The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase
| Autor: | Masami Yoshimura, Ratna Gupta, Emily Qualls-Creekmore |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Gs alpha subunit Recombinant protein Biophysics Alcohol cAMP Cyclic AMP Biochemistry law.invention Adenylyl cyclase lcsh:Biochemistry 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine KO Knock out law Gsα lcsh:QD415-436 lcsh:QH301-705.5 Forskolin Ethanol DKO Double knock out 3. Good health 030104 developmental biology PMSF phenylmethylsulfonyl fluoride GppNHp Guanosine 5′-[β γ-imido]triphosphate chemistry lcsh:Biology (General) Recombinant DNA AC Adenylyl cyclase PMSF Signal transduction 030217 neurology & neurosurgery Research Article |
| Zdroj: | Biochemistry and Biophysics Reports, Vol 10, Iss C, Pp 157-164 (2017) Biochemistry and Biophysics Reports |
| ISSN: | 2405-5808 |
| Popis: | 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 AC in vitro. AC recombinant proteins containing only the C1a or C2 domains 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 C1a or C2 domains 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 EC50 were 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. Highlights • The activity of catalytic domains of mammalian adenylyl cyclase respond to ethanol in an isoform specific manner. • The alcohol cutoff effect of the recombinant adenylyl cyclase type 7 is same as that observed for the native protein. • Alcohol appears to interact with adenylyl cyclase type 7 at multiple sites with positive cooperativity. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|