in a Solubilized Preparation of Myocardial Adenylate Cyclase”

SUMMARY A solubilized preparation of myocardial adenylate cyclase was chromatographed on Sephadex G-100. Binding of 1251-glucagon and fluoride-stimulatable adenylate cyclase activity occurred in the elution fractions excluded from the gel suggesting a molecular weight greater than 100,000 for the enzyme-receptor site complex. Prior incubation of the binding peak with glucagon shifted its elution pattern on Sephadex G-100 to a smaller molecular weight peak of approximately 20,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The findings are consistent with a dissociable receptor site for glucagon on myocardial adenylate cyclase and may provide a mechanism for activation-inactivation of the enzyme following hormone binding.


SUMMARY
A solubilized preparation of myocardial adenylate cyclase was chromatographed on Sephadex G-100. Binding of 1251-glucagon and fluoride-stimulatable adenylate cyclase activity occurred in the elution fractions excluded from the gel suggesting a molecular weight greater than 100,000 for the enzyme-receptor site complex. Prior incubation of the binding peak with glucagon shifted its elution pattern on Sephadex G-100 to a smaller molecular weight peak of approximately 20,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The findings are consistent with a dissociable receptor site for glucagon on myocardial adenylate cyclase and may provide a mechanism for activation-inactivation of the enzyme following hormone binding.
The mechanism by which hormones bind to membrane receptor sites and effect subsequent activation of adenylate cyclase is of great interest. Rodbell et al. (1) have proposed a threecomponent model of the hormone-responsive, membrane-bound adenylate cyclase. The regulatory site (receptor) located on the external surface of the cell membrane serves as the binding site for the hormone.
The catalytic site, on the interior of the cell membrane, has access to ATP and generates adenosine 3':5'monophosphate.
,411 intermediate coupler serves to transmit a message initiat,ed by the binding event to the catalytic site resulting in activation of adenylate cyclase. Although it is not clear how these three subunits interact several investigations have demonstrated a clear separation of binding from activation of the enzyme (2, 3). We have been utilizing a solubilized preparation of cat myocardial adenylate cyclase in or&r to study the interrelationships of these various components (4-7). The data in this report provide evidence for a smaller molecular weight glucagon binding site which is dissociable both from a larger molecular weight component and catalytic adenylate cyclase activity.
Normal cats were anesthetized with pentobarbital, 25 to 35 mg per kg intraperitoneally, the heart was quickly excised, and the left ventricle was dissected fret of endocardium and epicardium.
About 300 mg of muscle were homogenized in 4.5 ml of a cold solution containing 0.25 31 sucrose, 10 rnlf Tris-HCl, pH 7.7, 20 mM Lubrol-PX, and 1 mM EDTA-magnesium chIoritle. The homogenate was centrifuged at 12,000 x g for 10 min at 4" and the supernatant was used for Sephadex chromatography tlescribed in the legend to Fig. 1.
Glucagon binding to the solubilized myocardial receptor was determined using l%labeled glucagon. Glucagon was iodinated by the method of Hunter and Greenwood (8). ~251-Glucagon was purified on a column of cellulose powder as described by Rodbell et al. (9). The 1251-glucagon was applied to a 2.5.cm cellulose column prepared in a Pasteur pipette, inside diameter 0.6 cm, washed with 3.0 ml of a solution of 1 To albumin in 10 rnhf sodium phosphate adjusted to pH 7.5, and then cluted with 0.6 ml of the same solution adjusted to pH 10.0 with concentrated ammonium hydroxide.
The 12jI-glucagon eluted in this manner was biologically active as determined by its ability to act,ivate the particulate myocardial adenylate cyclase (10). The specific fractions of solubilized enzyme referred to in the test mere incubated at 37" in a final volume of 100 ~1 containing 1.0% albumin in 10 mM Tris-HCl, pH 7.7, and 1251-glucagon (0.370 PCi per pmole). After 90 min the incubation mixture was added to 2.5.cm cellulose columns in disposable Pasteur pipettes (inside diameter 0.6 cm) and washed with 1.4 ml of 1 y. albumin in 10 m&I Tris-HCl, pl-I 7.7. Bound 1251-glucagon did not adsorb to the column whereas free (unbound) 12"I-glucagon did. The eluate was then counted in a Nuclear-Chicago Autogamma. This method removes more than 90% of the free (unbound) '%glucagon as determined by the number of counts found in the control samples of identical composition incubated simultaneously using 10 InM Tris-HCl, pH 7.7, in place of the enzyme fraction (10). Similar control values are obtained when Lubrol-PX is included in the incubation mixture.
Each value represents the mean of duplicate esperimerits.
The assay was initiated by adding the enzyme-fluoride mixture prepared at 1" to the other components which were at 23". After 5 min at 37" the incubations were stopped and the cyclic [Vladenosine 3': 5'.monophosphate accumulated was determined as previously described (7). Protein was measured by the method of Lowry et al. (la), using bovine serum albumin as a standard.
Sodium dodecyl sulfate polyacrylamide gel electrophorcsis was performed utilizing a modification of the method of Weber and Osborn (13,14). Acrylamide concentration was 7.5% with a ratio of bisacrylamide to acrylamide of 1: 37. The SDS1 concentration in the gels and buffer was 0.1%.
Prior to electrophoresis the samples were incubated at 37" for 30 min in 0.01 in phosphate, pH 7.0, containing 0.1% SDS and 0.1% mercaptoethanol.
We have previously demonstrated that almost all of the sohl- bilizetl myocardial adenylatc cyclase applied to a Sephades G-100 column is excluded from the gel (5). i'%Glucagon binding is found in this fraction also, although over a somewhat broader columii volume.
When this fraction is rechromatographed on a 2.8.ml Sephatles G-100 column (Fig. l/l) the i2%glucagon bind ing arca is more sharply defined and appears in the eluate betweeii 0.9 and 1.8 ml with a peak at approximately 1.0 to 1.2 ml. L~luc tlestran (mol wt apl~rosin-iately 2,000,OOO) clutes over the range 0.9 to 1.5 ml on these same columns with a peak similar to that of the binding activity.
Fluoride-stimulatable adenylate cyclase activity is found in this fraction in a narrower elution pattern between 0.9 and 1.3 ml. In addition, a minor peak of fluoride-stimulated adenylate cyclase activity occurred in the later fraction (3.0 ml).
Following incubation with i2jI-glucagon the bound and free glucagon are separated by cellulose chromatography as described earlier in the text. When the cellulose effluent containing the bound glucagon is chromatographed on Sephades G-100 (Fig. IB) the bound i2jI-glucagon appears in the eluate at 2.5 to 3.3 ml consistent with a smaller molecular weight binding site. The specificity of the binding is shown in Fig. 1C in which unlabeled glucagon displaced bound 1251-glucagon from the binding site. During the process of Sephades chromatography the specific activity of the glucagon binding increases about 25-fold as compared to the crude extract.
In order to define more precisely the molecular weight of the binding material shown in Figure IB, we fractionated an aliquot on SDS acrylamide gels. The bound i2%glucagon migrated in an area consistent with a molecular weight of approximately 20,000. When we incubated the original solubilized heart muscle, which had not been chromatographed on Sephadex G-100, with l%glucagon, a similar electrophoretic pattern emerged. All of t,he bound 12%glucagon was found in an area which corresponded to the Sephades fraction run on a companion gel. Unbound (free) i2+glucagon migrated with the leading edge of the dye front in both control and experimental gels, well ahead of the binding peak. The small amount of protein in the Sephades fraction precluded visualization of bands with either Coomassie blue or periodic acid-Schiff stains. The crude heart muscle preparat.ion showed a comples pattern of over 20 peptide bands.
It is also noteworthy that the peak of adenylate cyclase activity did not shift aft'er incubation with glucagon (Fig. lB), all of the activity appearing in the eluate between 0.9 and 1.3 ml. The minor peak of adenylate cyclase activity in the small molecular weight area (Fig. IA) was not observed after glucagon preincubation (Fig. 1B). Furthermore, in experiments in which the solubilized preparation was freed of detergent by DEAE-cellulose chromatography (5) no adenylate cyclase activity was found in the area of the small molecular weight binding peak under any conditions.
The binding profile, including the shift upon prior incubation with glucagon, was identical whether the det,ergent, was present or absent.
Hormones which exert specific effects upon certain cells and tissues are presumed to act by attaching to the cell surface receptors and in many cases to activate subsequently membranebound adenylate cyclase systems (15). Glucagon is thought to increase cardiac inot.ropy through such a sequence of events. Little is known about the nature of the interaction between the receptor and the adenylate cyclaye that yields increased enzyme activity.
It is not certain if the receptor is a discrete molecule or just a functional part of the catalytic unit.
Current theory based upon the work of Rodbell et al. points to a three-component model comprising the hormone-responsive, membrane-bound adenylate cyclase system (1). In their model the receptor or discriminat,or locat.ed on t,he external cell surface binds certain hormones and initiates the process leading to activation.
Evidence from a number of laboratories suggests that the process of binding and that of act,ivat.ion are separate events which can be dissociated by several esperiment.al interventions (2, 3). Thus, while the activation of the solubilized adenylate cyclase by glucagon requires the presence of phospholipids, the binding of gluca-by guest on March 24, 2020 http://www.jbc.org/ Downloaded from gon to this same preparation occurs in the absence of such lipid (10).
The present report describes the physical dissociation of a small molecule of approximate molecular weight 20,000 from a fraction of solubilized cat myocardium that contains adenylate cyclase. The dissociation occurs only under conditions where the larger molecular weight fraction is first incubated with glucagon at concentrations that arc capable of stimulating the adenylate cyclase. In the absence of preincubation with glucagon, all of the glucagon binding activity is found in a fraction of the Sephades eluate which is excluded from the column and which also contains almost all of the adenylate cyclase activity.
The catalytic activity of the enzyme remains in the large molecular weight fraction even after dissociation of the glucagon binding peak. It is also of interest that SDS is capable of preventing the initial binding of glucagon Iv-hen present in the incubation but does not dissociate bound i"jI-glucagon.
This finding is consistent with the observation of Krug et al. that glucagon bound to its specific recept,or represents a strong bond (16) and may reflect hormone (ligand)-induced allosteric interaction as postulated by Koshland (17).
Results similar to the findings in this investigation have recently been reported in studies of the adenosine 3':5'monophosphatedependcnt protein kinase from the laboratories of Garren et al. (18) and Tao (19), in which they demonstrated the dissociation of a smaller molecular weight adenosine 3': 5'.monophosphate binding protein from the larger molecular weight catalytic unit.
Their observations suggested that in the process of binding, the receptor, whose association with the enzyme was inhibitory to catalytic function, was dissociated from the protein kinase and the enzyme was rendered active. The data in this study are consistent with such a sequence of events occurring in the solubilized adenylate cyclase system. However, whether the smaller molecular weight glucagon binding peak described in these experiments is inhibitory to the adenylat,e cyclase or interacts directly with the catalytic moiety in some other manner cannot be determined from the present results. Further studies with a more purified preparation of solubilized adenylate cyclase arc required to define this interaction more precisely.