Fukuzawa H, Fujiwara S, Yamamoto Y, Dionisio-Sese M L, Miyachi S
Institute of Applied Microbiology, University of Tokyo, Japan.
Proc Natl Acad Sci U S A. 1990 Jun;87(11):4383-7. doi: 10.1073/pnas.87.11.4383.
cDNA clones for the periplasmic carbonic anhydrase (CA; carbonate hydro-lyase, EC 4.2.1.1) of Chlamydomonas reinhardtii cells were isolated and characterized. The fact that the cloned cDNA fragments encoded a 377-amino acid polypeptide (41,626 Da) consisting of an NH2-terminal hydrophobic signal peptide of 20 amino acids, a large (35,603 Da) subunit polypeptide, and a small (4144 Da) subunit polypeptide indicates that the two subunits are cotranslated as a precursor polypeptide. The amino acid sequence of mature subunits deduced from the nucleotide sequence showed 20-22% homology with human CA isozymes (CAI, CAII, and CAIII). Three zinc-liganded histidine residues and those forming a hydrogen-bond network to zinc-bound solvent molecules were highly conserved in the plant and animal enzymes. Three possible asparagine-linked glycosylation sites were found in the large subunit. Northern blot analysis was carried out using the cDNA fragment as a probe. The level of 2.0-kilobase CA mRNA increased within 1 hr when CO2 concentration of the bubbling gas was changed from 5% to 0.04% (air level) in the presence of light. On the other hand, CA mRNA did not accumulate when CO2 concentration was lowered in the dark. Experiments using 3-(3,4-dichlorophenyl)-1,1-dimethylurea showed that photosynthesis is absolutely required for the accumulation of CA mRNA. These results indicate that CA biosynthesis is regulated by changes in environmental CO2 concentration as well as light at the level of mRNA abundance.
莱茵衣藻细胞周质碳酸酐酶（CA；碳酸水解酶，EC 4.2.1.1）的cDNA克隆已被分离和鉴定。克隆的cDNA片段编码一个由20个氨基酸的NH2末端疏水信号肽、一个大的（35,603 Da）亚基多肽和一个小的（4144 Da）亚基多肽组成的377个氨基酸的多肽，这一事实表明这两个亚基作为前体多肽共翻译。从核苷酸序列推导的成熟亚基的氨基酸序列与人碳酸酐酶同工酶（CAI、CAII和CAIII）显示出20 - 22%的同源性。在植物和动物酶中，三个与锌配位的组氨酸残基以及那些与锌结合的溶剂分子形成氢键网络的残基高度保守。在大亚基中发现了三个可能的天冬酰胺连接的糖基化位点。使用该cDNA片段作为探针进行了Northern印迹分析。在光照下，当鼓泡气体中的CO2浓度从5%变为0.04%（空气水平）时，2.0千碱基的CA mRNA水平在1小时内升高。另一方面，在黑暗中降低CO2浓度时，CA mRNA不会积累。使用3 -（3,4 - 二氯苯基）- 1,1 - 二甲基脲的实验表明，CA mRNA的积累绝对需要光合作用。这些结果表明，CA的生物合成在mRNA丰度水平上受到环境CO2浓度变化以及光照的调节。
