Cell-Free Synthesis of Functional Type IV Adenylyl Cyclase
| Autor: | R V Rebois, D R Warner, N S Basi |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Gs alpha subunit
Transcription Genetic Protein Conformation Biophysics Biochemistry Antibodies ADCY10 Cell-free system Adenylyl cyclase chemistry.chemical_compound GTP-Binding Proteins Enzyme Stability Cyclic AMP Animals Molecular Biology Brain Chemistry ADCY6 Forskolin Cell-Free System Colforsin ADCY9 Cell Biology ADCY3 Molecular Weight Kinetics chemistry Protein Biosynthesis Cattle Electrophoresis Polyacrylamide Gel Dimyristoylphosphatidylcholine Adenylyl Cyclases |
| Zdroj: | Analytical Biochemistry. 232:31-36 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.1995.9965 |
| Popis: | Type IV adenylyl cyclase was synthesized in a cell-free coupled transcription and translation system. Radiolabeled type IV adenylyl cyclase was specifically immune precipitated with anti-ACIV antibodies. The molecular weight of in vitro translated type IV adenylyl cyclase was 110,000, similar to that for type IV adenylyl cyclase produced in the baculovirus system [B. Gao and A. G. Gilman, (1991) Proc. Natl. Acad. Sci. USA 88, 10178-10182]. Dimyristoyl phosphatidylcholine was required for efficient stimulation of activity by both forskolin and GS, with a maximum specific activity of 700 +/- 100 nmol cAMP.min-1.mg-1 attained with both effectors combined. Both bovine brain GS and in vitro translated GS alpha activated in vitro translated type IV adenylyl cyclase; however, G beta gamma only enhanced stimulation in the presence of in vitro translated GS alpha. Forskolin maximally activated at concentrations from 200 to 400 microM in the absence or presence of GS. The in vitro translated product was very stable as production of cAMP by forskolin/GS activated type IV adenylyl cyclase was linear for up to 90 min at 30 degrees C. |
| Databáze: | OpenAIRE |
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