Biswas E E
Department of Laboratory Sciences, Program in Biotechnology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Biochemistry. 2001 Jul 27;40(28):8181-7. doi: 10.1021/bi0106686.
Members of the ATP binding cassette (ABC) superfamily are transmembrane proteins that are found in a variety of tissues which transport substances across cell membranes in an energy-dependent manner. The retina-specific ABC protein (ABCR) has been linked through genetic studies to a number of inherited visual disorders, including Stargardt macular degeneration and age-related macular degeneration (ARMD). Like other ABC transporters, ABCR is characterized by two nucleotide binding domains and two transmembrane domains. We have cloned and expressed the 522-amino acid (aa) N-terminal cytoplasmic region (aa 854-1375) of ABCR containing nucleotide binding domain 1 (NBD1) with a purification tag at its amino terminus. The expressed recombinant protein was found to be soluble and was purified using single-step affinity chromatography. The purified protein migrated as a 66 kDa protein on SDS-PAGE. Analysis of the ATP binding and hydrolysis properties of the NBD1 polypeptide demonstrated significant differences between NBD1 and NBD2 [Biswas, E. E., and Biswas, S. B. (2000) Biochemistry 39, 15879-15886]. NBD1 was active as an ATPase, and nucleotide inhibition studies suggested that nucleotide binding was not specific for ATP and all four ribonucleotides can compete for binding. Further analysis demonstrated that NBD1 is a general nucleotidase capable of hydrolysis of ATP, CTP, GTP, and UTP. In contrast, NBD2 is specific for adenosine nucleotides (ATP and dATP). NBD1 bound ATP with a higher affinity than NBD2 (K(mNBD1) = 200 microm vs K(mNBD2) = 631 microm) but was less efficient as an ATPase (V(maxNBD1) = 28.9 nmol min(-)(1) mg(-)(1) vs V(maxNBD2) = 144 nmol min(-)(1) mg(-)(1)). The binding efficiencies for CTP and GTP were comparable to that observed for ATP (K(mCTP) = 155 microm vs K(mGTP) = 183 microm), while that observed for UTP was decreased 2-fold (K(mUTP) = 436 microm). Thus, the nucleotide binding preference of NBD1 is as follows: CTP > GTP > ATP >> UTP. These studies demonstrate that NBD1 of ABCR is a general nucleotidase, whereas NBD2 is a specific ATPase.
ATP结合盒（ABC）超家族的成员是跨膜蛋白，存在于多种组织中，以能量依赖的方式跨细胞膜转运物质。通过遗传学研究发现，视网膜特异性ABC蛋白（ABCR）与多种遗传性视觉疾病有关，包括Stargardt黄斑变性和年龄相关性黄斑变性（ARMD）。与其他ABC转运蛋白一样，ABCR的特征是具有两个核苷酸结合结构域和两个跨膜结构域。我们克隆并表达了ABCR含核苷酸结合结构域1（NBD1）的522个氨基酸（aa）的N端胞质区域（aa 854 - 1375），其氨基末端带有纯化标签。发现表达的重组蛋白是可溶的，并通过单步亲和层析进行纯化。纯化后的蛋白在SDS - PAGE上以66 kDa蛋白的形式迁移。对NBD1多肽的ATP结合和水解特性分析表明，NBD1和NBD2之间存在显著差异[Biswas，E. E.，和Biswas，S. B.（2000）Biochemistry 39，15879 - 15886]。NBD1作为一种ATP酶具有活性，核苷酸抑制研究表明，核苷酸结合对ATP不具有特异性，所有四种核糖核苷酸都能竞争结合。进一步分析表明，NBD1是一种能水解ATP、CTP、GTP和UTP的通用核苷酸酶。相比之下，NBD2对腺苷核苷酸（ATP和dATP）具有特异性。NBD1比NBD2以更高的亲和力结合ATP（K（mNBD1） = 200 μM vs K（mNBD2） = 631 μM），但作为ATP酶的效率较低（V（maxNBD1） = 28.9 nmol min⁻¹ mg⁻¹ vs V（maxNBD2） = 144 nmol min⁻¹ mg⁻¹）。CTP和GTP的结合效率与ATP相当（K（mCTP） = 155 μM vs K（mGTP） = 183 μM），而UTP的结合效率降低了2倍（K（mUTP） = 436 μM）。因此，NBD1的核苷酸结合偏好如下：CTP > GTP > ATP >> UTP。这些研究表明，ABCR的NBD1是一种通用核苷酸酶，而NBD2是一种特异性ATP酶。
