Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme.
Adenylate cyclase can be resolved into at least two proteins, a thermolabile, N-ethylmaleimide-sensitive component and a second protein (or proteins) that is more stable to either of these treatments. Neither component by itself catalyzes the formation of cyclic AMP using MgATP as substrate. However, mixture of the two reconstitutes MgATP-dependent fluoride- and guanyl-5'-yl imidodiphosphate (Gpp(NH)p)-stimulatable adenylate cyclase activity. The more stable component can be resolved from the first in various tissues or cultured cells by treatment of membrnes or detergent extracts with heat or N-ethylmaleimide. The two proteins have also been resolved genetically in two clonal cell lines that are deficient in adenylate cyclase activity. An adenylate cyclase-deficient variant of the S49 lymphoma cell (AC-) contains only the thermolabile activity, while the activity of the more stable protein is found in a complementary hepatoma cell line (HC-1). In addition, AC-S49 cell plasma membranes contain MnATP-dependent adenylate cyclase activity. The protein that catalyzes this reaction appears to be the same as that which can combine with the thermostable component to reconstitute Mg2+-dependent enzyme activity because both activities co-fractionate by gel exclusion chromatography and sucrose density  gradient centrifugation, both activities have identical denaturation kinetics at 30 degrees C, and both activities are stabilized at 30 degrees C and labilized at 0 degree C by various nucleotides and divalent cations with similar specificity. It is thus hypothesized that the thermolabile factor is the catalytic subunit of the physiological adenylate cyclase and that the Mn2+-dependent activity is a nonphysiological expression of the catalytic protein. The thermostable moiety of the enzyme, which is proposed to serve a regulatory function, appears to consist of two functional components, based upon differential thermal lability of its ability to reconstitute hormone-, NaF-, or Gpp(NH)p-stimulated adenylate cyclase activity. These components have not, however, been physically separated. The thermolabile and thermostable components can interact in detergent solution or in a suitable membrane. Mixing of the detergent-solubilized regulatory component with AC-membranes that contain only the catalytic protein and beta-adrenergic receptors reconstitutes catecholamine-stimulatable adenylate cyclase activity; however, addition of the catalytic protein to membranes that contain receptor and the regulatory component yields MgATP-dependent enzymatic activity that is unresponsive to hormone.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004355:Drug Stability; D005033:Ethylmaleimide / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006358:Hot Temperature; D007545:Isoproterenol / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*
D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; D011956:Receptors, Cell Surface; D006165:Guanylyl Imidodiphosphate; D008345:Manganese; D000262:Adenylyl Cyclases; D008274:Magnesium; D007545:Isoproterenol; D005033:Ethylmaleimide; D005459:Fluorides