The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase.
The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase.
复制标题
酒精对哺乳动物腺苷酸环化酶重组蛋白的影响。
DOI：
10.1016/j.bbrep.2017.03.011
复制
发表时间：
2017
影响因子：
2.7
通讯作者：
Yoshimura,Masami
中科院分区：
文献类型：
--
作者：
Qualls-Creekmore,Emily;Gupta,Ratna;Yoshimura,Masami
The cyclic AMP (cAMP) signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant ACin vitro. AC recombinant proteins containing only the C1aor C2domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C1aor C2domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl2, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC50were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.
