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'-dioxygen ase. This enzyme (EC1.13.11.21) converts p-carotene to retinal (precur sor of retinol and retinoic acid). To assess activity, cellular enzyme preparations were incubated with p-carotene for 60 min; retinal forme d 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, res pectively). Using the enzyme preparation of TC7 cells, retinal formati on was linear with incubation time and protein concentration; Km and V m values were 1.6 mu M and 23.8 pmol/h.mg, respectively. In addition, when TC7 cells were maintained in serum-free medium, activity was incr eased 8.2-fold after 19 days of postconfluency. Finally, 48 h incubati on with beta-carotene (delivered to TC7 cells in Tween 40) resulted in a 1.7-fold increase of dioxygenase activity and the appearance of vit amin A (9.3 pmol/mg protein). However, retinoic acid was not detected under our experimental conditions. In sum, the TC7 subclone of the Cac o-2 cell line possesses p-carotene 15,15'-dioxygenase activity and thu s can be useful in future investigations of human carotenoid metabolis m. (C) 1998 Academic Press.