Retinoylation reaction of proteins in Leydig (TM-3) cells

摘要

The covalent incorporation of [H-3]all-trans-retinoic acid into proteins has been studied in Leydig (TM-3) cells. The maximum retinoylation activity of Leydig cells proteins was 570 +/- 27 fmoles/8 x 10(4) cells at 37 degrees C. About 95% of [H-3]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion or after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation process was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC50 values of 0.6 and 1.2 mu M respectively. Dibutyryl-cAMP and forskolin increased the retinoylation activity by 75 and 81% at 500 and 25 mu M respectively. Also hCG increased the retinoylation binding activity of 110% at 250 ng/mL. After cycloheximide treatment of the Leydig cells the binding activity of [H-3]RA was about the same that in the control, suggesting that the bond occurs on proteins in pre-existing cells. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (500 mu M); on the contrary, there was an increase of the binding activity of about 60 and 50% respectively.