Hoffenberg S, Nikolova L, Pan J Y, Daniel D S, Wessling-Resnick M, Knoll B J, Dickey B F
Baylor College of Medicine, Department of Medicine, Houston, TX 77030, USA.
Biochem Biophys Res Commun. 1995 Oct 4;215(1):241-9. doi: 10.1006/bbrc.1995.2459.
Rab5 is a Ras-related GTPase which regulates endosomal fusion. The D136N mutant of Rab5, which was predicted to switch specificity from guanine to xanthine nucleotides, was expressed in E. coli, extracted with urea, purified by column chromatography, and refolded by stepwise dialysis against buffer containing XDP. The purified protein bound xanthine nucleotides with considerably higher affinity than guanine nucleotides. In vitro prenylation of the mutant protein was highly dependent on xanthosine diphosphate. In contrast, both the wild type and mutant proteins were protected from proteolysis equally well by non-cognate and cognate triphosphate nucleosides at high concentration. The D136N Rab5 mutant appears to be a valuable reagent in conjunction with xanthine nucleotides for the study of protein-nucleotide interactions in systems in which multiple GTPases are active, although interactions with non-cognate nucleotides should be evaluated if they are present at high concentration.
Rab5是一种与Ras相关的GTP酶，可调节内体融合。Rab5的D136N突变体预计会将特异性从鸟嘌呤核苷酸转换为黄嘌呤核苷酸，该突变体在大肠杆菌中表达，用尿素提取，通过柱色谱纯化，并通过逐步透析含XDP的缓冲液进行复性。纯化后的蛋白与黄嘌呤核苷酸结合的亲和力明显高于鸟嘌呤核苷酸。突变蛋白的体外异戊二烯化高度依赖于二磷酸黄苷。相比之下，野生型和突变型蛋白在高浓度下均能被非同源和同源三磷酸核苷同样有效地保护免受蛋白水解。D136N Rab5突变体似乎是一种有价值的试剂，与黄嘌呤核苷酸一起用于研究多种GTP酶活跃的系统中的蛋白质-核苷酸相互作用，不过如果高浓度存在非同源核苷酸，应评估它们之间的相互作用。
