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Biomedical subjects

M Rodbell

Publications and source records attributed to M Rodbell.

At least 55 records · Page 3Linked to original sources
PubMed1979

The fat cell adenylate cyclase system. Characterization and manipulation of its bimodal regulation by GTP.

GTP evoked both an activatory and an inhibitory response from adipocyte adenylate cyclase. This paper describes the persistence of the bimodal response under a variety of assay conditions. Additionally, manipulations are described which eliminate one or other of these actions. Treatment of adipocyte plasma membranes with cholera toxin A1 peptide and NAD+ abolishes the inhibitory phase of GTP action while preserving the activating phase. Treatment of the membranes with p-hydroxymercuriphenylsulfonic acid eliminates the activatory phase while maintaining the inhibitory processes mediated by GTP in adipocytes normally coexist and operate through different pathways since either phase can be abolished leaving the other intact. Adenosine and its purine-modified analogs inhibit fat cell adenylate cyclase in the GTP inhibitory phase (Londos, C., Cooper, D. M. F., Schlegel, W., and Rodbell, M. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5362-5366). When this effect of GTP is abolished by either cholera toxin or Gpp(NH)p pretreatment, the inhibitory action of adenosine analogs is also lost. These data suggest a central role for GTP in mediating both activation and inhibition of adenylate cyclase by agents which act through cell surface receptors.

Adenylyl Cyclases↗
PubMed1979

Effects of phospholipase A2 and filipin on the activation of adenylate cyclase.

Rat liver plasma membranes were incubated with phospholipase A2 (purified from snake venom) or with filipin, a polyene antibiotic, followed by analysis of the binding of glucagon to receptors, effects of GTP on the glucagon-receptor complex, and the activity and responses of adenylate cyclase to glucagon + GTP, GTP, Gpp(NH)p, and F-. Phospholipase A2 treatment resulted in concomitant lossess of glucagon binding and of activation of cyclase by glucagon + GTP. Greater than 85% of maximal hydrolysis of membrane phospholipids was required before significant effects of phospholipase A2 on receptor binding and activity response to glucagon were observed. The stimulatory effects of Gpp(NH)p or F- remained essentially unaffected even at maximal hydrolysis of phospholipids, whereas the stimulatory effect of GTP was reduced. Detailed analysis of receptor binding indicates that phospholipase A2 treatment affected the affinity but not the number of glucagon receptors. The receptors remain sensitive to the effects of GTP on hormone binding. Filipin also caused marked reduction in activation by glucagon + GTP. However, in contrast to phospholipase A2 treatment, the binding of glucagon to receptors was unaffected. The effect of GTP on the binding process was also not affected. The most sensitive parameter of activity altered by filipin was stimulation by GTP or Gpp(NH)p; basal and fluoride-stimulated activities were least affected. It is concluded from these findings that phospholipase A2 and filipin, as was previously shown with phospholipase C, are valuable tools for differentially affecting the components involved in hormone, guanyl nucleotide, and fluoride action on hepatic adenylate cyclase.

Adenosine Triphosphate↗
PubMed1979

Glucagon1-6 binds to the glucagon receptor and activates hepatic adenylate cyclase.

A fragment of glucagon encompassing its first six NH2-terminal residues (His-Ser-Gln-Gly-Thr-Phe) binds to the glucagon receptor and stimulates adenylate cyclase activity in rat liver plasma membranes. Glucagon1-6 is a partial agonist since it stimulates, at saturating concentrations, to the extent of 75% of the maximal activity given by the native hormone. The binding affinity and potency of glucagon1-6 are 0.001% the native hormone. Discussed are the implications of these findings on the structure-function relationships required for the action of glucagon and for preparing clinically useful analogs of the hormone.

Adenylyl Cyclases↗
PubMed1978

A reassessment of structure-function relationships in glucagon. Glucagon1-21 is a full agonist.

Glucagon1-21 has been prepared by treating native glucagon with carboxypeptidase A. Purified glucagon1-21 did not contain detectable methionine (less than 0.001 residue/mol) and the activity of the compound did not change after treatment with cyanogen bromide as has been shown with native glucagon. Glucagon1-21 stimulates hepatic adenylate cyclase activity to the same extent as native glucagon but with 0.1% the potency. Glucagon1-21 also displayed 0.1% the binding affinity of native glucagon to the glucagon receptor in hepatic membranes. Glucagon22-29 alone or in combination with glucagon1-21 did not activate adenylate cyclase or displase 125I-glucagon from its receptor. The finding that glucagon1-21 is a full agonist on adenylate cyclase is discussed in relation to the structure-function relationships required for the biological action of glucagon.

Adenylyl Cyclases↗
PubMed1978

Adenosine analogs inhibit adipocyte adenylate cyclase by a GTP-dependent process: basis for actions of adenosine and methylxanthines on cyclic AMP production and lipolysis.

Adenylate cyclase in purified membranes from rat adipocytes is inhibited by low concentrations of purine-modified adenosine analogs, particularly those modified in the N6 position. Such inhibition is antagonized competitively by methylxanthines, but not by other cyclic nucleotide phosphodiesterase inhibitors, and it is dependent on "inhibitory" concentrations of GTP in the assay medium. Ribose-modified adenosine analogs inhibit adenylate cyclase through a process that is neither dependent upon the GTP concentration nor antagonized by methylxanthines. These results explain the potent effects of adenosine and methylxanthines on fat cell metabolism and demonstrate the importance of GTP in mediating inhibition by agents that act at cell surface receptors.

Adenosine↗
PubMed1978

The actions of hormones on adenylate cyclase systems.

The glucagon-sensitive adenylate cyclase system, viewed from the perspective of its behavior with isolated membrane preparations, displays far more complex regulatory characteristics than could have been envisioned from its behavior in the intact cell. What has emerged from our studies with isolated hepatic membranes is that glucagon can exert at least three actions which we believe are interdependent: desentization of the receptor, activation of adenylate cyclase, and promotion of adenosine inhibition of adenylate cyclase activity. Although the molecular basis remains unknown, GTP is intimately involved in the three processes. Undoubtedly, further levels of complexity will develop when the enzyme system is dissected and its components become amenable to study at the molecular level. At the moment, it is clear that adenylate cyclase systems are provided with a plethora of regulatory processes for controlling cyclic AMP production both in the absence and presence of hormones.

Adenosine↗
PubMed1977

GTP stimulates and inhibits adenylate cyclase in fat cell membranes through distinct regulatory processes.

GTP and hormones activate, synergistically, adenylate cyclase in purified plasma membranes from rat adipocytes. Addition of chelating reagents (EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) or thiol-reducing reagents (dithiothreitol or 2-mercaptoethanol) results in marked inhibition of enzyme activity without altering the synergistic stimulatory effects of GTP and hormones. The inhibitory effects of the reagents required the presence of GTP, indicating that inhibition involves a GTP-dependent process. This process is separate from the GTP-dependent process responsible for activation of the enzyme since it is selectively abolished by pretreatment of fat cell membranes with trypsin. It is suggested that inhibition and activation of fat cell adenylate cyclase by GTP occur through distinct regulatory processes.

Adenylyl Cyclase Inhibitors↗
PubMed1977

Activation of hepatic adenylate cyclase by guanyl nucleotides. Modeling of the transient kinetics suggests an "excited" state of GTPase is a control component of the system.

A three-state model developed originally from analysis of the steady state kinetics of hepatic adenylate cyclase has been extended to account for the transient kinetics of activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). In contrast to activation by Gpp(NH)p, activation of the enzyme by GTP proceeds not only without a lag phase but is of considerably lower magnitude. These differences between Gpp(NH)p and GTP can be explained by the hypothesis that GTP is hydrolyzed at the nucleotide regulatory site(s) associated with adenylate cyclase and that GTPase activity is revealed uniquely when the enzyme system is in its state of highest adenylate cyclase activity. With this hypothesis, the characteristics of activation by GTP could be simulated. The implications of this model are discussed with respect to the actions of hormones and cholera toxin on adenylate cyclase activity.

Adenylyl Cyclases↗
PubMed1977

Solubilization and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide-sensitive states.

Adenylate cyclase in liver membranes was solubilized with Lubrol PX and partially purified by gel filtration. The partially purified enzyme was susceptible to activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Studies on the binding of [3H]Gpp(NH)p to various fractions eluted from the gels revealed that an upper limit of 1% of the Gpp(NH)p binding sites is associated with adenylate cyclase activity stimulated by the nucleotide. The glucagon receptor, pretagged with 125I-glucagon in the membranes, solubilized with Lubrol PX, and fractionated on the same gel columns, eluted in a peak fraction that overlaps with, but is separate from, adenylate cyclase in its Gpp(NH)p-stimulated form. Addition of GTP to the solubilized glucagon-receptor complex caused complete dissociation of the complex, as has been shown with the membrane-bound form of the complex. Since the GTP-sensitive form of the glucagon receptor complex separates from the Gpp(NH)p-sensitive form of adenylate cyclase, it is concluded that the receptor and the enzyme are separate molecules, each associated with a distinct nucleotide regulatory site or component. These findings are discussed in terms of the possible structure of the hormone-sensitive state of adenylate cyclase.

Adenylyl Cyclases↗
PubMed1977

Reversible activation of hepatic adenylate cyclase by guanyl-5'-yl-(alpha,beta-methylene)diphosphonate and guanyl-5'-yl imidodiphosphate.

Guanyl-5'-yl-(alpha,beta-methylene)di[gamma-32P]phosphonate (Gp-(CH2)pp) is not hydrolyzed by rat liver membranes under conditions in which GTP and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) are hydrolyzed. Gp(CH2)pp activates adenylate cyclase in hepatic membranes with characteristics similar to those of Gpp(NH)p activation but with lower potency and effectiveness. The analogs, although with lower potency than GTP, also share the ability to change the glucagon receptor from a high to a low affinity state. Both Gp(CH2)pp and Gpp(NH)p stimulate adenylate cyclase activity following a lag period of about 1 min addition of GTP after steady state rates are achieved results in reduction in the rate following a lag period of 6 min from the time of addition of GTP. Pretreatment of the enzyme with Gpp(NH)p or Gp(CH2)pp, followed by washing the membranes, leads to a high activity state of the enzyme which slowly decays in rate unless the analogs are continuously present in the medium. These data suggest that the guanosyl nucleotide analogs act on the enzyme system by a slowly reversible process that possibly reflects slow binding and dissociation from different transition states of the enzyme system and suggest that activation of adenylate cyclase by GTP, Gpp(NH)p, and Gp(CH2)pp does not involve covalent modification of the enzyme.

Adenylyl Cyclases↗
PubMed1977

Effects of GTP on binding of (3H) glucagon to receptors in rat hepatic plasma membranes.

In this study, we report the preparation of [3H]glucagon and its characteristics of binding to receptors in the rat liver plasma membrane. Binding of the labeled hormone is optimal at pH 7.0. In the absence of GTP, [3H]glucagon binding to receptors is slow and the time of equilibration is inversely proportional to the hormone concentration. In the presence of GTP, equilibrium is reached within 30 s regardless of hormone levels, and the kinetics of binding are in accord with the kinetics of activation of adenylate cyclase by native glucagon in the presence of the nucleotide. Equilibrium binding measurements indicate that, in the absence of GTP, the binding isotherm is sigmoidal with an apparent Kd of 2 nM. The addition of GTP results in a complex binding isotherm with about 90% of the binding sites having a considerably lower apparent dissociation constant (greater than 10 nM) and a small population of sites having high affinity for the hormone. The binding properties of [3H]glucagon are compared with those of 125I-glucagon, and the implications of the actions of GTP on glucagon binding are discussed in relation to the overall regulation of adenylate cyclase by hormone and the nucleotide.

Animals↗