Ohara Tomomi, Ohashi-Kobayashi Ayako, Maeda Masatomo
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
Biol Pharm Bull. 2008 Jan;31(1):1-5. doi: 10.1248/bpb.31.1.
The ATP-binding cassette (ABC) transporter, transporter associated with antigen processing (TAP)-like (TAPL) tagged with a histidine cluster was overexpressed, amounting to as much as 1-2% of total membrane proteins in Drosophila cell line S2. TAPL was effectively solubilized from membranes by Triton X-100, NP-40 and n-dodecyl-beta-D-maltoside. Solubilized TAPL bound ATP-agarose and adenosine 5'-diphosphate (ADP)-agarose but not adenosine 5'-monophosphate (AMP)-agarose. The binding was competed for by excess free ATP, ADP, guanosine 5'-triphosphate (GTP) and dATP but not by AMP. Pyrimidine nucleotides such as uridine 5'-triphosphate (UTP) and cytidine 5'-triphosphate (CTP) were less effective competitors, suggesting that purine nucleotide triphosphates are substrates for TAPL. The ATP-binding of TAPL required Mg(2+), and was observed at neutral pH. Chemical cross-linking experiments suggested that TAPL forms a homodimer in the membrane and under the solubilized conditions.
带有组氨酸簇标记的ATP结合盒（ABC）转运蛋白、与抗原加工相关的转运蛋白（TAP）样蛋白（TAPL）在果蝇细胞系S2中过表达，其含量高达总膜蛋白的1-2%。TAPL可通过Triton X-100、NP-40和正十二烷基-β-D-麦芽糖苷从膜中有效溶解。溶解的TAPL与ATP琼脂糖和5'-二磷酸腺苷（ADP）琼脂糖结合，但不与5'-单磷酸腺苷（AMP）琼脂糖结合。这种结合可被过量的游离ATP、ADP、5'-三磷酸鸟苷（GTP）和脱氧三磷酸腺苷（dATP）竞争，但不能被AMP竞争。嘧啶核苷酸如5'-三磷酸尿苷（UTP）和5'-三磷酸胞苷（CTP）作为竞争剂的效果较差，这表明嘌呤核苷酸三磷酸是TAPL的底物。TAPL的ATP结合需要Mg(2+)，且在中性pH条件下可观察到。化学交联实验表明，TAPL在膜中和溶解条件下形成同源二聚体。
