Molecular and biochemical characterization of the bicarbonate-sensing soluble adenylyl cyclase from a bony fish, the rainbow trout Oncorhynchus mykiss.
Molecular and biochemical characterization of the bicarbonate-sensing soluble adenylyl cyclase from a bony fish, the rainbow trout Oncorhynchus mykiss.
复制标题
来自硬骨鱼虹鳟鱼 Oncorhynchus mykiss 的碳酸氢盐感应可溶性腺苷酸环化酶的分子和生化特征。
DOI：
10.1098/rsfs.2020.0026
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发表时间：
2021
期刊：
影响因子：
4.4
通讯作者：
Salmerón C
中科院分区：
文献类型：
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作者：
Salmerón C
可溶性腺苷环化酶(SAC)是一种受刺激的酶，可产生无处不在的信号分子cAMP，被认为是进化上保守的酸碱传感器。然而，它在脊椎动物中含量最多、种类最多的硬骨鱼中的存在尚未得到证实。在这里，我们鉴定了不同种类的软骨鱼、鳍鱼和裂鳍鱼的SAC基因。接下来，我们专注于彩虹鲑鱼SAC(RtsAC)，并确定了20个可能的剪接mRNAs，编码大小从28 kDa到186 kDa的蛋白质异构体。对纯化的重组rtsAC蛋白进行生化和动力学分析，以确定对鱼内液(EC50∼7 mM)生理相关水平的刺激。RtsAC活性对来自其他生物的SAC抑制剂Kh7、LRE1和DIDS敏感，而对跨膜腺苷酸环化酶调节剂Forsklin和2，5-二脱氧腺苷不敏感。Western印迹和免疫细胞化学显示rtsAC在鳃离子转运细胞、肝细胞、红细胞、心肌细胞和心肌细胞中高表达。对RTgill-W1细胞系的分析表明，一些较长的rtsAC亚型可能优先定位于细胞核、高尔基体和足体。这些结果表明，SAC有望在多骨鱼类中介导多种酸碱平衡反应，并为哺乳动物潜在的新功能提供线索。
Soluble adenylyl cyclase (sAC) is a-stimulated enzyme that produces the ubiquitous signalling molecule cAMP, and deemed an evolutionarily conserved acid–base sensor. However, its presence is not yet confirmed in bony fishes, the most abundant and diverse of vertebrates. Here, we identified sAC genes in various cartilaginous, ray-finned and lobe-finned fish species. Next, we focused on rainbow trout sAC (rtsAC) and identified 20 potential alternative spliced mRNAs coding for protein isoforms ranging in size from 28 to 186 kDa. Biochemical and kinetic analyses on purified recombinant rtsAC protein determined stimulation byat physiologically relevant levels for fish internal fluids (EC50∼ 7 mM). rtsAC activity was sensitive to KH7, LRE1, and DIDS (established inhibitors of sAC from other organisms), and insensitive to forskolin and 2,5-dideoxyadenosine (modulators of transmembrane adenylyl cyclases). Western blot and immunocytochemistry revealed high rtsAC expression in gill ion-transporting cells, hepatocytes, red blood cells, myocytes and cardiomyocytes. Analyses in the cell line RTgill-W1 suggested that some of the longer rtsAC isoforms may be preferentially localized in the nucleus, the Golgi apparatus and podosomes. These results indicate that sAC is poised to mediate multiple acid–base homeostatic responses in bony fishes, and provide cues about potential novel functions in mammals.