

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.CrossRefGoogle ScholarPubMed
Fidge, N. H. & Goodman, D. S. (1969). The enzymatic reduction of retinal to retinol in rat intestine. Journal of Biological Chemistry 243, 4372–4379.CrossRefGoogle Scholar
Fidge, N. H., Smith, F. R. & Goodman, D. S. (1969). Vitamin A and carotenoids. Enzymic conversion of β-carotene into retinal in hog intestinal mucosa. Biochemical Journal 114, 689–694.CrossRefGoogle ScholarPubMed
Folch, J., Lees, M. & Sloane Stanley, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497–509.CrossRefGoogle ScholarPubMed
Ganguly, J. & Sastry, P. S. (1985). Mechanism of conversion of β-carotene into vitamin A-central cleavage versus random cleavage. World Review of Nuirition and Dietetics 45, 198–220.CrossRefGoogle ScholarPubMed
Goodman, D. S. & Huang, H. S. (1965). BiosynThesis of vitamin A with rat intestinal enzymes. Science 149, 879–880.CrossRefGoogle ScholarPubMed
Goodman, D. S., Huang, H. S., Kanai, M. & Shiratori, T. (1967). The enzymatic conversion of all-trans β-carotene into retinal. Journal of Biological Chemistry 242, 3543–3554.CrossRefGoogle Scholar
Goodman, D. S., Huang, H. S. & Shiratori, T. (1966). Mechanism of the biosynThesis of vitamin A from β-carotene. Journal of Biological Chemistry 241, 1929–1932.CrossRefGoogle ScholarPubMed
Goodman, D. S. & Olson, J. A. (1969). The conversion of all-trans β-carotene into retinal. Methods in Enzymology 15, 462–475.CrossRefGoogle Scholar
Hansen, S. & Maret, W. (1988). Retinal is not formed in vitro by enzymatic central cleavage of p-carotene. Biochemistry 27, 200–206.CrossRefGoogle ScholarPubMed
Harrison, E. H., Smith, J. E. & Goodman, D. S. (1979). Unusual properties of retinyl palmitate hydrolase activity in rat liver. Journal of Lipid Research 20, 760–771.CrossRefGoogle ScholarPubMed
Hjelmeland, L. M. & Chrambach, A. (1984). Solubilization of functional membrane proteins. Methods in Enzymology 104, 305–318.CrossRefGoogle ScholarPubMed
Kozub, J., Milborrow, B. V. & Netting, A. G. (1988). Separation of serum retinol from carotenoids using normal phase disposable cyano columns. Annals of Clinical Biochemistry 25, 114–115.CrossRefGoogle ScholarPubMed
Lakshmanan, M. R., Chansang, H. & Olson, J. A. (1972). Purification and properties of carotene 15, 15′-dioxygenase of rabbit intestine. Journal of Lipid Research 13, 477–482.CrossRefGoogle Scholar
Lakshmanan, M. R., Mychkovsky, I. & Attlesey, M. (1989). Enzymatic conversion of all-trans-β-carotene to retinal by a cytosolic enzyme from rabbit and rat intestinal mucosa. Proceedings of the National Academy of Sciences, USA 86, 9124–9128.CrossRefGoogle Scholar
Ldkshmanan, M. R., Pope, J. L. & Olson, J. A. (1968). The specificity of a partially purified carotenoid cleavage enzyme of rabbit intestine. Biochemical and Biophysical Research Communications 33, 347–352.CrossRefGoogle Scholar
Olson, J. A. (1961). The conversion of radioactive pkarotene into vitamin A by the rat intestine in vim. Journal of Biological Chemistry 236, 349–356.CrossRefGoogle Scholar
Olson, J. A. (1968). Some aspects of vitamin A metabolism. Vitamins and Hormones 26, 1–63.Google ScholarPubMed
Olson, J. A. (1989). Provitamin A function of carotenoids: The conversion of β-carotene into Vitamin A. Journal of Nutrition 119, 105–108.CrossRefGoogle ScholarPubMed
Olson, J. A. & Hayaishi, O. (1965). The enzymatic cleavage ofb-carotene into Vitamin A by soluble enzymes of rat liver and intestine. Proceedings of the National Academy of Sciences, USA 54, 1365–1370.CrossRefGoogle ScholarPubMed
Prystowsky, J. H., Smith, J. E. & Goodman, D. S. (1981). Retinyl palmitate hydrolase activity in normal rat liver. Journal of Biological Chemistry 256, 4498–4503.CrossRefGoogle ScholarPubMed
Renswoude, J. V. & Kempf, C. (1984). Purification of integral membrane proteins. Methods in Enzymology 104, 329–339.CrossRefGoogle ScholarPubMed
Roberts, A. B., Nichols, M. D., Frolik, C. A., Newton, D. L. & Sporn, M. B. (1978). Assay of retinoids in biological samples by reverse-phase high-pressure liquid chromatography. Cancer Research 38, 3327–3332.Google ScholarPubMed
Sharma, R. V., Mathur, S. N., Dmitrovskii, A. A., Das, R. C. & Ganuly, J. (1977). Studies on the metabolism of β-carotene and apo-β-carotenoids in rats and chickens. Biochimica et Biophysica Acta 486, 183–194.CrossRefGoogle Scholar
Sharma, R. V., Mathur, S. N. & Ganuly, J. (1976). Studies on the relative biopotencies and intestinal absorption of different apo-β-carotenoids in rats and chickens. Biochemical Journal 158, 377–382.CrossRefGoogle ScholarPubMed
Simpson, K. L. (1983). Relative values of carotenoids as precursors of vitamin A. Proceedings of the Nutrition Society 42, 7–17.CrossRefGoogle ScholarPubMed
Singh, H. & Carna, H. R. (1974). Enzymatic cleavage of carotenoids. Biochimica et Biophysica Acta 370, 49–61.CrossRefGoogle ScholarPubMed
Sklan, D. (1983 a). Carotene cleavage in the corpus luteum of cattle. International Journal of Vitamin and Nutrition Research 53, 23–26.Google ScholarPubMed
Sklan, D. (1983 b). Carotene-cleavage activity in chick intestinal mucosa cytosol: association with a high-molecular-weight lipid-protein aggregate fraction and partial characterization of the activity. British Journal of Nutrition 50, 417–425.CrossRefGoogle ScholarPubMed
Sklan, D., Blaner, W. S., Adachi, N., Smith, J. E. & Goodman, D. S. (1982). Association of cellular retinol-binding protein and several lipid hydrolase activities with a vitamin A-containing high molecular-weight lipid-protein aggregate from rat liver cytosol. Archives of Biochemistry and Biophysics 214, 3544.CrossRefGoogle ScholarPubMed
Sklan, D. & Halevy, O. (1984). Vitamin A metabolism in chick liver: some properties of the cytosolic lipid-protein aggregate. British Journal of Nutrition 52, 107–114.CrossRefGoogle ScholarPubMed
Vartapetian, B. B., Dmitrovsky, A. A., Alkhasov, D. G., Lemberg, I. H., Girshin, A. B., Gusinsky, G. M., Starikova, N. A., Eropheeva, N. N. t., Bogdanova, I. P. (1966). A new approach to the study of mechanism of vitamin A biosynthesis from carotene: O18 activation by nuclear reaction O18 (αnγ)Ne21 using cyclotron-accelerated α-particles. Biokhimiya 31, 881–886.Google Scholar