Molecular resolution and reconstitution of the GPP(NH) and NAF sensitive adenylate cyclase system

The mechanism of activation of adenylate cyclase by guanylyl-5'-imidodiphosphate [Gpp(NH)p] and NaF has been investigated by studying the reconstitution of Gpp(NH)p and NaF sensitivity of an enzyme rendered insensitive to these agents by differential detergent extraction of a particulate brain enzyme. Such reconstitution can be achieved by the addition of macromolecular regulatory factors from membranes of various tissues. Trypsin digestion and thermal inactivation provide evidence for the existence of two distinct regulatory functions, one capable of restoring the Gpp(NH)p response and another the NaF response. The regulatory protein(s) seem to interact with their respective activators in an easily reversible, divalent cation-independent reaction. This appears to be followed by a high-affinity interaction between the catalytic and regulatory components of adenylate cyclase in a slow, temperature-dependent, divalent cation-dependent process that produces the persistently activated state of the enzyme. The enzyme activation can be reversed by methods that separate catalytic from regulatory components and the resulting enzyme activity can be restimulated by the reconstitution technique.The molecular mechanisms of activation of adenylate cyclase by NaF and guanylyl-5'-imidodiphosphate [Gpp(NH)p], and the relevance of such mechanisms to the hormonal modulation of this enzyme, have been difficult to investigate due to the lack of appropriate techniques for the resolution and reconstitution of the various molecular components of the adenylate cyclase system. We have reported (1) the development of such a technique which involves the preparation of a Gpp(NH)p-and NaF-insensitive brain particulate adenylate cyclase by differential detergent extraction. The enzyme regained its characteristic Gpp(NH)p and NaF responses upon the addition of one or more proteins solubilized from membrane preparations of various tissues (1). These regulatory proteins can be partially separated from solubilized adenylate cyclase by gel filtration on Ultrogel AcA 34 columns. We report here the utilization of this reconstitution technique to help elucidate the mode of interaction of the various components of this multifactorial system. Thermal and trypsin inactivation of the Gpp(NH)p-and NaF-reconstituting activities suggests that the two activities are functionally separable. Gpp(NH)p and NaF appear to interact reversibly with the corresponding regulatory proteins in a divalent cation-independent step, followed by a divalent cation-dependent step that leads to the persistent state of enzyme activation. Techniques that separate regulatory from catalytic components can also reverse the enzyme activation.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3693 The Gpp(NH)p-and NaF-insensitive adenylate cyclase was prepared by a modification of a published procedure (1, ...

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See 1 more Smart Citation

Properties of the interaction of fluoride- and guanylyl-5'-imidodiphosphate-regulatory proteins with adenylate cyclase.

Hebdon¹,

Vine²,

Sahyoun³

et al. 1978

“…The gradients contained 25 mM Tris-HCl (pH 7.6), 0.1% Triton X-100 (all detergent concentrations are expressed as wt/vol), and 5 mM MgCl2. Samples (0.2 ml) were loaded and the gradients were centrifuged in a Beckman SW 50.1 rotor at 45,000 rpm for [14][15][16] hr at 40C, and 0.2-ml fractions were collected. Gel permeation chromatography was performed by applying 0.5-ml samples to 26-ml Ultrogel AcA-34 columns and elutingwith 50 mM Tris-HCl, pH 7.6/0.1% Triton X-100/5 mM MgCl2; 0.8-ml fractions were collected.…”

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Molecular complexes involved in the regulation of adenylate cyclase.

Sahyoun¹,

LeVine²,

Davis³

et al. 1981

Selective extraction of the adenylate cyclase regulatory protein (N-protein) from pigeon erythrocyte plasma membranes provided evidence for its cytoskeletal association. Cholate, but not Triton X-100 or digitonin, was effective in solubilizing the ADP-ribosylated N-protein. The labeled protein complex or components thereof that were associated with the Triton-insoluble cytoskeleton (shells) could be partly released by 0.1 mM EDTA; 1 M KCI in the presence of Triton X-100 achieved complete solubilization. 5'-Guanylyl imidodiphosphate (p[NH]ppG) and NaF, activators of adenylate cyclase, promoted the release of the regulatory protein from the cytoskeleton but MnC12, an "uncoupler" of the adenylate cyclase system, had the opposite effect. The solubilized, labeled N-protein was able to bind specificially to rat eryth-ocyte inside-out vesicles in the presence of divalent cations. A proteolytic product of inside-out vesicles inhibited the binding of the N-protein to fresh vesicles. Three molecular species which contained the Mr 45,000 polypeptide component of the N-protein were identified by gel permeation chromatography and by sucrose density gradient velocity sedimentation. p[NH]ppG appeared to convert the two larger molecular complexes to a smaller molecular entity. Such a molecular dissociation might be relevant to the effects of guanyl nucleotides on the activity of adenylate cyclase and on the affinity of hormone receptors.Activation ofthe adenylate cyclase system by hormones or GTP analogs appears to involve changes in the molecular associations among the receptor, the regulatory nucleotide-binding protein (N-protein), and the catalytic component. Gel permeation chromatography (1), sucrose density gradient sedimentation (2, 3), and target-irradiation analysis (4) have provided evidence for these molecular changes. Furthermore, selective extraction (5) and reconstitution (6) experiments suggested that constituents of the adenylate cyclase complex were bound to the cytoskeleton in a manner that depended on the state of activation of the enzyme (6). This report focuses on several molecular interactions of the N-protein and on their regulation by agents that activate adenylate cyclase. We have also attempted to solubilize, separate, and characterize the putative molecular complexes that result from these interactions. This approach may help to elucidate further the mechanism of activation of the enzyme as well as the mechanism of the regulation ofthe number and the affinity of hormone receptors that act on adenylate cyclase.Our (125-150 g) and from White Carneau pigeons. Rat reticulocytes were produced by the phenylhydrazine injection method (1). Pigeon erythrocytes were lysed by freeze-thawing, and the plasma membrane fraction was separated as reported (9). Rat reticulocyte and erythrocyte ghosts were made by hypotonic lysis (10) and they were resealed in the presence of MgCl2 (11). Inside-out vesicles were prepared by incubation of the erythrocyte ghosts in 0.1 mM sodium phosphate buffer (pH 7.6) at 37°C ...

“…The response of adenylate cyclase from mammalian brain to 5'-guanylyl imidodiphosphate (p[NH]ppG) can be abolished by two different procedures (7,8) and subsequently restored by addition of a diluted unfractionated detergent extract of brain that contains both the CU and the G/F. Because the G/F is not separated from the CU, the interpretation of the experiments is complicated.…”

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

Strittmatter¹,

Neer²

1980

102

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.