CELL-FREE SYNTHESIS OF FUNCTIONAL TYPE-IV ADENYLYL-CYCLASE

Type IV adenylyl cyclase was synthesized in a cell-free coupled transc ription and translation system. Radiolabeled type TV adenylyl cyclase was specifically immune precipitated with anti-ACIV antibodies. The mo lecular weight of in vitro translated type IV adenylyl cyclase was 110 ,000, similar to that for type IV adenylyl cyclase produced in the bac ulovirus system [B. Gao and A. G;. Gilman, (1991) Proc. Natl. Acad. Sc i. 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 effecters combined, Both bovine brain Gs and in vit ro translated Gs alpha activated in vitro translated type IV adenylyl cyclase; however, G beta gamma only enhanced stimulation in the presen ce of in vitro translated Gs alpha. Forskolin maximally activated at c oncentrations from 200 to 400 mu M in the absence or presence of Gs Th e in vitro translated product was very stable as production of cAMP by forskolin/G(s) activated type IV adenylyl cyclase was linear for up t o 90 min at 30 degrees C. (C) 1995 Academic Press, Inc.