Properties of the separated catalytic and regulatory units of brain adenylate cyclase.

Abstract

Adenylate cyclase from bovine brain cortex was solubilized with 14 mM cholate and 1 M (NH4)2SO4. Gel filtration over a column of Sepharose 6B separated the catalytic unit (CU) from a factor (G/F) that confers responsiveness to 5'-guanylyl imidophosphate (p[NH]ppG) or fluoride. The separated CU, which elutes with a Kav, of 0.48 +/- 0.01 (n=5), is not responsive to p[NH]ppG or fluoride and is relatively inactive when Mg . ATP is the substrate but activated 8-15-fold by Mn2+. The separated G/F elutes with a Kav of 0.70 +/- 0.02 (n=4). It restores the responsiveness of the CU to p[NH]ppG and fluoride. Activation of the enzyme by p[NH]ppG before solubilization does not decrease the amount of G/F eluting with a Kav of 0.7. Therefore, the G/F is probably present in brain cortex in excess over the CU. p[NH]ppG stabilizes the G/F but not the CU against thermal inactivation, suggesting that it interacts with G/F and not with CU. Incubation of the G/F with p[NH]ppG before addition of CU markedly increases the rate of activation of the reconstituted enzyme by p[NH]ppG. We propose, therefore, that the rate-limiting step in adenylate cyclase activation is a process in G/F alone and not a slow conformational change in CU or a slow association of G/F with CU. Binding of p[NH]ppG to the isolated G/F appears to be readily reversible; the ability of fully activated G/F to stimulate CU can be blocked if GDP is added before CU. In contrast, after the CU has been activated by interaction with G/F, GDP cannot reverse the activation. This suggests that association with the CU increases the affinity of G/F for p[NH]ppG.

Age-related changes in beta-adrenergic neuroeffector systems in the human heart.

“…In contrast, there was no age-related change in maximum adenylyl cyclase stimulation by Mn2+, an adenylyl cyclase catalytic unit activator whose action is not influenced by G proteins. 48 In older explanted hearts, we observed a marked decrease in the maximum tension response to isoproterenol as well as a reduced isoproterenol/Ca2`ratio, similar to what has recently been reported in isolated human ventricular myocytes.49 Additionally, the EC50 value for isoproterenol was markedly increased (by 10-fold) with age. Despite these changes in the fl-receptor pathway, younger and older hearts had similar myocardial levels of the adrenergic neurotransmitters norepinephrine and NPY and similar levels of dopamine and epinephrine.…”

Beta-adrenergic neuroeffector abnormalities in the failing human heart are produced by local rather than systemic mechanisms.

“…In human ventricular myocardium, adenylate cyclase re- Values are maximum net increase in tension±SEM (n refers to number of trabeculae). These data suggest that PPH failing right ventricle exhibits a chamber-specific abnormality ofthe adenylate cyclase catalytic subunit (C) which leads to a reduction in activity when G, is "activated" by NaF (38) or C is stimulated by Mn2" (39). However, when GTP and Mg2" are added to the reconstituted, standard adenylate cyclase assay condition the response of PPH right ventricular preparations to certain agonists (VIP, histamine, and NaF) is normal, suggesting that the catalytic subunit defect can be overcome by the addition of moderate amounts of guanine nucleotides and Mg2+.…”

Activation and attenuation of adenylate cyclase. The role of GTP-binding proteins as macromolecular messengers in receptor–cyclase coupling

“…Speigel & Aurbach, 1974;Lefkowitz, 1975 hormone and nucleotide effectors impinging on the system. This possibility has recently been strengthened in studies demonstrating that the apparently 'excess' G-proteins can restore the sensitivity to guanine nucleotides of isolated catalytic subunits in reconstitution experiments (Strittmatter & Neer, 1980). Genetic manipulation and reconstitution have provided another avenue for functionally and physically isolating the G-protein, as well as for beginning to identify the nature of its interaction with other components of the adenylate cyclase systems.…”

“…Bimbaumer and colleagues (Birnbaumer et al, 1980a,b;Iyengar et al, 1980) (Strittmatter & Neer, 1980 (Cassel & Selinger, 1977b, 1978, and also regulate the expression of adenylate cyclase activity.…”

“…This coincides with the ability of guanine nucleotides to stabilize the interaction of the Gprotein with AC-and UNC acceptor preparations in reconstitution studies (Ross et al, 1978;Howlett et al, 1979;Sternweis & Gilman, 1979). Concomitant with this interaction of G-protein with C is an increase in the affinity of the G-protein for bound guanine nucleotides Strittmatter & Neer, 1980). Fig.…”