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The adenylate cyclase enzymes from pig kidney medulla, rat liver and rat brain membranes were solubilized with Triton X-100 into three different states : basal, NaF-activated and guanosine 5'-(β,γ-imino)triphosphate- [GuoPP(NH)P]-activated. The effects of 2-mercaptoethanol and other thiol-protecting reagents on the response of solubilized adenylate cyclase to NaF and GuoPP(NH)P and on its apparent sedimentation constants were examined. In the absence of 2-mercaptoethanol, GuoPP(NH)P produced very little stimulation of the adenylate cyclase activity in 200 000 x g supernatant fractions of detergent-treated membranes. 2-Mercaptoethanol markedly enhanced the response to GuoPP(NH)P and to a lesser extent to NaF. When membranes were solubilized and ultracentrifuged without 2-mercaptoethanol, the adenylate cyclases in all of the three states studied had the same apparent sedimentation coefficient (7.4 S, 7.6 S and 7.9 S for the enzymes from pig kidney, rat liver and rat brain respectively). In the presence of 2-mercaptoethanol the apparent sedimentation constant of the adenylate cyclase solubilized in the basal activity state fell by about 1 S, that of the GuoPP(NH)P-preactivated state remained unchanged and that of the NaF-preactivated state dropped with a concomitant loss in activity. The reduction in size and activity were abolished when NaF was added to the sucrose density gradients. Stimulation by GuoPP(NH)P or NaF of the light form of adenylate cyclase (basal state solubilized in the presence of 2-mercaptoethanol) raised the apparent sedimentation constant, and the values obtained were identical to those found for the preactivated forms of the enzyme. [ABSTRACT FROM AUTHOR]

Specific vasopressin receptors in rat liver membranes were recently characterized [Cantau et al., Journal of Receptors Research, in the press]. They differ from the receptors characterized earlier in kidney membranes as regards coupling with adenylate cyclase and specificity towards vasopressin structural analogues [Rajerison et al. (1974) J. Biol. Chem. 249, 6390–6400; Butlen et al. (1978) Mol. Pharmacol. 14, 1006–1017]. The object of the present work was to see whether these differences reflect a difference in the molecular size of liver and kidney vasopressin receptors. For this purpose, rat liver and kidney membranes were incubated with [³H]vasopressin and solubilized with Triton X-100 (0.3%). The properties of the macromolecular components of soluble extracts to which labelled vasopressin remained bound were observed to resemble those of the intact membrane receptors as regards binding reversibility at 30–37°C and sensitivity to guanyl nucleotides. The hydrodynamic parameters of the soluble hormone-receptor complexes were estimated from Ultrogel column filtration experiments and from sucrose density gradient ultracentrifugation experiments. The following values were obtained for liver and kidney receptors respectively: Stokes' radii: 5.4 and 5.6 nm; standard sedimentation coefficients: 3.7 and 3.7 S; partial specific volumes: 0.75 and 0.78 ml · g-1; molecular weight: 83000 and 80000. These results indicate that the marked functional differences between liver and kidney receptors are not accompanied by appreciable differences in molecular size. [ABSTRACT FROM AUTHOR]

The effects of 17 non-ionic detergents on pig kidney plasma membrane adenylate cyclase were tested (digitonin; detergents from the Triton series: X45, X100, X102, X114, X165, X305, X405, N101; the Brij series: 35, 56, 96 and the Tween series: 20, 40, 60, 80, 85). Membranes (2.66 mg protein/ml) were treated with increasing amounts of detergent for 150 min at 0 °C. The adenylate cyclase activities present in treated membranes and in their corresponding non-sedimentable fractions were measured at 30 °C. Only detergents having hydrophilic lipophilic balance values from 12 to 14 were effective solubilizing agents. For these detergents, the solubilization yield was dependent on their structure. When solubilized adenylate cyclase kept at 0 °C was incubated at 30 °C, the enzyme catalytic activity exponentially decreased towards an equilibrium value with a half-time of 5 min. This temperature-dependent adjustment of enzyme activity was reversible. Its magnitude was dependent on the detergent used. A more progressive but less pronounced effect was observed with non-solubilizing detergents. The micellar form of detergent in the incubation medium was found to be responsible for the appearance of microheterogeneity in the population of solubilized enzyme molecules differing by the substrate accessibility. This effect was suppressed when reducing the detergent concentration in the incubation medium. [ABSTRACT FROM AUTHOR]

In this study, we investigated the mechanism by which a peptide mimicking the third cytoplasmic loop of the vasopressin V2 receptor inhibits signaling. This loop was synthesized as a cyclic peptide (i3 cyc) that adopted defined secondary structure in solution. We found that i3 cyc inhibited the adenylyl cyclase activity induced by vasopressin or a nonhydrolyzable analog of GTP, guanosine 5'-O-(3-thio)triphosphate. This peptide also affected the specific binding of [³H]AVP by converting vasopressin binding sites from a high to a low affinity state without any effect on the global maximal binding capacity. The inhibitory actions of i3 cyc could also be observed in the presence of maximally uncoupling concentration of guanosine 5'-O-(3-thio)triphosphate, indicating a direct effect on the receptor itself and not exclusively on the interaction between the Gs protein and the V2 receptor (V2-R). Bioluminescence resonance energy-transfer experiments confirmed this assumption, because i3 cyc induced a significant inhibition of the bioluminescence resonance energy-transfer signal between the Renilla reniformis luciferase and the enhanced yellow fluorescent protein fused V2-R. This suggests that the proper arrangement of the dimer could be an important prerequisite for triggering Gs protein activation. In addition to its effect on the receptor itself, the peptide exerted some of its actions at the G protein level, because it could also inhibit gnanosine 5'-O-(3thio)triphosphate-stimulated AC activity. Taken together, the data demonstrate that a peptide mimicking V2-R third intracellular loop affects both the dimeric structural organization of the receptor and has direct inhibitory action on Gs. [ABSTRACT FROM AUTHOR]