Binding of all-trans-retinoic acid to MLTC-1 proteins

The covalent incorporation of [3H]all-trans-retinoic acid into proteins has been studied in tumoural Leydig (MLTC-1) cells.The maximum retinoylation activity of MLTC-1 cell proteins was 710 ± 29 (mean ± SD) fmoles/8 × 104 cells at 37 ◦C. About90% of [3H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion and about 65–75% after hydrolysis withhydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation reaction was inhibitedby 13-cis-retinoic acid and 9-cis-retinoic acid with IC50 values of 0.9μM and 0.65μM, respectively. Retinoylation was notinhibited by high concentrations of palmitic or myristic acids (250μM); but there was an increase of the binding activity ofabout 25% and 130%, respectively. On the other hand, the retinoylation reaction was inhibited (about 40%) by 250μM lauricacid. After pre-incubation of the cells with different concentrations of unlabeled RA, the retinoylation reaction with 100nM[3H]RA involved first an increase at 100nM RA and then a decrease of retinoylation activity between 200 and 600nM RA.After cycloheximide treatment of the tumoural Leydig cells the binding activity of [3H]RA was about the same as that in thecontrol, suggesting that the bond occurred on proteins in pre-existing cells.