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Cell-free synthesis of functional type IV adenylyl cyclase.
- Source :
-
Analytical biochemistry [Anal Biochem] 1995 Nov 20; Vol. 232 (1), pp. 31-6. - Publication Year :
- 1995
-
Abstract
- 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.
- Subjects :
- Adenylyl Cyclases chemistry
Adenylyl Cyclases metabolism
Animals
Antibodies immunology
Brain Chemistry
Cattle
Cell-Free System
Colforsin pharmacology
Cyclic AMP metabolism
Dimyristoylphosphatidylcholine pharmacology
Electrophoresis, Polyacrylamide Gel
Enzyme Stability
GTP-Binding Proteins metabolism
Kinetics
Molecular Weight
Protein Biosynthesis genetics
Protein Conformation
Transcription, Genetic genetics
Adenylyl Cyclases biosynthesis
Subjects
Details
- Language :
- English
- ISSN :
- 0003-2697
- Volume :
- 232
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Analytical biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 8600828
- Full Text :
- https://doi.org/10.1006/abio.1995.9965