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

The covalent incorporation of [H-3]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 x 10(4) cells at 37 degrees C. About 90% of [H-3]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion and about 65-75% after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation reaction was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC50 values of 0.9 mu M and 0.65 mu M, respectively. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (250 mu M); but there was an increase of the binding activity of about 25% and 130%, respectively. On the other hand, the retinoylation reaction was inhibited (about 40%) by 250 mu M lauric acid. After pre-incubation of the cells with different concentrations of unlabeled RA, the retinoylation reaction with 100 nM [H-3]RA involved first an increase at 100 nM RA and then a decrease of retinoylation activity between 200 and 600 nM RA. After cycloheximide treatment of the tumoural Leydig cells the binding activity of [H-3]RA was about the same as that in the control, suggesting that the bond occurred on proteins in pre-existing cells.