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 ...

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Concepts and criteria that have been developed for the study of the molecular organization of membrane-associated proteins are employed here to investigate the interaction of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with other membrane components. Detergent-solubilized adenylate cyclase can be shown to bind to erythrocyte-derived Triton X-100 shells containing cytoskeletal elements. This binding appears to be saturable with respect to adenylate cyclase concentration, and it is enhanced by the presence of divalent cations. Preactivation of the enzyme with 5'-guanylyl imidodiphosphate and isoproterenol, or with NaF, is a prerequisite for effective binding. Two exceptions to this general observation are noted: rat brain adenylate cyclase, which binds without prestimulation, and rat testicular cytosolic adenylate cyclase, which fails to bind under any of the conditions tried. The binding sites on the Triton X-100 shells are inactivated or released by treatment with various concentrations of trypsin or KCl. Moreover, exposure of the Triton X-100 shells to increasing temperatures results in a progressive loss of the adenylate cyclase binding capacity. On the basis ofthese and other findings, it is suggested that the adenylate cyclase complex possesses two principal domains that allow it to interact with both cytoskeletal elements and the lipid bilayer. The specific modulation of these interactions may be involved in the hormonal regulation of adenylate cyclase activity.An association between components of the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] system and the cytoskeleton has been postulated recently (1). Supporting evidence was obtained from the differential solubilization ofthe enzyme from rat erythrocyte ghosts and pigeon erythrocyte plasma membranes with Triton X-100 or low ionic strength buffers (1). Moreover, the effects of cytochalasin B, colchicine, and vinblastine on intracellular levels of 3',5'-cyclic AMP (cAMP) indicate that microfilaments and microtubules may influence the hormonal or cholera toxin stimulation of adenylate cyclase in whole cells (2-4). The morphological co-alignment (5, 6) and the biochemical coprecipitation (7, 8) of actin and certain crosslinked surface antigens suggests a more general role for cytoskeletal components in transmembrane signaling.Not only do membrane proteins that are linked to the cytoskeleton resist solubilization by nonionic detergents but also they can be separated from and reconstituted with other cytoskeletal components. This approach has been employed to investigate the cytoskeletal interactions of band 3 [a transmembrane erythrocyte protein (9)], spectrin [a peripheral membrane protein (10, 11)], and ankyrin [which bridges these two types of proteins (12) Here we have adapted similar techniques to study the binding of adenylate cyclase to the rat erythrocyte cytoskeleton. Our results demonstrate that detergent-solubilized adenylate cyclase binds in a saturable and specific manner to the shells der...

(5,6). Examples exist where this modulation seems to reflect changes in membrane fluidity (7,8) and others where there appears to be a requirement for certain specific phospholipids for enzyme activity (9-11). Such investigations have been extended to the study of adenylate cyclase principally by the direct addition of phospholipids' (or cholesterol) to cells or membranes by lipic4 fusion or exchange or by supplemention of the medium of auxotrophic mutant cells with phospholipid precursors (12)(13)(14)(15). Although these studies have yielded much new and interesting information, the added lipids may be exerting uncontrolled effects on the metabolism of the cells or they may not partition equally throughout the membrane.We have reported the solubilization of adenylate cyclase from rat brain and its subsequent incorporation into liposomes of defined phospholipid composition (16). These-studies showed that the enzyme activity in the liposomes was dependent on the particular phospholipids present. One drawback to this study is the use of nonionic detergents which, themselves, are capable of stimulating activity. In the present investigation we have used sodium deoxycholate to solubilize the enzyme; this preparation is totally dependent on addition of certain specific phospholipids or nonionic detergent for activity. The results further support the hypothesis that the activity of adenylate cyclase may be dependent on the presence of specific phospholipids.MATERIALS AND METHODS Materials. [a-32P]ATP and cyclic [2,8-3H]AMP were from New England Nuclear; alumina, activity 1, was from ICN. Triton X-100 was obtained from Packard. Pyruvate kinase was purchased from Boehringer Mannheim. Phosphatidyl-N-methylethanolamine (no. 835-8125) and phosphatidylglycerol (no. 835-8126) were supplied by GIBCO. The following naturally derived lipids were from Sigma: phosphatidylcholine (P 5763), lysophosphatidylcholine (L 4129), sphingomyelin (S 7004), phosphatidylserine (P 6641), phosphatidic acid (P 9511), phosphatidylinositol (P 0639), phosphatidylethanolamine (P 4513), phosphatidyl-N,N-dimethylethanolamine (P 1634), cholesterol (CH-S), cholesterol acetate (CH-SA), cholesterol stearate (CH-SS), and cholesterol oleate (CH-SO). ATP, phosphoenolpyruvate, and sodium deoxycholate were also obtained from Sigma. Triolein and 1,3-diolein were purchased from P-L Biochemicals.Preparation of Solubilized Adenylate Cyclase; Male rats (Sprague-Dawley, 120-170 g) were decapitated and their brains were removed. Subsequent operations were performed at 0°C. Each brain was homogenized in 8 vol (vol/wt) of 3 mM MgCl2/ 3 mM dithiothreitol/50 mM Tris-HCl, pH 8.2, and centrifuged for 10 min at 40,000 X gm. This procedure was repeated once and the pellet was then homogenized in 8 vol of 1 mM MgCl2/ 3 mM dithiothreitol/0.5% deoxycholate/50 mM Tris HCl, pH 8.2. The homogenate was incubated at 0°C for 20 min and centrifuged for 40 min at 300,000 x gm.; further centrifugation of the supernatant for 45 min at 300,000 X gm. did not sediment any further enzyme ac...

The proteins of sarcoplasmic reticulum were cross-linked by rapid oxidation of thiol groups with I2. About two-thirds of the thiols were oxidized without any significant cross-linking, implying an extensive formation of intramolecular disulphide bonds. When the thiols were completely oxidized at room temperature a series of oligomers containing up to five molecules were observed, as well as large aggregates which were excluded from the gels. Complete oxidation at -10 degrees C left most of the ATPase (adenosine triphosphatase) as monomer. Similar results were obtained when copper-phenanthroline complexes or dimethyl suberimidate were used as cross-linking reagents. We conclude that most of the cross-linked species arise by linking of randomly colliding ATPase molecules which are present in the membrane at very high concentration.