Steinert K, Kroth-Pancic P G, Bickel-Sandkötter S
Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, Germany.
Biochim Biophys Acta. 1995 Jun 12;1249(2):137-44. doi: 10.1016/0167-4838(95)00033-q.
Using PCR with degenerate oligonucleotides, we amplified two conserved regions of the plasma membrane ATPase A (alpha)- and B (beta)-subunit-encoding genes from Haloferax volcanii, a halophilic archaeon. The amplified fragments were cloned and sequenced, and then used as homologous probes to clone genomic restriction fragments, one of which contained the nearly complete atpA- and atpB-gene. To complete the latter one, we sequenced a region of an overlapping fragment using synthetic oligonucleotides as primers. The deduced amino-acid sequence showed a high degree of similarity with the A and B sequences of Halobacterium halobium-ATPase, and a relatively high degree with Daucus carota V-ATPase, but less similarity to F-ATPase alpha- and beta-subunits. Like in V-ATPases, the A-subunit is more related to the catalytic F-ATPase beta-subunit, whereas B corresponds to alpha. Cross-reaction of antibodies against CF1-alpha (synthetic peptide) with B and CF1-beta with A of Haloferax volcanii confirmed this finding. The ATPase of Haloferax volcanii is a halophilic enzyme, the amino-acid sequences contain about 20% negatively charged residues. This is discussed in terms of adaption to hypersaline conditions. The enzyme is specific for ATP, we determined KM values for ATP in presence of Mn2+ and Mg2+, respectively. Other nucleotides, including GTP and ADP are competitive inhibitors of ATP hydrolysis. Despite of the high degree of similarity to the Halobacterium enzyme, the ATPase showed no sensitivity to most of the tested inhibitors of ATP hydrolysis. NEM, a specific inhibitor for V-ATPases inhibited the enzyme only slightly. Bafilomycin, NBD-Cl and nitrate showed no effect. These results will be discussed in context with some specific differences in the primary structure of the Haloferax A-subunit.
我们使用简并寡核苷酸通过聚合酶链反应（PCR），从嗜盐古菌沃氏嗜盐菌（Haloferax volcanii）中扩增了质膜ATP酶A（α）-和B（β）-亚基编码基因的两个保守区域。将扩增片段进行克隆和测序，然后用作同源探针来克隆基因组限制性片段，其中一个片段包含几乎完整的atpA和atpB基因。为了完成后者，我们使用合成寡核苷酸作为引物对重叠片段的一个区域进行测序。推导的氨基酸序列与嗜盐盐杆菌（Halobacterium halobium）ATP酶的A和B序列具有高度相似性，与胡萝卜（Daucus carota）V-ATP酶具有较高相似性，但与F-ATP酶的α-和β-亚基相似性较低。与V-ATP酶一样，A亚基与催化性F-ATP酶的β-亚基关系更密切，而B亚基对应于α亚基。针对CF1-α（合成肽）的抗体与沃氏嗜盐菌的B亚基以及针对CF1-β的抗体与A亚基的交叉反应证实了这一发现。沃氏嗜盐菌的ATP酶是一种嗜盐酶，其氨基酸序列包含约20%的带负电荷残基。从适应高盐条件的角度对此进行了讨论。该酶对ATP具有特异性，我们分别测定了在存在Mn2+和Mg2+时ATP的KM值。其他核苷酸，包括GTP和ADP是ATP水解的竞争性抑制剂。尽管与嗜盐盐杆菌的酶具有高度相似性，但该ATP酶对大多数测试的ATP水解抑制剂不敏感。NEM，一种V-ATP酶的特异性抑制剂，仅轻微抑制该酶。巴弗洛霉素、NBD-Cl和硝酸盐没有作用。将结合沃氏嗜盐菌A亚基一级结构中的一些特定差异来讨论这些结果。
