Martin Rodbell

A Highly Sensitive Adenylate Cyclase Assay

Abstract A highly sensitive adenylate cyclase assay method has been developed which employs sequential chromatography on columns of Dowex cation exchange resin and aluminum oxide. With the use of [α-32P]ATP as substrate, this method permits the nearly complete separation of cyclic [32P]AMP formed from the substrate and other 32P-containing compounds, i.e., 32P in the assay blanks was barely detectable. In comparative studies, this method was found to be considerably more sensitive than previously reported methods. The high sensitivity of this method permits detection of the small amounts of cyclic AMP formed at low enzyme concentrations or at early time points in kinetic studies.

Glucagon-sensitive adenyl cylase in plasma membrane of hepatic parenchymal cells.

The plasma membrane of hepatic parenchymal cells contains an adenyl cyclase system that is stimulated by glucagon. Adrenocorticotropin and epinephrine do not stimulate this adenyl cyclase, and very little cyclic phospho-diesterase activity is present in the membrane. These findings support the concept that glucagon exerts its regulatory action in the liver by stimulating adenyl cyclase activity in the plasma membrane.

Opiate Receptor-Mediated Inhibition of Adenylate Cyclase in Rat Striatal Plasma Membranes

Abstract: Plasma membranes from rat striatum contain adenylate cyclase activity that is subject to dual regulation by GTP. Low concentrations (up to 30 nM) of the nucleotide increase activity whereas higher concentrations evoke a steady decline in activity; such behavior characterizes dually regulated adenylate cyclase systems. The opiates, morphine sulfate and D‐Ala‐Met‐enkephalin, produce naloxone‐reversible inhibition of the enzyme that is dependent on “inhibitory concentrations” of GTP (above 50 nM). In the absence of GTP no inhibition is observed. Sodium ions decrease the inhibition of activity promoted by GTP alone, but amplify the degree of inhibition seen in the presence of the opiates and GTP. The potencies of the opiates in mediating these effects mirror their affinities for 8 opiate receptors in striatum. It is suggested that this action of the opiates may represent their primary action in striatum.

Characteristics of the guanine nucleotide regulatory component of adenylate cyclase in human erythrocyte membranes

This study probes the structure and mutual interactions of the components of adenylate cyclase. We use a complementation assay which involves the addition of an adenylate cyclase-related guanine nucleotide-binding protein component to a membrane lacking this component to measure guanine nucleotide-stimulated-adenylate cyclase. Instead of using detergent extracts we were able to achieve full complementation by mixing intact membrane preparations in the presence of the nucleotide component. Of particular interest was the human erythrocyte membrane which contains very low amounts of catalytic activity and no measurable beta-adrenergic receptor but has normal amounts of the nucleotide component. This component appears to be the same, by several criteria, as components found in pigeon and turkey erythrocytes and in rat liver plasma membrane. The component confers Gpp(NH)p, fluoride, and GTP stimulation of adenylate cyclase along a single reconstitution curve. It is labeled with NAD by cholera toxin, and has an apparent molecular weight of 39 000 upon sodium dodecyl sulfate gel electrophoresis. The presence of the nucleotide unit in the virtual absence of the active catalytic unit allowed us to determine those properties intrinsic to each unit and those conferred by the association of the units. The nucleotide component binds guanine nucleotides weakly in the human erythrocyte membrane, yet produces persistent activation of adenylate cyclase and tight binding (of Gpp(NH)p) upon combination with the catalytic unit. Treatment of the human erythrocyte membrane with N-ethylmaleimide causes a simultaneous diminution in both Gpp(NH)p and fluoride stimulation in reconstituted activities, suggesting that both activities are conferred by the same component.

The Role of GTP-Binding Proteins in Signal Transduction: From the Sublimely Simple to the Conceptually Complex

Publisher Summary This chapter presents an admittedly personalized description of the development of the role of GTP-binding proteins in signal transduction. It provides an overview of the currently accepted views of G-protein structure and function and discusses the role of the cytoskeleton in signal transduction. The chapter presents speculations on the structure and dynamics of the GTP-binding proteins based on the evidence that they may exist in association with surface receptors as multimeric structures. It has been pointed out that microtubules and F-actin behave in a paradoxical fashion with regard to classical thermodynamics. While systems generally evolve toward greater stability, microtubules form from GTP-tubulin, GTP is hydrolyzed following the incorporation of tubulin, Pi is released into the medium, and the resulting GDP-microtubules are unstable and rapidly lose subunits upon dilution; in contrast, in a regime of growth, the dissociation rate constant of terminal subunits is very low.

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.

The complex regulation of receptor-coupled G-proteins

Heterotrimeric G-proteins are associated with the cytoplasmic surface of the cell membrane as oligomeric structures. The oligomeric structures were deduced from a variety of studies including target (irradiation) analysis, hydrodynamic evaluation of detergent extracted material, and cross-linking of G-proteins in their membrane environment. From the functional mass determined by target analysis, it was estimated that one receptor (for glucagon) is associated with 8-10 units of Gs, the heterotrimeric G-protein that stimulates adenylyl cyclase. It is proposed that the receptor associates with each monomer of the chain via weak and strong binding forces that are dictated according to whether either GTP or GDP is bound to the alpha-subunits (weak forces) or, due to the hormone-induced release of the nucleotides during the exchange reaction, these subunits become transiently devoid of nucleotides (strong forces). The hormone-induced changes in type and degree of nucleotide binding allow for movement of the receptor along the oligomeric chain and filling of the nucleotide binding sites with the activating nucleotide, GTP. In this manner, the receptor catalytically activates Gs. It is suggested that the dynamic instability of the oligomeric chain produced by the asymmetric distribution of GTP and GDP along the chain results in release of a GTP-monomer from one end and association of a GDP-monomer at the opposite end. Adenylyl cyclase associates with the released GTP-monomer inducing a transient state of the coupled proteins. In a Mg-dependent fashion, hydrolysis of GTP occurs resulting in re-organization of the coupled proteins such that alpha and beta gamma interact with distinct domains of the cyclase molecule. The final state of the coupled process determines the degree of cyclase activity. Release of Pi from its binding site restores association of alpha and beta gamma to the GDP-bound form of the heterotrimer. The latter associates with the oligomeric structure of G-proteins to complete the cycle of events in the overall process of hormonal activation of the system.

Inhibition and activation of fat cell adenylate cyclase by GTP is mediated by structures of different size

Abstract The adenylate cyclase system in the plasma membrane of fat cells contains regulatory components that either stimulate or inhibit activity in response to ligands acting at the cell surface. GTP is required for both the stimulation by various hormones (catecholamines and peptide hormones) and the inhibition by adenosine. We have analyzed the effects of high-energy radiation on the stimulatory and inhibitory processes and conclude that these processes are mediated by structures of different functional size. Moreover, the fat cell cyclase system, when analyzed under conditions in which the inhibitory action of GTP is minimally expressed, displays targets of the same size as those previously observed for those involved in the activation of the hepatic enzyme by glucagon and guanine nucleotides ( W. Schlegel, E. S. Kempner, and M. Rodbell, 1979 , J. Biol. Chem.254, 5168–5176). These findings extend our recent evidence for the nonidentity of the two GTP-mediated processes ( D. M. F. Cooper, W. Schlegel, M. C. Lin, and M. Rodbell, 1979 , J. Biol. Chem.254, 8927–8931).

Adenylate cyclase in polyacrylamide gel electrophoresis: solubilized but active.

Abstract An intrinsic “insoluble” membrane-bound enzyme, adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) was analyzed in a detergent solubilized but active form by polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis provided improved resolution of the enzyme from contaminant proteins compared to previously used column chromatographic procedures, and allowed the analysis of much smaller quantities of material. The success of this approach depended on a particular choice of the nature and concentration of the detergent, of pH, of buffer constituents, of temperature, and of gel architecture. Using either the basal enzyme or enzyme pretreated in the membrane with guanyl-5′-yl imidodiphosphate (Gpp(NH)p) in the presence or absence of glucagon, up to 90% of enzymatic activity was recovered. However, the basal enzyme lost sensitivity to stimulation by Gpp(NH)p but not fluoride during electrophoresis. Ferguson plot analysis was used to determine the size and charge parameters of the enzyme, and bandwidth to estimate the degree of polydispersity. Increasing detergent concentrations reduced the effective size and degree of polydispersity of the enzyme, although this also led to progressive inactivation. The size of the enzyme was independent of preactivation by Gpp(NH)p alone or Gpp(NH)p plus glucagon. The “smallest and least polydisperse” size to which the active enzyme could be reduced was 3.6 ± 0.8 to 4.0 ± 0.8 nm (geometric mean radius).

The characteristics of lubrol-solubilized adenylate cyclase from rat liver plasma membranes

Abstract Adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) was solubilized from rat hepatic plasma membranes with Lubrol PX. The enzyme could be stabilized during solubilization by pretreating the membrane with glucagon and 5′-guanylimidodiphosphate (Gpp(MH)p) or Gpp(NH)p alone but not by glucagon alone or in the presence of GTP or other guanine nucleotides. The solubilized enzyme was purified 3–4-fold by molecular exclusion chromatography on Ultrogel AcA22; the presence both 0.01% Lubrol and 25% sucrose were required to maintain both the solubility as well as stability of the enzyme. The enzyme was purified from 70% of the other solubilized membrane proteins and 85% of the solubilized phospholipids, and was completely separated from 5′-nucleotidase. In addition, the enzyme was purified 2-fold with respect to non-specific nucleotide phosphohydrolase and pyrophosphohydrolase activities but not with respect to ‘specific’ GTPase activity. No perceptible change in molecular size of partially purified adenylate cyclase was observed after it had been pre-activated in its membrane-bound form with hormone and Gpp-(NH)p. The solubilized enzyme also retained its ability to be activated by the guanine nucleotide analogues Gpp(NH)p and Gp(CH2)pp with similar concentration and time-dependent characteristics displayed by the membrane-bound form of the enzyme. Attempts to further purify the solubilized enzyme by a variety of size- and charge-based chromatography techniques proved unsuccessful. As an explanation for this phenomenon we suggest that, after solubilization with Lubrol, the enzyme is probably but a fraction of the total protein in the complex. That is, judging from the highest specific activity obtained for the partially purified liver enzyme (1.2 nmol./min per mg) and assuming that its turnover number is that of the highly purified bacterial adenylate cyclase [54], the enzyme comprises less than 0.1% of the protein present in the adenylate cyclase-containing fraction from the Ultrogel column. The idea of determining the physical properties of Lubrol-solubilized adenylate cyclase becomes even more difficult when one considers that properties, such as Stokes radii on Ultrogel columns depend upon the concentrationof detergent used in the column elution buffer. For example, in the absence of detergent we have observed that Lubrol-solubilized adenylate cyclase aggregates and elutes in the void volume of the column. Thus, knowledge of the aggregation characteristics of the enzyme further suggests that caution must be used in assigning values to the intrinsic physical properties of the enzyme.