Research suggests that the cyclic AMP (cAMP) signaling pathway is implicated in the development of alcoholism. Previous work in our laboratory has demonstrated that alcohol enhances the activity of adenylyl cyclase (AC) in an isoform specific manner; human type 7 AC (AC7) is most enhanced by ethanol as measured by cAMP accumulation assay in whole cells. We hypothesize that alcohol enhances AC activity by directly interacting with the protein and that alcohol effects on AC can be studied using recombinant AC expressed in bacteria. Our objectives include: 1) design and optimization of the conditions for protein expression and protein purity for recombinant AC proteins; 2) identification of the importance of each cytoplasmic domain to the alcohol effect through chimeric analysis; and 3) investigation of the interaction between alcohol and AC stimulators such as MnCl2, forskolin, and Gsá through concentration-response experiments. To examine these objectives, we expressed in bacteria, and purified recombinant AC proteins carrying the C1a and/or C2 domains of AC2, AC7, and AC9. We present the optimal conditions for the expression and purification of multiple recombinant AC proteins and show that purified recombinant AC proteins retain enzymatic activity and alcohol responsiveness. Through chimeric analysis, we found that the C1a and C2 domains both contribute to the alcohol effect on AC7, however, AC7 C1a may play a stronger role. We also find that recombinant AC responds to alcohol differently under varied conditions of AC activation by MnCl2, forskolin, and Gsá. Through concentration-response experiments we found that there is some interaction between alcohol and forskolin or Gsá, but alcohol does not appear
to be competing with forskolin or Gsá at an allosteric site. Overall the results suggest that alcohol interacts with AC proteins directly, independent of stimulants examined, and causes a conformational change, which results in either enhancement or inhibition depending on stimulation conditions.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-10302013-142308 |
| Date | 02 December 2013 |
| Creators | Creekmore, Emily Qualls |
| Contributors | Yoshimura, Masami, Strain, George, Li, Shisheng, Figueiredo, Marxa, Elzer, Philip |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-10302013-142308/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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