Cell-free synthesis of functional type IV adenylyl cyclase.
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.
Warner
D R
DR
National Institutes of Health, Bethesda, Maryland 20892, USA.
Basi
N S
NS
Rebois
R V
RV
D000262:Adenylyl Cyclases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001923:Brain Chemistry; D002417:Cattle; D002474:Cell-Free System; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D019204:GTP-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D014176:Protein Biosynthesis / Q000235:genetics; D011487:Protein Conformation; D014158:Transcription, Genetic / Q000235:genetics
D000906:Antibodies; D005576:Colforsin; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D004134:Dimyristoylphosphatidylcholine