Kaufman J F, Krangel M S, Strominger J L
J Biol Chem. 1984 Jun 10;259(11):7230-8.
Exogenous radioactive palmitic acid is incorporated post-translationally into the HLA-B and -DR heavy chains, but not HLA-A heavy chains or -DR light chains of the human B lymphoblastoid cells JY and T51 . Protease digestions localize the label to the transmembrane region of the B7 heavy chain. Both B7 and DR heavy chains have a cysteine in the transmembrane hydrophobic region, while the A2 heavy chain and DR light chains have none. Palmitic acid is covalently linked to these transmembrane cysteines via a thioester bond since: 1) the label is not removed by organic extraction or boiling in sodium dodecyl sulfate and dithiothreitol, but is released at room temperature by methanolic KOH as methyl palmitate, and by hydroxylamine as palmitohydroxamate . 2) The pH sensitivity and kinetics of release by hydroxylamine and Tris are similar to those of palmitoyl-CoA (thioester linkage) and unlike those of methyl palmitate and palmitoyllysophosphatidylcholine ( hydroxyester linkages). 3) Neutral hydroxylamine treatment (but not neutral Tris treatment) generates sites that can be reduced and alkylated in the transmembrane region of B7 heavy chain and to a lesser extent in DR heavy chain. 4) Organic extraction of pronase digests of labeled B7 yields peptides containing palmitate and cysteine (but not serine or threonine) which co-migrate by thin layer chromatography. A population of beta 2-microglobulin molecules not associated with heavy chains is palmitylated , but not via a thioester linkage.
外源性放射性棕榈酸在翻译后被掺入人B淋巴母细胞系JY和T51的HLA - B和 - DR重链中，但不掺入HLA - A重链或 - DR轻链。蛋白酶消化将标记定位到B7重链的跨膜区域。B7和DR重链在跨膜疏水区域都有一个半胱氨酸，而A2重链和DR轻链则没有。棕榈酸通过硫酯键与这些跨膜半胱氨酸共价连接，原因如下：1）该标记不能通过有机萃取或在十二烷基硫酸钠和二硫苏糖醇中煮沸去除，但在室温下可被甲醇氢氧化钾释放为棕榈酸甲酯，被羟胺释放为棕榈羟肟酸。2）羟胺和Tris释放的pH敏感性和动力学与棕榈酰辅酶A（硫酯键）相似，与棕榈酸甲酯和棕榈酰溶血磷脂酰胆碱（羟酯键）不同。3）中性羟胺处理（但不是中性Tris处理）在B7重链的跨膜区域产生可被还原和烷基化的位点，在DR重链中程度较小。4）对标记的B7进行胰蛋白酶消化后的有机萃取产生含有棕榈酸和半胱氨酸（但不含丝氨酸或苏氨酸）的肽，这些肽通过薄层色谱共迁移。一群未与重链结合的β2微球蛋白分子被棕榈酰化，但不是通过硫酯键。
