Specific phospholipids are required to reconstitute adenylate cyclase solubilized from rat brain.

Hebdon, George; LeVine, Harry; Sahyoun, Naji; Schmitges, Claus J.; Cuatrecasas, Pedro

doi:10.1073/pnas.78.1.120

Cited by 47 publications

(12 citation statements)

References 19 publications

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“…(37). Solubilized adenylate cyclase was reported to require phosphatidylcholine or sphingomyelin, whereas phosphatidylserine was without effect (38). A similar pattern was observed for UDP-glucuronosyltransferase from guinea pig liver (39).…”

Section: Methodsmentioning

confidence: 51%

Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester.

Delclos¹,

Yeh²,

Blumberg³

1983

Proc. Natl. Acad. Sci. U.S.A.

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As part of our effort to characterize receptors for the phorbol ester tumor promoters, a phorbol ester photoaffinity probe, [20-3H]phorbol 12-p-azidobenzoate 13-benzoate (PaBzBz), was synthesized. In the dark, PaBzBz bound reversibly to brain particulate fractions with a dissociation constant (Kd) of 0.81 ± 0.09 x 10-9 M. Specific binding of PaBzBz, at a concentration equal to its Kd, represented 85% of the total bound. At saturation, 24 ± 5 pmol of PaBzBz were bound per mg of brain protein, a level similar to that observed with [20-3H]phorbol 12,13-dibutyrate. Under the conditions used (concentrations greater than the Kd for PaBzBz), irradiation caused 45% of the PaBzBz binding to become irreversible. Most of the binding (=60%), including most of the specific irreversible binding, was to phospholipid rather than to protein. Based on susceptibility to enzymatic digestion and on chromatographic mobility, the specifically labeled phospholipids were identified as phosphatidylserine, phosphatidylethanolamine, and phosphatidylethanolamine plasmalogen. Although the PaBzBz specifically labeled lipid, labeling was blocked by pretreatment of membranes at 100IC for 5 min or by papain digestion. Therefore, it seems likely that the identified lipids are specifically associated with a protein receptor and are preferentially labeled either because of the location or reactivity of the nitrene generated on the photoaffinity probe.The phorbol esters are potent tumor promoters, which have also been shown to induce dramatic cellular and biochemical changes in vivo and in vitro (1-4). This laboratory has demonstrated specific, saturable binding of the phorbol esters to tissue particulate preparations and intact cells from a variety of organisms (5-10). The structure-activity requirements for binding are consistent with the receptors mediating the biological responses to the phorbol esters. The highest level of binding activity, 28 pmol/mg of protein, is found in brain (7). This level, which is considerably higher than that typical for hormone or neurotransmitter receptors, led us to suggest that the phorbol esters may be binding to a modulatory site on an enzyme or transport protein (7).We previously have demonstrated that phorbol ester binding in brain was sensitive to heat, protease, and phospholipase A2 treatments (7). In addition, we have shown that low concentrations of ascorbate inactivated binding with a time course and dose-response curve paralleling that for induction of lipid peroxidation (11). These results suggested either that the phorbol ester receptor is dependent on its specific lipid environment or else that lipid plays a role in the binding site itself.In an effort to obtain fiurther evidence on the nature of the phorbol ester binding site, we prepared a radioactive phorbol ester photoaffinity derivative, phorbol 12-p-azidobenzoate 13-benzoate (PaBzBz). This derivative contains an aryl azide, which is one of the widely used classes of photoaffinity labels (12, 13). We describe here specific, covalent bind...

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Section: Methodsmentioning

confidence: 51%

Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester.

Delclos¹,

Yeh²,

Blumberg³

1983

Proc. Natl. Acad. Sci. U.S.A.

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“…Reconstitution of Guanylate Cyclase into Phosphatidylcholine Vesicles-Guanylate cyclase was reconstituted into membrane vesicles using a dilution method similar to that of Hebdon et al (24). Phosphatidylcholine, NaCl, MgCl 2 , and Hepes, pH 7.6, were added to a concentrated fraction of purified guanylate cyclase in 2.5 mM DDM, 0.025% phosphatidylcholine, 20% glycerol, 120 mM NaCl, 6 mM EGTA, 10 mM Hepes, pH 7.6, to make a final concentration of 5 mg/ml phosphatidylcholine, 120 mM NaCl, 5 mM MgCl 2 , and 10 mM Hepes.…”

Section: Materials-8-n 3 [␣-mentioning

confidence: 99%

The Photoreceptor Guanylate Cyclase Is an Autophosphorylating Protein Kinase

Aparicio¹,

Applebury²

1996

Journal of Biological Chemistry

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The membrane guanylate cyclase of vertebrate photoreceptor rod outer segments plays a key role in controlling the levels of cGMP, the intracellular second messenger that mediates visual signaling (1-5). In the dark-adapted photoreceptor, the resting cGMP levels are set by the balance of synthesis and degradation due to the basal activities of the guanylate cyclase and the cGMP phosphodiesterase. Throughout the processes of phototriggering, photorecovery, and light adaptation, the activity of the guanylate cyclase appears to be modulated to control cGMP levels. Several factors are known to regulate the guanylate cyclase activity. Intracellular calcium concentration in the range of 0.1 to 1 M inhibits cyclase activity in the presence of small calcium-binding proteins, GCAPs 1 (6 -8). In contrast, a protein of 40 kDa, composed of a multimer of 6 -7-kDa peptides, activates the cyclase in a calcium-dependent manner with halfmaximal activation occurring between 2 and 5 M (9). In addition, guanylate cyclase activity has been reported to be modulated by ATP (10, 11) and by protein kinase C (12). The calcium regulation mediated by GCAPs is thought to be responsible for cyclase activation during photorecovery and light adaptation; however, the physiological significance of regulation by the other factors is unclear. In all cases, the mechanisms by which these factors control the activity of the photoreceptor guanylate cyclase are not understood.The primary structure of the photoreceptor membrane guanylate cyclase has been deduced from cDNA sequences isolated from human, bovine, and rat retinas (13-15). 2 The information indicates that it is a member of the family of membrane guanylate cyclases that function as receptors for peptide ligands. Four functional domains are characteristic of these proteins: an amino-terminal "extracellular" domain, a transmembrane region, an intracellular protein kinase-like domain, and a carboxylterminal guanylate cyclase catalytic domain (17)(18)(19). In other members of the family, binding of peptide ligands to the extracellular amino-terminal domains induces stimulation of cytoplasmic guanylate cyclase activity. Molecules known to function in this manner include receptors for ANP (GC-A) and CNP (GC-B), guanylin and Escherichia coli enterotoxin (GC-C), and the sea urchin egg peptides, speract and resact. Although no ligand has been identified for the photoreceptor guanylate cyclase, its structural similarity to the receptor guanylate cyclases suggests that it may be regulated in a similar manner.In this work, we have focused on the role of the kinase-like domain of the photoreceptor guanylate cyclase. Studies of other members of the receptor guanylate cyclase family clearly indicate that the kinase homology domain is required to transduce the ligand-binding signals and activate the guanylate cyclases (20 -22). ATP is required for ligand-mediated activation, but nonhydrolyzable ATP analogues work as well (17). No kinase activity has been measured for any receptor guanylate cyclase, and autoph...

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“…True, but it must be recalled that (i) detergents, in contrast to phospholipids, uncouple the functional units of adenylate cyclase (38,45), whereas phospholipids augment their interaction (39,40). Thus, if we find that both Ns and C are associated in detergent, in all likelihood they are associated even tighter within the lipid bilayer, and (ii) the shuttle model is based on experiments performed in digitonin extracts in the absence of added phospholipids to stabilize the components (12,44).…”

Section: C8ec9mentioning

confidence: 99%

“…This value is similar to the size of S49 lymphoma adenylate cyclase (38 NsC, however, is less stable than N' p[NH]ppG-C' on DEAE-Sephacel, probably because active N, confers more stability on C than native inactive Ns. The presence of phospholipids during the purification probably augments the Nsto-C interaction (39,40). To resolve Ns from C, the combination of detergent with high salt is required (15,41).…”

Section: C8ec9mentioning

confidence: 99%

Stimulatory GTP regulatory unit Ns and the catalytic unit of adenylate cyclase are tightly associated: mechanistic consequences.

Arad¹,

Rosenbusch²,

Levitzki³

1984

Proc. Natl. Acad. Sci. U.S.A.

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Turkey erythrocyte membranes were solubilized in the mild detergent octylpenta(oxyethylene) [CH3(CH2)7-(OCH2CH2)50H], which possesses a high critical micelle concentration (=6 mM) and forms small, dynamic micelles. Both the native enzyme N,(GDP)-C and the p [NH] Receptors for hormones and neurotransmitters interact with an effector system within the plane of the membrane to generate the primary biochemical response. A major class of receptors consists of those that activate adenylate cyclase upon agonist binding (refs. 1 and 2; ref. 3 and references therein). The activation of the catalytic unit of adenylate cyclase depends not only on the occupancy of the receptor by an agonist but also on the simultaneous occupancy of the GTP regulatory protein Ns by GTP (4). A number of mechanisms have been proposed for the sequence of events leading from the step of agonist occupancy on the receptor to the production of cAMP from ATP by the catalytic unit C. Currently three types of mechanisms are being discussed (Fig. 1).Although there is considerable evidence that hormone activation of adenylate cyclase by 8-agonists (5-7), glucagon (8, 9), and neurohypophyseal hormone (10) occurs by the "collision coupling" mechanism, counter arguments have been raised. These arguments are based on two types of observations: (i) guanyl nucleotides reduce the affinity of fadrenergic receptors towards their agonists (11) and, therefore, Ns and R are associated (12), and (ii) the n-receptor in the presence of guanyl-5'-yl-imidodiphosphate (p[NH]ppG) can activate Ns in the total absence of C (13). These observations were taken to mean (12, 13) that Ns "shuttles" between R and C (Fig. 1).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. other at all times. This model predicts (i) linear dependence of the rate constant of adenylate cyclase activation on the concentration of receptor R; (ii) the interaction between N, and C is never rate limiting; (iii) noncooperative binding of H. Model 3: N, shuttle. In this model the receptor R, the GTP stimulatory unit N,, and the catalytic unit C are all separate and interact sequentially as depicted. This model predicts noncooperative binding of H. if HR-N'(GTP) does not accumulate, and complex kinetics of adenylate cyclase activation. In the extreme case in which one of the components is in great excess, the kinetics will approach first-order kinetics.In fact, these two observations do not contradict the collision coupling theory. First, although the f3-adrenergic receptor forms a stable complex with Ns, in the presence of a f3-agonist and in the absence of guanyl nucleotide, it readily decomposes upon GTP or p[NH]ppG binding (12). Second, the fact that R and Ns interact with each other in the absence of C does not disprove that in the native situation Ns and C always function as a complex. Indeed, to separate Ns from C, a combination of ...

show abstract

Specific phospholipids are required to reconstitute adenylate cyclase solubilized from rat brain.

Cited by 47 publications

References 19 publications

Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester.

Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester.

The Photoreceptor Guanylate Cyclase Is an Autophosphorylating Protein Kinase

Stimulatory GTP regulatory unit Ns and the catalytic unit of adenylate cyclase are tightly associated: mechanistic consequences.

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