Characterization of beta-carotene 15,15'-dioxygenase activity in TC7 clone of human intestinal cell line Caco-2.
The purpose of this study was to identify mammalian cell line(s) which possess intrinsic enzymatic activity of beta-carotene 15, 15'-dioxygenase. This enzyme (EC1.13.11.21) converts beta-carotene to retinal (precursor of retinol and retinoic acid). To assess activity, cellular enzyme preparations were incubated with beta-carotene for 60 min; retinal formed was quantified by HPLC. Activity was not detected in IPEC-1, HepG2, HL60, Wurzburg, or parent Caco-2 cell lines. However, two subclones of Caco-2, PF11 and TC7, possessed activity (2.5 and 14.7 pmol/h.mg, respectively). Using the enzyme preparation of TC7 cells, retinal formation was linear with incubation time and protein concentration; Km and Vm values were 1.6 microM and 23.8 pmol/h.mg, respectively. In addition, when TC7 cells were maintained in serum-free medium, activity was increased 8.2-fold after 19 days of postconfluency. Finally, 48 h incubation with beta-carotene (delivered to TC7 cells in Tween 40) resulted in a 1.7-fold increase of dioxygenase activity and the appearance of vitamin A (9.3 pmol/mg protein). However, retinoic acid was not detected under our experimental conditions. In sum, the TC7 subclone of the Caco-2 cell line possesses beta-carotene 15, 15'-dioxygenase activity and thus can be useful in future investigations of human carotenoid metabolism.
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Beltsville Human Nutrition Research Center, USDA-ARS, Beltsville, Maryland, 20705, USA.
Albaugh
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Smith
J C
JC
D018938:Caco-2 Cells / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D002999:Clone Cells / Q000201:enzymology*; D016895:Culture Media, Serum-Free; D006801:Humans; D007422:Intestines / Q000201:enzymology*; D010105:Oxygenases / Q000378:metabolism*; D012172:Retinaldehyde / Q000096:biosynthesis; D019207:beta Carotene / Q000378:metabolism; D050559:beta-Carotene 15,15'-Monooxygenase
D016895:Culture Media, Serum-Free; D019207:beta Carotene; D010105:Oxygenases; C493358:BCO1 protein, human; D050559:beta-Carotene 15,15'-Monooxygenase; D012172:Retinaldehyde