Fisher M, Gokhman I, Pick U, Zamir A
Biochemistry Department, Weizmann Institute of Science, Rehovot 76100, Israel.
J Biol Chem. 1996 Jul 26;271(30):17718-23. doi: 10.1074/jbc.271.30.17718.
The mechanisms allowing proliferation of the unicellular green alga Dunaliella salina in up to saturating NaCl concentrations are only partially understood at present. Previously, the level of a plasma membrane Mr 60,000 protein, p60, was found to increase with rising external salinities. Based on cDNA cloning and enzymatic assays, it is now shown that p60 is an internally duplicated carbonic anhydrase, with each repeat homologous to animal and Chlamydomonas reinhardtii carbonic anhydrases, but exceptional in the excess of acidic over basic residues. Increasing salinities, alkaline shift, or removal of bicarbonate induced in D. salina parallel increases in the levels of p60, its mRNA, and external carbonic anhydrase activity. Moreover, purified p60 exhibited carbonic anhydrase activity comparable to other carbonic anhydrases. A p60-enriched soluble preparation showed maximal carbonic anhydrase activity at approximately 1.0 M NaCl and retained considerable activity at higher salt concentrations. In contrast, a similar preparation from C. reinhardtii was approximately 90% inhibited in 0.6 M NaCl. These results identified p60 as a structurally novel carbonic anhydrase transcriptionally regulated by CO2 availability and exhibiting halophilic-like characteristics. This enzyme is potentially suited to optimize CO2 uptake by cells growing in hypersaline media.
目前，对于单细胞绿藻杜氏盐藻（Dunaliella salina）在高达饱和浓度的氯化钠环境中实现增殖的机制，我们仅了解其中一部分。此前，人们发现一种分子量为60,000的质膜蛋白p60的水平会随着外部盐度的升高而增加。基于cDNA克隆和酶活性测定，现已表明p60是一种内部重复的碳酸酐酶，其每个重复序列都与动物和莱茵衣藻（Chlamydomonas reinhardtii）的碳酸酐酶同源，但在酸性残基超过碱性残基方面表现特殊。盐度增加、pH值碱性偏移或去除碳酸氢盐会导致杜氏盐藻中p60及其mRNA水平以及外部碳酸酐酶活性平行增加。此外，纯化后的p60表现出与其他碳酸酐酶相当的碳酸酐酶活性。富含p60的可溶性制剂在约1.0 M氯化钠浓度下表现出最大碳酸酐酶活性，并且在更高盐浓度下仍保留相当的活性。相比之下，来自莱茵衣藻的类似制剂在0.6 M氯化钠中约90%受到抑制。这些结果表明p60是一种结构新颖的碳酸酐酶，其转录受二氧化碳可用性调控，并表现出嗜盐样特征。这种酶可能适合优化在高盐培养基中生长的细胞对二氧化碳的摄取。
