Takahashi N, Breitman T R
Laboratory of Biological Chemistry, National Cancer Institute, Bethesda, Maryland 20892.
J Biol Chem. 1989 Mar 25;264(9):5159-63.
all-trans-Retinoic acid is a potent inducer in vitro of the differentiation of the human acute myeloid leukemia cell line HL60 and of fresh cells from patients with acute promyelocytic leukemia. The recent discovery of nuclear retinoic acid receptors provides a basis for understanding how retinoic acid acts at the genetic level. We have now found that retinoic acid is incorporated into HL60 cells in a form that is not removed by extraction with CHCl3:CH3OH. About 90% of this labeled retinoic acid is trichloroacetic acid-soluble after digestion with proteinase K or after hydrolysis with either NH2OH or CH3OH:KOH under mild conditions. Methyl retinoate is the major product of hydrolysis with CH3OH:KOH. These results are consistent with retinoylation of protein with the formation of an ester, probably thioester, bond. The extent of the retinoylation of HL60 protein is dependent on both time and retinoic acid concentration. A major fraction of the retinoylation is of protein that has a molecular mass of 55 kDa after reduction with dithiothreitol. On two-dimensional gels, the retinoylated protein has a pI of about 4.9 and a molecular mass of 55-60 kDa. These characteristics and its localization in the cell nucleus are consistent with retinoylation of the HL60 nuclear retinoic acid receptor or a closely related protein.
全反式维甲酸在体外是人类急性髓系白血病细胞系HL60以及急性早幼粒细胞白血病患者新鲜细胞分化的有效诱导剂。核维甲酸受体的最新发现为理解维甲酸在基因水平的作用提供了基础。我们现已发现，维甲酸以一种不能被氯仿：甲醇萃取去除的形式掺入HL60细胞。在用蛋白酶K消化后，或在温和条件下用羟胺或甲醇：氢氧化钾水解后，约90%的这种标记维甲酸可溶于三氯乙酸。维甲酸甲酯是甲醇：氢氧化钾水解的主要产物。这些结果与蛋白质的视黄酰化反应一致，形成了酯键，可能是硫酯键。HL60蛋白的视黄酰化程度取决于时间和维甲酸浓度。视黄酰化的主要部分是在用二硫苏糖醇还原后分子量为55 kDa的蛋白质。在二维凝胶上，视黄酰化蛋白的pI约为4.9，分子量为55 - 60 kDa。这些特征及其在细胞核中的定位与HL60核维甲酸受体或密切相关蛋白的视黄酰化一致。
