Glucagon Binding Sites Research Articles

Inhibitors of glucagon inactivation: Effect on glucagon-receptor interactions and glucagon-stimulated adenylate cyclase activity in liver cell membranes

The specific binding of 125I-labeled glucagon to liver cell microsomal membranes is accompanied by a rapid loss in the biological activity of the hormone present in the medium. Inhibitors of glucagon inactivation have been identified and the effects of such compounds on hormone-binding and hormone-stimulated adenylate cyclase activity have been examined. 1. 1. Compounds which inhibit inactivation of glucagon by microsomal membranes include: (a) peptide hormones of low molecular weight which have little or no tertiary structure, such as adrenocorticotropic hormone; (b) peptide antibiotics, such as bacitracin; (c) bile salts and synthetic detergents, such as sodium deoxycholate and Triton X-100; (d) polyene antibiotics, such as filipin; and (e) metal-complexing agents, such as 1,10-phenantroline. 2. 2.Under appropriate conditions, inhibitors of glucagon inactivation cause a significant (20–80%) increase in glucagon-binding to liver microsomal membranes when subsaturating concentrations of hormone are used. The ability of the various inhibitors to suppress inactivation correlates well with their ability to enhance hormone binding. The effects of the inhibitors on binding result from an enhanced affinity of the glucagon-membrane interaction, and not from the exposure of new glucagon-binding sites. 3. 3. Adrenocorticotropic hormone and bacitracin increase the apparent affinity of adenylate cyclase of liver membranes for glucagon, but do not affect the maximal activity of the enzyme. Such effects are not apparent with other inhibitors tested, probably because some of these compounds perturb unfavorably the catalytic properties of adenylate cyclase. 4. 4. When inactivation of inhibited the glucagon-membrane interaction obeys kinetics of simple, dissociable processes. In this case the rate constant of glucagon-membrane association (10 6 M −1 · −1) and of dissociayion (2 · 10 −4 s −1) can be measured independently, and the dissociation constant (2 · 10 −10 M) determined from these rate constants is similar to that (2.5 · 10 −10 M) calculated separately from equilibrium data. 5. 5. Selective inhibition of the glucagon-inactivating components of liver cell membranes allows more meaningful kinetic characterization of the hormone-receptor interaction in these membranes and may also aid in further studies designed to isolate and purify the receptors.

Evidence for Interdependent Action of Glucagon and Nucleotides on the Hepatic Adenylate Cyclase System

Abstract The kinetic characteristics of glucagon action and binding as functions of hormone and nucleotide concentrations have been investigated in the hepatic adenylate cyclase system. Either App(NH)p (5'-adenylylimidodiphosphate), 0.1 mm, or ATP, 2 mm, was used as substrate. The formation of adenosine 3' : 5'-monophosphate (cyclic AMP) in response to glucagon exhibits lag times as long as 4 min in the presence of about 0.1 nm glucagon. The lag times diminish as the hormone or substrate concentrations increase. Addition of GTP (0.1 mm) abolishes the lags at all concentrations of glucagon tested in the presence of 0.1 mm App(NH)p and causes a decrease in the concentration of glucagon required for half-maximal activation of the enzyme. In the presence of GTP, the apparent Km for glucagon action ranges from 0.2 to 0.5 nm glucagon; in its absence, the Km ranges from 2 to 7 nm. Pretreatment of the enzyme system with glucagon for 15 min prior to addition of substrate also abolishes the lags seen at low concentrations of the hormone. When the pretreated enzyme system is incubated with limiting concentrations of substrate (0.1 mm App(NH)p) in the absence of free hormone, the velocity of enzyme activity is dependent upon GTP. Stimulatory effects are observed with 30 nm GTP; GTP exerts maximal effects at 1 µm. The lag times observed in onset of glucagon activation appear to reflect a change in state of the enzyme induced by glucagon binding to its receptor. The time dependency is changed simply by increasing the concentration of glucagon; GTP acts subsequent to or attendant upon this change in state induced by glucagon. Glucagon binding exhibits Hill coefficients of about 1.5, suggesting cooperative interactions between two or more subunits of the system. GTP, in addition to its effects on glucagon action, stimulates the rate of dissociation of glucagon from its receptor and, as a consequence, causes a decrease in the steady state levels of bound glucagon. The release of hormone seems not to be causally related to enzyme activation since the rates of dissociation are increased by addition of 0.1 mm GTP without concomitant increases in enzyme velocity. A hyperbolic relationship is observed between the levels of receptors occupied by glucagon and adenylate cyclase activity generated in the presence of GTP. Complete activation of the enzyme requires full occupation of the specific binding sites in hepatic plasma membranes (about 3 pmoles per mg of membrane protein). This finding shows that all of the specific binding sites for glucagon in these membranes represent receptors functionally and structurally linked to the adenylate cyclase system. The finding that glucagon and GTP activate adenylate cyclase by a concerted or interdependent mechanism is discussed in relation to the characteristics of the adenylate cyclase system as an allosteric regulatory enzyme system.

The Role of Acidic Phospholipids in Glucagon Action on Rat Liver Adenylate Cyclase

Abstract We have examined the effects of phospholipase C from Bacillus cereus (Bc) and from Clostridium perfringens (Cp) on various parameters involved in the activity and response of adenylate cyclase to glucagon in rat liver plasma membranes. A crude preparation of Cp-phospholipase C hydrolyzes neutral phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin) in these membranes. In contrast, highly purified Bc-phospholipase C hydrolyzes acidic phospholipids (phosphatidylserine, phosphatidylinositol) but not sphingomyelin. Treatment of the membranes with either type of phospholipase does not alter basal adenylate cyclase activity or the stimulatory effects of fluoride ion on the enzyme system. Bc-phospholipase C abolishes the effects of glucagon on adenylate cyclase whereas Cp-phospholipase C causes only partial loss of glucagon response even after hydrolysis of 60% of the membrane phospholipids. These findings provide evidence that acidic phospholipids are more specifically involved in glucagon activation of adenylate cyclase. Acidic phospholipids are not directly involved in the binding of glucagon to its receptor. Treatment with Bc-phospholipase C results in a 10-fold reduction in the affinity but not the quantity of specific binding sites for glucagon. Binding of Des-His-glucagon, a competitive, inactive analogue of glucagon is unaffected by Bc-phospholipase C treatment and displays the same apparent affinity as does glucagon for the binding sites in phospholipase-treated membranes. These findings suggest that acidic phospholipids are involved in the liganding of the histidine residue of glucagon to a regulatory site responsible for glucagon action. GTP, which is required for glucagon action on adenylate cyclase and which increases the rate of dissociation of glucagon from its receptor, does not exert these effects in Bc-phospholipase C-treated membranes. GTP also does not alter the rate of dissociation of Des-His-glucagon from the binding sites in either untreated or treated membranes, indicating that the histidine residue of glucagon is required for the effects of GTP to be expressed. It appears, therefore, that GTP and the histidine residue of glucagon bind to a common site involved both in the activation of adenylate cyclase and in the dissociation of glucagon from its receptor. Acidic phospholipids are required for the concerted effects of glucagon and GTP at this site.

The Glucagon-sensitive Adenyl Cyclase System in Plasma Membranes of Rat Liver: V. AN OBLIGATORY ROLE OF GUANYL NUCLEOTIDES IN GLUCAGON ACTION

Abstract A method is described for the enzymatic synthesis of 5'-adenylyl imidodiphosphate labeled with 32P at the α position (AMP-PNP-α-32P), an analogue of ATP containing nitrogen substituted for oxygen between the terminal phosphates. The nucleotide is only slowly hydrolyzed during incubation with rat liver plasma membranes and is a substrate for adenyl cyclase in these membranes. In the presence of 0.2 mm AMP-PNP, glucagon and fluoride ion stimulate adenyl cyclase activity; linear rates are maintained for at least 10 min of incubation at 30°. GTP enhanced the initial rate of basal and glucagon-stimulated adenyl cyclase activity. Reduction in concentration of Mg++ in the assay medium or incubation of liver membranes for 5 min at 30° prior to addition of glucagon results in loss of response of adenyl cyclase to glucagon and in reduction in the effects of GTP on basal activity. Under these conditions GTP, GDP, or GMP-PCP are required for glucagon stimulation of the enzyme even though the specific binding sites for glucagon are saturated with hormone; as little as 10 nm GTP or GDP is required. UTP and CTP exert smaller effects than the guanyl nucleotides and act only at concentrations higher than 0.1 mm. The guanyl nucleotides inhibited the response of adenyl cyclase to fluoride ion (10 mm) over the same concentration range over which they stimulate the response of the enzyme to glucagon. This action of the nucleotides is observed in plasma membranes treated with phospholipase A under conditions that result in loss of glucagon binding and of hormonal response. It is concluded that guanyl nucleotides play a specific and obligatory role in the activation of adenyl cyclase by glucagon. The nucleotides bind at sites, distinct from the glucagon binding sites, that appear to regulate both the response of adenyl cyclase to glucagon, and, possibly by a related mechanism, the actions of fluoride ion on this system.

The Glucagon-sensitive Adenyl Cyclase System in Plasma Membranes of Rat Liver: IV. EFFECTS OF GUANYL NUCLEOTIDES ON BINDING OF 125I-GLUCAGON

Abstract Studies have been made of the effects of nucleotides on binding of 125I-glucagon at its specific binding sites in plasma membranes of rat liver. GTP and GDP, equally and at a minimal concentration of 0.05 µm, stimulate the rate and degree of dissociation of bound labeled hormone, decrease uptake of glucagon by the membranes, and decrease the affinity of the binding sites for glucagon. These effects of the nucleotides are concentration-dependent, reversible, and rapid in onset. Divalent metal ions are not required for the actions of the nucleotides which act equally at 0° or 30°. 5'-Methylene guanylyl-diphosphonate, a nonphosphorylating analogue of GTP, mimicked the effects of GTP or GDP on glucagon binding although at 100 times the concentration of the natural nucleotides. Based on these observations and the finding that the nucleotides do not act competitively with glucagon, it is suggested that GTP or GDP regulate glucagon binding by an allosteric type of action. This action of the guanyl nucleotides is inhibited by a sulfhydryl reagent (p-chloromercuri-benzoate), which also inhibits binding of glucagon. Sodium fluoride, which stimulates adenyl cyclase activity in liver membranes, has no effect on either the binding of glucagon or on the actions of guanyl nucleotides on this process. ATP, ADP, UTP, and CTP act similarly to the guanyl nucleotides on the glucagon binding process but only at concentrations greater than 0.1 mm. Cyclic 3', 5'-GMP, 5'-GMP, and the corresponding adenine nucleotides are inactive on the glucagon-binding process.