Karcz S R, Galatis D, Cowman A F
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Mol Biochem Parasitol. 1993 Apr;58(2):269-76. doi: 10.1016/0166-6851(93)90048-3.
The Plasmodium falciparum P-glycoprotein homologue 1 (PGH1) is structurally similar to several members of the ATP-binding cassette (ABC) superfamily of membrane transporters. We have examined whether the nucleotide binding domains predicted from the deduced amino sequence are functional by photoaffinity labeling of purified parasite digestive vacuoles with the analogue 8-azido-alpha-[32P]ATP (8-N3-ATP). This reagent labels a 160-kDa protein in vacuoles from both a chloroquine sensitive and a chloroquine-resistant parasite isolate. The 160-kDa protein could be immunoprecipitated with affinity-purified antibodies against the P. falciparum P-glycoprotein homologue (PGH1). Inhibition of photoaffinity labeling of PGH1 could be achieved with ATP, ADP, GTP and GDP but not with AMP or GMP. In order to map the 8-N3-ATP binding sites on PGH1, photoaffinity-labeled PGH1 was digested with trypsin and immunoprecipitated with site-specific antibodies. Taken together, these results indicate that 8-N3-ATP specifically labels PGH1 and that one binding site resides within the amino terminal half of the molecule. This supports the contention that PGH1 is involved in a nucleotide-regulated transport function across the membrane of the digestive vacuole.
恶性疟原虫P -糖蛋白同源物1（PGH1）在结构上与膜转运蛋白ATP结合盒（ABC）超家族的几个成员相似。我们通过用类似物8-叠氮基-α-[³²P]ATP（8-N3-ATP）对纯化的寄生虫消化液泡进行光亲和标记，研究了从推导的氨基酸序列预测的核苷酸结合结构域是否具有功能。该试剂在来自氯喹敏感和氯喹抗性寄生虫分离株的液泡中标记了一种160 kDa的蛋白质。160 kDa的蛋白质可以用针对恶性疟原虫P -糖蛋白同源物（PGH1）的亲和纯化抗体进行免疫沉淀。ATP、ADP、GTP和GDP可以抑制PGH1的光亲和标记，但AMP或GMP则不能。为了绘制PGH1上的8-N3-ATP结合位点，用胰蛋白酶消化光亲和标记的PGH1，并用位点特异性抗体进行免疫沉淀。综上所述，这些结果表明8-N3-ATP特异性标记PGH1，且一个结合位点位于该分子的氨基末端一半内。这支持了PGH1参与消化液泡膜上核苷酸调节的转运功能这一论点。
