CHARACTERIZATION OF SOLUBLE FORMS OF NONCHIMERIC TYPE-V ADENYLYL CYCLASES

Type V adenylyl cyclase (ACV) belongs to the family of Ca2+- inhibited cyclases, We have generated two soluble forms of the enzyme containin g the C1 or C1a region (which lacks the C-terminal 112 amino acids) li nked to the C2 domain and compared their regulation with the full-leng th ACV. All three forms of ACV were stimulated by the alpha subunit of the stimulatory G protein G(s) (G(s alpha)) and forskolin, However, t he synergistic stimulation by both these activators was markedly enhan ced in the soluble enzymes, Moreover, the cu subunit of the inhibitory G protein G(i) (G(i alpha)) inhibited all forms of the enzyme, indica ting that the regions for G(s alpha) and G(i alpha) interaction are pr eserved in the soluble forms, Ca2+ inhibited forskolin-stimulated aden ylyl cyclase (AC) activity of the full-length and C1-C2 forms of ACV b ut did not alter the activity of the C1a-C2 form, Maximal stimulation of AC activity by combination of G(s alpha) and forskolin obliterated the Ca2+-mediated inhibition of the full-length and C1-C2 forms of ACV . In Ca-45(2+) overlay experiments, the C1-C2 but not the C1a-C2 solub le ACV bound Ca2+. Moreover, proteins corresponding to the C1a and C2 domains did not bind calcium, On the other hand, the proteins correspo nding to C1 and its C-terminal 112 amino acids (Gib) bound Ca-45(2+), To our knowledge, this is the first report of nonchimeric soluble form s of AC In which regulation by G(s alpha) and C-i alpha is preserved, Moreover, we demonstrate that the 112 amino acid C1b region of ACV is responsible for the binding of Ca2+ and inhibition of enzyme activity.