Characterization of 125I-Glucagon Binding in a Solubilized Preparation of Cat Myocardial Adenylate Cyclase

A solubilized preparation of cat myocardium, which contains adenylate cyclase, has been shown to specifically bind biologically active 1251-glucagon. lZZI-glucagon binding was observed over the concentration range 1 x lop7 to 1 x lo-” M. Unlabeled glucagon displaced 129-glucagon over a similar concentration range. The binding specificity of this preparation was also shown by the fact that parathyroid hormone did not bind to cardiac receptor sites in this preparation nor did it displace W-glucagon from its binding site. The binding of ‘*“I-glucagon at 37 or 25” is linear for approximately 30 min until maximum binding is reached. In contrast, the activatiol; of adenylate cyclase is maximal within 5 min, indicating the presence of additional glucagon binding sites over and above those required for activation of the enzyme. The binding material was stable at 4” for 4 days and indefinitely when stored in liquid nitrogen. Boiling the binding material for 15 min or incubating it with 1 N HCI for 15 min destroyed most of its glucagon-binding ability. Optimal binding was observed over a broad pH range from 3.6 to 8.5, with a decline above pH 9.0. Preincubation of the binding material with trypsin decreased binding about twothirds. Phospholipases A and C, DNase, RNase, neuraminidase, urea (1 M), GTP, and ATP were without effect on the binding. Solubilized myocardial adenylate cyclase has been shown to have a molecular weight of about 100,000 to 200,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the crude solubilized preparation incubated with lzaIglucagon suggested a molecular weight for the binding fraction of approximately 26,000. Chromatography of the 1251-glucagon-receptor complex on either Sephadex G-100 or

Unlabeled glucagon displaced 129-glucagon over a similar concentration range.
The binding specificity of this preparation was also shown by the fact that parathyroid hormone did not bind to cardiac receptor sites in this preparation nor did it displace W-glucagon from its binding site. The binding of '*"I-glucagon at 37 or 25" is linear for approximately 30 min until maximum binding is reached. In contrast, the activatiol; of adenylate cyclase is maximal within 5 min, indicating the presence of additional glucagon binding sites over and above those required for activation of the enzyme. The binding material was stable at 4" for 4 days and indefinitely when stored in liquid nitrogen.
Boiling the binding material for 15 min or incubating it with 1 N HCI for 15 min destroyed most of its glucagon-binding ability. Optimal binding was observed over a broad pH range from 3.6 to 8.5, with a decline above pH 9.0. Preincubation of the binding material with trypsin decreased binding about twothirds.
Phospholipases A and C, DNase, RNase, neuraminidase, urea (1 M Bio-Gel P-30 produced a dissociation of the receptor from catalytic adenylate cyclase activity, which apparently represents a larger molecular weight component (> 100,000). The 12"I-glucagon-receptor complex eluted from the gels at an elution volume consistent with the salt peak. However, this eluate was shown by hydrodynamic flow electrophoresis to be neither free glucagon or iodine.
Moreover, when it was subjected to electrophoresis on sodium dodecyl sulfate polyacrylamide gels, the complex had a molecular weight of about 26,000, identical with what was obtained with the crude material.
It appeared, therefore, that the complex of glucagon to its receptor had a strong positive charge which resulted in adsorption of the complex to the gel. Following neutralization of the charge-gel interaction by Sephadex and Bio-Gel chromatography of the 1251-glucagon-receptor complex in 0.01 or 0.025 N NaOH, the binding fraction eluted in a more appropriate location for its apparent size. The property of adsorption to Sephadex G-100 and Bio-Gel P-30 may prove useful in purification of the glucagon receptor site.
The interaction of hormones with the membrane-bound enzyme adenylate cyclase resulting in increased intracellular levels of adenosine 3':5'-monophosphate has been the subject of intensive study in many tissues (1). Current evidence pertinent to the heart suggests that cyclic AAlP1 mediates the inotropic and chronotropic effects of several hormones including the catecholamincs (2)) glucagon (3-5)) histamine (6)) and thyroxine and triiodothyronine (7). It is thought that the initial step in hormone-induced activation of adenylate cyclase is binding to a cell membrane receptor site (8,9). Lefkowitz and his co-workers have demonstrated catecholaminc binding to specific fl adrenergic receptor sites in a preparation of microsomes from dog heart (10-12).
In addition, the binding process has been shown to be separate and distinct from the activation of adenylate cyclase (13,14). Using solubilized preparations of cat heart, we have demonstrated a critical requirement for acidic phospholipids in the activation process following binding. Phosphatidylserine was shown to be required for the glucagon and histamine activation of the detergent-free myocardial adenylate cyclase (15,16) and monophosphatidylinositol for the catecholamine activation (17). Recently we reported preliminary data concerning the binding of lY-glucagon to myocardial receptors (18) and demonstrated a glucagon binding site (mol wt approximately 24,000 to 28,000) which was dissociable from the larger molecular weight (mol wt greater than 100,000) catalytic subunit of the adenylate cyclase (19). The purpose of the present investigation was to more fully characterize the binding of 12"I-glucagotl in this soluble preparation of cat heart and to provide further information relative to the dissociable receptor site.

RESULTS
Biological Activity of 1z5Z-GZucagon-Rodbell et al. showed that glucagon labeled with lz51 retained biological activity as determined by its ability to activate particulate liver adenylate cyclase (23). 1251-G1ucagon, prepared in a similar manner to that described by these workers activates the particulate myocardial adenylate cyclase. The solubilized myocardial adenylate cyclase, freed of detergent by DEAE-cellulose chromatography is unresponsive to glucagon unless phosphatidylserine is added to the incubation misture (15,16). Since the binding studies in this investigation utilized the solubilized heart preparation, it seemed important to demonstrate that the 12jI-glucagon would activate the detergent-free solubilized adenylate cyclase. Fig. 1 shows that '*"I-glucagon at 1 x lo+ M activates the solubilized myocardial adenylate cgclase in the presence of phosphatidylserine and is, therefore, biologically active in the contest of this particular system. 1251-Glucagon Binding to Solubilized Myocardial Preparation-1251-Glucagon binds to the solubilized preparation of myocardium both in the presence and absence of detergent (Fig. 2). The binding is concentration-related over the range 1 x 10-T to 1 x 10e5 RI, half-maximal binding occurring at about 5 x lo-7 M in both. The addiCon of unlabeled glucagon to the incubation produces a displacement in the amount of 1251-glucagon bound A  2. Binding of 1251-glucagon to solubilized myocardium in the presence and absence of detergent.
Approximately 300 mg of cat heart muscle were solubilized in a solutidn containing 20 mM Lubrol-PX as described under "Methods." The homogenate was centrifuged at 12,000 X g for 10 min at 4". Approximately 1.3 ml of the supernatant fluid (protein concentration, 3.5 mg per ml) were applied to a DEAE-cellulose column (1.0 X 12.0 cm) equilibrated at 4' with 10 mM Tris-HCl, pH 7.7. The flow rate was approximately 0.2 ml per min. The column was washed with 15 to 20 volumes of 10 mM Tris HCl, pH 7.7. The protein fraction containing the glucagon-binding activity and adenylate cyclase was eluted with 1 M Tris-HCl, pH 7.7. This fraction has been shown to be free of detergent (21). The incubation conditions for 1z61glucagon binding and separation of bound from free 12KI-glucagon have been described in the text. Each value represents the mean f S.E. of five experiments from five cats. to its receptor site (Fig. 3). The decrease in binding proceeds over a concentration range similar to that observed for binding, half-maximal displacement occurring at a concentration of unlabeled glucagon approximately 5 X 10-T M. When unlabeled glucagon is added to the incubation in final concentration greater than 1 x 10-S M, a marked (3-to 4-fold) increase in the control values (glucagon incubated in the absence of solubilized preparation) is noted. The reason for this is unclear.
It should also be noted that about 10 to 20% of the bound 1251-glucagon is not displaced by unlabeled glucagon.
Presumably, this remnant represents nonspecific binding.
In order to further demonstrate the specificity of glucagon binding in this system, we examined the effectiveness of another polypeptide hormone, parathyroid hormone, in displacing bound glucagon.
Parathyroid hormone did not displace 1251-glucagon. Furthermore, specific binding of 1251-parathyroid hormone to the solubilized heart preparation could not be demonstrated.
Binding of 1251-Glucagon as Function of Time and Temperature-The binding of 1251-glucagon at 25 or 37" is linear for approximately 30 min until maximal binding is achieved (Fig. 4). At lower temperatures, 18 and O", binding also occurs although at slower rates, the maximum not being reached after 120 min of incubation.
Correlalion of 1251-Glucagon Binding with Adenylate Cyclase Activation-As noted in Fig. 5, glucagon binding and activation of the particulate heart adenylate cyclase are closely correlated in terms of molar concentrations.
However, maximal activation of the enzyme occurs within 5 min in both t,he particulate and solubilized (in the presence of phosphatidylserine) systems, whereas binding is only approximately 15 to 20% complete at this time point.
This suggests the presence of an excess of binding sites over and above what is required for the activation of the adenylate cyclase. An aliquot of the solubilized myocardial preparation, containing approximately 100 pg of protein, was added to the incubation mixture described in the text. Control samples were incubated simultaneously for each point in time. Labeled glucagon was present at a final concentration of 5 X low7 M. The values represent the mean of duplicate samples. stability of the binding material in the solubilized preparation of myocardium (Table I). The ability of the material to bind glucagon appears to be unaffected by successive freezing and thawing and by storage at 4" for 4 days. The material is stable 5. Correlation of Y-glucagon binding with adenylate cyclase activation.
The conditions for 1251-glucagon binding and assay of adenylate cyclase have been described in the text. Each adenylate cyclase incubation contained phosphatidylserine, 128 rg per ml. Phosphatidylserine was not added to the r251-glucagon binding tubes since we had previously demonstrated that phosphatidylserine is not required for the binding of glucagon in this system (18). Labeled glucagon was present at a final concentration of 5 X 10-r M. Each value represents the mean f S.E. of three samples for the adenylate cyclase experiments and the mean of duplicate samples for the W-glucagon binding experiments. Each value represents the mean of duplicate samples. E$ect of @-Optimal binding of 12SI-glucagon was observed over a very broad pH range from 3.6 to 8.5 (Fig. 6). A decrease in binding occurred above pH 9.1.
Sol ubi l i zed extract I '=I-glucagon bound pmoles/mg protein/60 min Eflect of Enzymes-The solubilized preparation of myocardium was incubated with a variety of enzymes in order to obtain information relative to the chemical nature of the binding material (Table II). Trypsin produced a 66% decrease in binding, indicating that the binding site was, at least partially, a protein.
Neuraminidase, which cleaves sialic acid and hence is destructive of some glycoproteins was without effect. Phospholipase A and C, DNase, and RNase were also without effect.

5.4
Eflect of Nucleotides- Table  IV shows that ATP had no effect on the binding of r251-glucagon.
In addition, GTP did not alter the binding of i%glucagon in contrast to what has been reported in liver membranes (26). Effect of Urea-Urea has been shown to decrease the binding of lZSI-glucagon to liver membranes (23) 6. Effect of pH on rz61-glucagon binding.
Acetate buffer was used for pH 3.6 to 5.5, phosphate buffer for pH 6.5, Tris-HCl for pH 7.0 to 9.1, and glycine buffer for pH 10.0 and 11.0. Labeled glucagon was present at a final concentration of 5 X 10-r M. Each value represents the mean of duplicate samples. Aliquots of the solubilized myocardial extract containing approximately 300 pg of protein were incubated with 60 pg of trypsin for 30 min at 37" followed by addition of 60 pg of trypsin inhibitor for 5 min at room temperature.
Other aliquots containing 300 pg of protein were incubated with 25 units of neuraminidase.
An aliquot of these mixtures containing 150 pg of protein was then incubated with i26I-glucagon for 60 min as described in the text. RNase and DNase were added directly to the incubations at final concentrations of 6.0 and 60 units per ml, respectively.   This procedure has been modified to utilize larger Sephadex G-100 (86 ml) and Bio-Gel P-30 (86 ml) columns in order to more thoroughly study the elution pattern from the gel (Fig. 7A).
Solubilized myocardial adenylate cyclase (mol wt approximately 100,000 to 200,000) is excluded from the Sephadex G-100 gel and appears in the fraction 20 to 40 ml similar to that seen with dextran blue (mol wt approximately 2,000,000), which is also excluded from the gel. i%Glucagon binding activity is also found in this fraction (Fig. 7A). In the procedure used to demonstrate glucagon binding in this system, bound and free glucagon were first separated by cellulose chromatography. When 5 ml of the cellulose effluent containing the bound lz51glucagon are chromatographed on the 86-ml Sephadex G-100 column (Fig. 7B), almost all of the labeled material appears in the salt volume (80 to 95 ml). This peak is clearly retarded behind the peak of two substances of similar molecular weight, growth hormone (approximately 32,000) and cytochrome c (approximately 12,800). A similar elution pattern was observed when the chromatography was performed on an 86-ml Bio-Gel P-30 column ( Fig. 8A ; compare with Fig. 7B). However, when the column eluate was subjected to electrophoreses on a sodium dodecyl sulfate polyacrylamide gel, the labeled fraction migrated in an area consistent with a molecular weight of about 26,000 as we previously reported (19). To resolve this apparent paradox, we subjected the Sephadex eluate to hydrodynamic flow electrophoresis in 0.1 M Verona1 buffer, pH 8.6, and demonstrated that the labeled material was distinct both from free glucagon and iodine.
It seemed likely, therefore, that the binding site-glucagon complex was interacting with the gel itself. In order to investigate this point we altered the chromatographic conditions in several ways. Increasing the ionic strength of the elution buffer to 100 mM Tris pH 7.7, did not alter the elution profile nor did elution with 200 DIM acetate buffer, pH 5.5. However, when elution was performed at strongly alkaline pH (0.01 N NaOH), a shift in the binding peak was observed toward the expected elution profile for a substance of its apparent molecular weight (Figs. 7C and 8B). This was particularly marked on the Bio-Gel P-30 column and suggested that the receptor-1251glucagon complex interacted more strongly with the Sephadex gel. Increasing the ionic strength of the NaOH (0.025 N) produced a further definition and migration of the peak on Sephadex G-100. These data appear to be similar to the gel interactions described for iodotyrosines and triiodothyronine and thyroxine on Sephadex (27,28).
Dissociable Glucagon Binding Site in Solubilized Preparation of Liver-It seemed of interest to determine whether the dissociation observed with the myocardial glucagon receptor would also be found in a preparation of liver since glucagon has been shown to both activate hepatic adenylate cyclase and bind to membrane receptor sites (23). Fig. 9 shows that the same dissociation was observed with a solubilized preparation of liver. It is also noteworthy that binding of iz51-glucagon in the Lubrol-PX solubilized liver homogenate is unimpaired in contrast to what has been reported in digitonin-treated liver membranes (23). JIolecular Weight of Glucagon Binding Site-Electrophoresis in polyacrylamide gels containing SDS was used to estimate the molecular size of the glucagon binding site in the solubilized preparations of cat myocardial adenylate cyclase. Results of a typical experiment are shown in Fig. 10. The complex is represented by the large peak of slower mobility on the upper panel. The small peak which ran with mobility identical to that of the marker dye is lz51-glucagon.
This peak dissociated during the experiment and corresponds to the single peak seen when 1251glucagon alone was subjected to electrophoresis (lower panel). Similar results were obtained with either crude solubilized preparations or after passage through cellulose or Sephadex.
The relative mobility (&) of the complex was determined by comparison to the mobility of marker dye after correction for gel length and its molecular weight was estimated according to the procedure described by Shapiro et al. (31) from plots of log molecular weight versus RM of standard proteins.
The molecular weight determined in this manner ranged from 24,000 to 28,000 at three acrylamide concentrations: 7.5, 10, and 12.5%. Neville (32) has pointed out that the use of RM in SDS gel electrophoresis to estimate molecular weight of a protein is valid only if its mobility and that of the reference proteins are the same at zero gel concentration. Fig. 11 shows Ferguson plots (33) of log RM versus gel concentration for the 1251-glucagon-binding fraction complex and for standard peptides.
Extrapolation of the lines indicates similar mobility at zero gel concentration.
The negative slope of the Ferguson plot represents the retardation of mobility by increasing gel concentration and is termed the retardation coefficient (KR). KR values for six standard peptides are plotted in Fig. 12 versus log RM at three gel concentrations and define three nonparallel lines which intersect at the y axis. The KR value for the complex falls on these lines. Thus it would seem that the behavior of the binding complex in this system is typical and permits estimation of its molecular weight.
Dissociation of '25Z-Glucagon from Its Binding Site-We have attempted to dissociate 1251-glucagon from its binding site utilizing prolonged dialysis in a solution of 1% albumin in 10 mM Tris-HCl, pH 7.7, at 4'. At 24 to 48 hours, approximately 70 to 80% of the 1251-glucagon is dissociable.
Of this amount approximately 50% of the glucagon is damaged and 50% is free glucagon as determined by rechromatography on cellulose and immunoassay techniques.
In addition, the receptor site, from which the n51-glucagon was dialyzed, can be reincubated with 1251-glucagon and shown to maintain its ability to bind glucagon.

DISCUSSION
The action of glucagon on the heart, like most of the actions of this hormone on other tissues, appears to be mediated by the adenylate cyclase-cyclic AMP system. Glucagon activates adenylate cyclase in particulate preparations of rat (3), cat (4), and human heart (4) and increases the intracellular level of I  7 (left). Sephadex G-100 chromatography and W-glucagon binding.
In the experiment shown in Panel A, 5 ml of the 12,000 X g supernatant of the solubilized myocardial homogenate prepared as described in the text were added to the Sephadex G-100 column of 86 ml volume equilibrated in 10 mM Tris, pH 7.7. The column was eluted with 10 mM Tris, pH 7.7, at a flow rate of approximately 1.2 ml per min at 25". Successive 2.5-ml fractions were assayed for '2%glucagon-binding activity as described in the text. The appearance of standard molecular weight markers for this column are shown by the arrows. In the experiment shown in Panel U, approximately 150 ~1 of solubilized myocardium (protein concentration, 3.3 mg per ml) were incubated at 37" for 60 min in a final volume of 600 ~1 containing 1.0% albumin in 10 mM Tris HCl, pH 7.7, and 1261-glucagon at a final concentration of 5 X 10u7 M. Upon completion of the incubation, the mixture was added to a dry S&cm cellulose column in a lo-ml serological pipette with an inside diameter of 0.8 cm and washed with 8.4 ml of 1% albumin in 10 mM Tris-HCl, pH 7.7. Five milliliters of the eluate, containing the bound 1251-glucagon were then added to the Sephadex column. Successive 2.5-ml fractions were counted in a Nuclear-Chicago Auto-Gamma counter. The conditions for the experiments shown in Panel C were identical to those detailed for Panel B except that cyclic AMP in the intact, isolated perfused rat heart (5). Its inotropic effects are potentiated by theophylline (34) which inhibits cyclic nucleotide phosphodiesterase, the enzyme catalyzing the breakdown of cyclic AMP to 5'-AMP. Furthermore dibutyryl adenosine 3') 5'-monophosphate exerts an inotropic effect on heart muscle similar to that of glucagon (35).
The complex interaction of glucagon with the adenylate cyclase in isolated liver plasma membrane has been the subject of intensive investigation in recent years by Rodbell et al. (23,26,[36][37][38][39][40][41]. These studies have provided critical information relative to the binding of 1251-glucagon to specific membrane receptor the Sephadex column was equilibrated in either 0.01 N NaOH (0) or in 0.025 N NaOH (0) prior to the application of 5 ml of the cellulose column eluate. FIG. 8 (center). Bio-Gel P-30 chromatography and lz61-glucagon binding.
The conditions of the experiments in A and B were identical to those described in the legend to Fig. 7, B and C. FIG. 9 (right). Dissociable glucagon binding site in a solubilized preparation of liver. In the experiment shown in Panel A, 0.2 ml of the 12,000 X g supernatant of solubilized cat liver prepared as described for the heart was added to a 2.8-ml Sephadex G-100 column equilibrated in 10 mM Tris, pH 7.7. Successive 0.25 ml fractions were assayed for 1261-glucagon binding activity as described in the text. In Panel B, 30 ~1 of solubilized liver (4.0 mg per ml) prepared as described above were incubated at 37" for 60 min in a final volume of 100 ~1 containing 1% albumin in 10 mM Tris-HCl, pH 7.7, and iZ51-glucagon at a final concentration of 5 X 10-r M. Upon completion of the incubation, the mixture was added to a dry 1.4-cm cellulose column, bound from free glucagon separated as described in the text, and 0.25 ml of the cellulose effluent was applied to the Sephadex G-100 column. Successive 0.25-ml fractions were counted in a Nuclear-Chicago Auto-Gamma counter.
sites, the role of nucleotides in the binding and activation process, the role of phospholipids in the activation process, and the elucidation of the structural requirements in the glucagon molecule required for binding and activation. Over the past several years we have undertaken somewhat parallel investigations in order to understand the molecular interaction of glucagon with the adenylate cyclase in cardiac tissue. solubilization procedure and, unlike the activation process, was seemingly independent of phospholipid as it did not require the addition of phosphatidylserine (18). Thus, the process of glucagon activation of myocardial adenylate cyclase was shown to be distinct from the binding process, a finding also observed for catecholamines and myocardial adenylate cyclase (14) and ACTH and adrenal adenylate cyclase (13).
The data in the present investigation more completely characterize the binding of 129-glucagon to its receptor site(s) in a solubilixed preparation of myocardium which contains, among other proteins, a solubilized adenylate cyclase. The preparation of ""I-glucagon utilized for these studies was shown to be biologically active in the context of this system, by its ability to activate both the membrane-bound adenylate cyclase and to activate the detergent-free, solubilized adenylate cyclase in the presence of phosphatidylserine.
The binding of '""I-glucagon in the solubilized preparation appeared to be in large part specific. Increasing concentrations of unlabeled glucagon decreased the binding of the 1251-glucagon to the site. Half-maximal displacement occurred at a concentration of unlabeled glucagon of about 5 X 10m7 M. Approximately 80 to 90% of the bound 1251-glucagon is displaceable, the remainder presumably representing nonspecific glucagon binding.
In addition to the displacement studies, specificity was demonstrated by the fact that another polypeptide hormone, parathyroid hormone, did not specifically bind to cardiac receptor sites (utilizing lZ51-parathyroid hormone) and did not displace bound 1251-glucagon. Finally, the concentration range over which 12"1-glucagon binds is quite similar to that observed for activation of adenylate cyclase in both the membrane-bound and soluble heart preparations. The latter data provide supporting evidence that the binding is related, at least in part, to the adenylate cyclase. However, as noted previously (18), there is additional binding of glucagon in this system over and above what is required to activate adenylate cyclase since the enzyme is maximally activated by 5 min, a point in time when binding is only about 25y0 complete.
In our early studies of the solubilized myocardial adenylate cyclase, we determined a molecular weight for the enzyme of approximately 100,000 to 200,000, using differential Sephadex chromatography.
This estimate of the molecular weight of the cat enzyme is similar to that reported by Lefkowitz cl al. (11) for the solubilized dog heart adenylate cyclase (160,000).
During the course of our attempts to purify the glucagon binding site, we subjected an aliquot of the incubation mixture containing 12"I-glucagon and the crude solubilizcd binding preparation to electrophoresis in an SDS polyacrylamide gel. Unexpectedly we found that the 12"I-glucagon-l~il~tling fraction appeared iu au area con&cut with a molecular weight of 24,000 to 28,000. In a I)rcliminary report we dcscribcd tllat the proc'rss of glucagoll bindiiig resulted in a dissociation of the '~~l-~luc~l~oll-l.ccc~~tol comples from the larger molecular wight catalytic moiety of the enzyme when the solubilizcd prcparatiou was iiicubated with 12~I-glucagon and then chromatographcd OH a Ycphades G-100 column (19). The labeled material from the Ycphades G-100 also had an approximate molcculnr weight of 24,000 to 28,000 when electrophoresed on SDS pol~~~ (,r~l:~i~li(le gels (19).
However, wlicii lvr: utilized larger, Win1 Sephadcs G-100 columns, it became apparent that the labclcd material was eluting at au elution volume consistent with the salt peak far behind two materials of similar molecular wight, growth hormone (in01 wt approximately 32,000), alltl cytochromc c (mol wt approsimately 12,800). Several lines of evidence suggested that this retardation rcpresentcd a direct iutcractiou of the 1251-glucagoli-receptor complex with the Scphades rather than a smaller molecular weight substance.
(CL) The iodinatcd material was repeatedly showu to have a molecular wight of about 26,000 on SDS polyacrylamide gel electrophoresis, and (b) hydrodynamic fiow electrophorcsis shoved that the iodinated material was ucither free glucagon (it did not rcmaiu at the origin) nor free iodiue, as determined by the migration of a free iodiue standard.
The intcractioil lvith the gel was not due to the low ionic strength of our eluting buffer (10 m11 Tris-I-ICl, pII 7.7) since increasing the conccntratioli of Tris-lIC1 to 100 mhI did not alter the elution profile.
From these data it seemed likclv that a strongly positive charged material was adsorbing to the gel, a phenomenon well descril~cd with Scphades and lsio-Gel, particularly with iodinatcd tyrosincs (27, 28). Furthermore, this property of adsorption to Scphatles gels is used iu the separation aud purification of misturcs of iodine, monoiodotyrosine, diiodotyrosine, triiodothyrollillc, and tctraiodothyronine (thyrosine) (27,28). \T%cn the Scphades G-100 and Bio-Gel P-30 columns were equilibrated with 0.01 or 0.025 s NaOII, the charge interaction was for the most part neutralized and the elution peak of iodinatcd material occurred at the volumc more appropriate for its molecular size.
This property of adsorption to Scphadcs G-100 and Isio-Gel I'-30 columns may ultimately prove cstrcmely useful in the purification of the receptor site. The values for protein in the tubes containing masimum amounts of bound material as mcasured by the method of Lowry et ~2. (24) arc quite low, ranging from 5 to 25 pg per ml. Thus, about 15.fold increases in specific activity for binding are noted when compared to the initial crude solubilized fraction.
The major obstacle to further purification at this time is the relative inability to remove bound glucagou from its receptor site. Khilc SDS blocks the biuding almost completely when it is added prior to the addition of ItsI-glucagon to the incubation misturc, when it is added after the binding occurs, as in SDS polgacrylamide gel clcctrophoresis, it does not dissociate the comples.
Similar tight binding has been described for glucagon with liver membraucs (43), luteinizing hormone and chorionic gonadotropin to testis and ovary (44)) and norepinephrine to cardiac receptors (I 2). Based upon the information acquired over the past decade it became possible to define a reasonable norkiug model of the hormone-sensitive adenylate cyclase as it esists in the cell mcmbrane. The most current model was proposed by Rodbell et al. (36) which is a modification of an earlier model proposed by Robison et al. (45). Rodbcll and his co-workers visualize the enzyme situated in the plasma iwmbrauc as c~onsisting of at least three subunits.
Oiie, the rcccptor site (discriminator) is situated on the csterior of the ccl1 and is the structural componcnt of the enzyme which serves as the biuding site for the hormone.
This site seems to interact, selectively with rcrtain hormones and to account for the tissue specificity of hormoiie activation of adcnylatc cyclase. Two, the catalytic site (amplifier) is situated on the interior of the ccl1 mcmbrnne, serves as the binding site for ATI' and magnesium, and is responsible for catalyzing the conversion of ATl' to cyclic Ahll'.
The third subunit is the coupler (trausduccr) which iu some mam1er connects the receptor subunit to the catalytic subunit, enabling the message of hormone binding to be trauslatcd into activation of the enzgmc.
The coupler subunit appears to be intimately related to certain mcmbranc 1~lios1~holi1~itls. Substantintioii for the presence of a coupler subunit comes from the observation that binding of hormone to the receptor site can be dissociated from activation of adenylate cyclasc (13,14,18). WC have shonn that the addition of spwific 1~l~os1~lioli1~ids, phosphatidylserinc for glucagon and histamine (I 5, lG), and mouophosphatidylinositol for catecholamincs (17) will rcstorc the activation of solubilized preparations of myocardial adcnylate cyclase under conditions iu which the cnzymc was not activated by, but could biud the hormone (18). Similar observations utilizing phospholipids have becu made in liver (38, 42) aud thyroid tissue (46). To date, howxcr, there is no definitive proof regarding the csistcnce of a distinct protein coupler subunit of the enzyme or whether the phospholipid itself is the coupler.
IYe proposc a somewhat modified version of the model of Rodbcll et al. (36) (Fig. 13). IIormonc (in this case glucagon) binding is followed by dissociation of the receptor site. Dissociation is thcu rapidly followcd by activation of the enzyme only when the critical phospholipid(s) is prcscnt t,o induce alteratioii in configuration of the catalytic site (36, 47). Wlieu &wagon biuding occurs in the dctergcnt-free solubilized preparation in the absence of added phospholipid, dissociation of the receptor site is also observed.
Jlowever, in this situation 13. Mode1 of hormone-sensitive adenylnte cyclase. R, receptor site; C, catalytic site; Cp, coupler; PI,, phospholipid. glucagon does not activate the enzyme, presumably due to the absence of phospholipid and the failure to achieve the necessary conformational changes in the catalytic site required for activation.
It is unclear whether this is analogous to the protein kinase system in which the binding of cyclic AMP to the receptor site results in a dissociation of the receptor site from the catalytic site, thus removing an inhibitory influence on the catalytic site (48)(49)(50).
In addition, the model does not clarify the potential role of guanosine triphosphate in hormone binding and activation of the membrane-bound adenylate cyclase reported in several tissues including liver (26), platelets (51), thyroid (52), frog bladder (53), and pancreas (54). It remains to be determined whether or not these effects are of physiological significance.