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J M Stadel

Publications and source records attributed to J M Stadel.

At least 73 records · Page 4Linked to original sources
PubMed1986

Solubilization of [3H]leukotriene D4 receptor complex from guinea pig lung membranes.

Guinea pig lung membrane leukotriene D4 (LTD4) receptors were prelabeled with [3H]LTD4 and solubilized using digitonin, 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane sulfonate, and other non-ionic, zwitterionic, and ionic detergents. [3H]LTD4 remains tightly associated with the receptor complex in the digitonin solubilized state. The dissociation rate of [3]LTD4 from the soluble receptor complex was increased in the presence of guanine nucleotides and sodium ions in a manner similar to that observed for the receptors in the membrane-bound state. The soluble [3H]LTD4 receptor complex was retained on wheat germ lectin affinity columns and destabilized by heat (40 +/- 4 degrees), trypsin, and chymotrypsin treatment, suggesting that the receptor is a glycoprotein. Size exclusion high pressure liquid chromatography of the soluble receptor complex showed that an apparent molecular weight of the soluble receptor complex, in the presence of digitonin, is in the range of 240,000-500,000. An approximately 20-fold enrichment of receptor-radioligand complex was achieved by passing the solubilized LTD4 receptor preparation successively through size exclusion and wheat germ lectin chromatography columns. These data provide the first step toward the purification and chemical characterization of LTD4 receptors.

Animals↗
PubMed1985

Molecular structure of the beta-adrenergic receptor.

The beta-adrenergic receptor from several tissues has been purified to homogeneity or photoaffinity radiolabeled and its subunit molecular weight determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In this study we have examined the oligomeric structure of nondenatured beta 1- and beta 2-adrenergic receptor proteins, as solubilized with the detergent digitonin. Model systems used were frog and turkey red blood cell as well as rat, rabbit, and bovine lung plasma membrane preparations. To correct for the effects of detergent binding, sedimentation equilibrium analysis in various solvents, as adapted for the air-driven ultracentrifuge, was used. With this approach an estimate of 6 g of digitonin/g of protein binding was determined, corresponding to a ratio of 180 mol of digitonin/mol of protein. Protein molecular weights estimated by this method were 43 500 for the turkey red blood cell beta 1 receptor and 54 000 for the frog red blood cell beta 2 receptor. Molecular weights of 60 000-65 000 were estimated for beta 1 and beta 2 receptors present in mammalian lungs. These values agree with estimates of subunit molecular weight obtained by SDS gel electrophoresis of purified or photoradiolabeled preparations and suggest beta-adrenergic receptors to be digitonin solubilized from the membrane as single polypeptide chains.

Amino Acids↗
PubMed1985

Characterization of guinea pig myocardial leukotriene C4 binding sites. Regulation by cations and sulfhydryl-directed reagents.

Using [3H]leukotriene C4 (LTC4) and radioligand-binding techniques, specific leukotriene C4 binding sites have been identified in membranes derived from guinea pig ventricular myocardium. High performance liquid chromatography analyses indicated that, in the presence of the gamma-glutamyl transpeptidase inhibitor L-serine-borate (80 mM), less than 2% of membrane-bound [3H]LTC4 was converted at 20 degrees to [3H]leukotriene D4 or [3H]leukotriene E4. The specific binding of 4 nM [3H]LTC4, in the presence of 80 mM L-serine-borate, reached a stable steady state within 15 min at 20 degrees (pH 7.5). A monophasic Scatchard plot of saturation binding data yielded a dissociation constant (Kd) of 27.5 +/- 6.0 nM and a maximum number of binding sites (Bmax) of 19.9 +/- 5.2 pmol/mg of membrane protein. Competition binding studies of [3H]LTC4 with synthetic leukotriene C4, leukotriene D4, and leukotriene E4 and the putative peptidoleukotriene antagonists FPL 55712, SKF 88046, and 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid revealed an order of potency of leukotriene C4 much greater than 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid greater than SKF 88046 greater than LTE4 greater than LTD4 greater than FPL 55712. The specific [3H]LTC4 binding was stimulated by the divalent cations Ca2+, Mg2+, and Mn2+ and to a lesser degree by the monovalent cations Na+, K+, Li+, and NH4+. CaCl2 (3 mM) and NaCl (150 mM) stimulated the LTC4 binding by increasing the Bmax to 42.6 +/- 5.9 and 35.0 +/- 2.0 pmol/mg, respectively, but had minimal effects on Kd. Pretreatment of the heart membranes with the sulfhydryl reagent N-ethylmaleimide decreased the specific [3H]LTC4 binding in a concentration-dependent manner. The N-ethylmaleimide-induced inactivation of [3H]LTC4 binding sites was protected by occupation of the binding site with the agonist leukotriene C4, but no protection was observed with the antagonist SKF 88046. Scatchard analyses of saturation isotherms indicated that 30 microM N-ethylmaleimide pretreatment reduced the Bmax of the [3H]LTC4 binding to 8.2 +/- 3.1 pmol/mg with minimal effects on Kd. The data provide direct biochemical evidence for specific [3H]LTC4 binding sites in the guinea pig heart membranes. The [3H]LTC4 binding sites appear to be modulated by divalent and monovalent cations and free sulfhydryl group(s) may be associated with the agonist-binding site. The results suggest that the physiological effects of the leukotrienes on the guinea pig heart may be mediated through membrane-bound receptors.

Animals↗
PubMed1985

Characterization of [3H]leukotriene D4 binding sites in guinea-pig ventricular myocardium.

[3H]Leukotriene (LT) D4 was used to identify specific LTD4 binding sites in guinea-pig ventricular myocardial membranes. High-performance liquid chromatography analyses indicated that, in the presence of the gamma-glutamyl transpeptidase inhibitor L-serine-borate (80 mM), less than 3% of membrane-bound [3H]LTD4 was converted to [3H]LTC4 or [3H]LTE4 at 30 degrees C. The specific [3H] LTD4 binding, assayed in the presence of 80 mM L-serine-borate, reached a stable steady state within 45 min at 30 degrees C (pH 7.5). A monophasic Scatchard plot of saturation binding data yielded an apparent dissociation constant (Kd) of 3.4 +/- 2.1 nM and a maximum number of binding sites of 850 +/- 91 fmol/mg of protein. Competition binding studies with [3H]LTD4, synthetic 5S, 6R-LTD4 (LTD4) and its diastereoisomer 5R,6S-LTD4, LTE4, LTC4 and the putative LT antagonists FPL 55712, 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid (2-nor-LTD1) and SKF 88046 demonstrated a potency order of LTD4 greater than LTE4 greater than LTC4 greater than 5R,6S-LTD4 much greater than 2-nor-LTD1. FPL 55712 and SKF 88046 were ineffective in displacing the specific [3H]LTD4 binding. Pretreatment of the heart membranes with the sulfhydryl reducing reagent dithiothreitol decreased the specific [3H]LTD4 binding in a concentration-dependent manner. Scatchard analyses of saturation isotherms indicated that 0.3 mM dithiothreitol pretreatment of heart membranes decreased the maximum number of binding sites of the [3H]LTD4 binding to 368 +/- 61 fmol/mg of protein with minimal effects on the apparent Kd.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Regulation of ligand binding to leukotriene D4 receptors: effects of cations and guanine nucleotides.

High affinity, stereoselective specific binding sites for [3H]leukotriene D4 [( 3H]LTD4) have been demonstrated in guinea pig lung membranes. Purine nucleotides quantitatively reduced [3H]LTD4 specific binding with a rank order potency of guanosine-5'-O-3-thiotriphosphate (GTP gamma S) = guanyl-5'-yl-imido-diphosphate [Gpp(NH)p] greater than GTP greater than ATP greater than GDP. In the presence of 1 microM Gpp(NH)p, the maximum number (Bmax) of [3H]LTD4 specific binding sites was reduced to 41 +/- 10 percent of the control level (950 +/- 150 fmol/mg membrane protein). In the presence of 3 microM Gpp(NH)p, the rate of association of [3H]LTD4 to the specific sites was estimated to have increased 2.5-fold. The rate of dissociation of [3H]LTD4 from the specific sites was also increased significantly in the presence of 50 microM Gpp(NH)p. The divalent cations, Ca2+ and Mg2+ (10 mM), increased the Bmax 2-fold and had minimal effects on the dissociation constant (Kd) of [3H]LTD4 specific binding. Sodium ions, at a concentration of 50 mM, reduced the Bmax, and had minimal effects on the Kd of [3H]LTD4 specific binding. These data indicate that guanine nucleotides, Na+, Mg2+ and Ca2+ regulate [3H]LTD4 binding to its receptors in guinea pig lung.

Animals↗
PubMed1984

Cell-free desensitization of catecholamine-sensitive adenylate cyclase. Agonist- and cAMP-promoted alterations in turkey erythrocyte beta-adrenergic receptors.

Conditions have been developed for desensitizing the beta-adrenergic receptor-coupled adenylate cyclase of turkey erythrocytes in a cell-free system. Desensitization is observed when cell lysates are incubated with isoproterenol or cAMP analogs for 30 min at 37 degrees C. Maximally effective concentrations of isoproterenol produce a 41.0 +/- 1.55% loss of iosproterenol-stimulated and a 15.0 +/- 2.35% loss of fluoride-stimulated enzyme activity. cAMP causes a 26.5 +/- 1.5% fall in isoproterenol-stimulated and a 21.5 +/- 4.4% fall in fluoride-sensitive activity. Desensitization by isoproterenol is dose-dependent, stereospecific, and blocked by the beta-adrenergic antagonist propranolol. Cell-free desensitization required ATP, Mg2+, and factor(s) present in the soluble fraction of the cell. Nonphosphorylating analogs of ATP did not support desensitization. Desensitization by agonist or cAMP in the cell-free system caused structural alterations in the beta-adrenergic receptor peptides apparent as an altered mobility of the photoaffinity labeled receptor peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As with the desensitization reaction, supernatant factors and ATP were also required for the agonist or cAMP-promoted receptor alterations. These data indicate that beta-adrenergic agonists promote a cAMP-mediated process which leads to receptor alterations and desensitization. The reactions involved in this process require ATP and soluble cellular factors. Additional processes must also occur to account for decreases in fluoride-sensitive enzyme activity. The availability of this cell-free system should facilitate elucidation of the molecular mechanisms involved in these processes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung.

A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [3H]-leukotriene D4 [( 3H]-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, we have identified specific binding sites for [3H]-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for [3H]-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [3H]-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with [3H]-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with [3H]-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung.

Animals↗
PubMed1983

Functional modification of the guanine nucleotide regulatory protein after desensitization of turkey erythrocytes by catecholamines.

Densensitization of turkey erythrocytes by exposure to the beta-adrenergic agonist (-)isoproterenol leads to decreased activation of adenylate cyclase by agonist, NaF, and guanyl-5'-yl imido diphosphate, with no reduction in the number of beta-adrenergic receptors. Interactions between the receptor and the guanine nucleotide regulatory protein (N protein) also seem to be impaired. These observations suggest that a component distal to the beta-adrenergic receptor may be a locus of modification. Accordingly we examined the N protein to determine whether it was altered by desensitization. The rate at which (-)isoproterenol stimulated the release of [3H]GDP from the N protein was substantially lower in membranes prepared from desensitized cells, providing further evidence for uncoupling of the receptor and the N protein. The amount of N protein in membranes from control and desensitized cells was compared by labeling the 42,000 Mr component of the N protein with [32P]NAD+ and cholera toxin; no significant difference was found. However, significantly more N protein (p less than .001) was solubilized by cholate extraction of desensitized membranes, suggesting an altered association of the N protein with the membrane after desensitization. The functional activity of the N protein was measured by reconstitution of cholate extracts of turkey erythrocyte membranes into S49 lymphoma cyc- membranes. Reconstitution of (-)isoproterenol stimulation of adenylate cyclase activity was reduced significantly (p less than .05) after desensitization. These observations suggest that desensitization of the turkey erythrocyte by (-)isoproterenol results in functional modifications of the guanine nucleotide regulatory protein, leading to impaired interactions with the beta-adrenergic receptor and reduced activation of adenylate cyclase.

Adenylyl Cyclases↗
PubMed1983

Functional integrity of desensitized beta-adrenergic receptors.

The adenylate cyclase-coupled beta 2-adrenergic receptor of the frog erythrocyte has served as a useful model system for elucidating the mechanisms of catecholamine-induced densensitization. In this system, it has been previously demonstrated that agonist-induced refractoriness is associated with sequestration of the beta-adrenergic receptors in vesicles away from the cell surface and from their effector unit, the adenylate cyclase system (Stadel, J.M., Strulovici, B., Nambi, P., Lavin, T.N., Briggs, M.M., Caron, M.G., and Lefkowitz, R.J. (1983) J. Biol. Chem. 258, 3032-3038). These internalized beta-adrenergic receptors appear to be structurally intact as assessed by photoaffinity labeling, but their functional status has previously been unknown. In the present studies, we sought to assess the functionality of the sequestered vesicular receptors by fusing them to Xenopus laevis erythrocytes. This cell is suitable for such studies, since it has almost no detectable beta-adrenergic receptor or catecholamine-sensitive adenylate cyclase, but contains prostaglandin E1-stimulable adenylate cyclase. Fusion of beta-adrenergic receptor-containing vesicles from desensitized frog erythrocytes with X. laevis erythrocytes results in a 30-fold stimulation of the hybrid adenylate cyclase by the beta-adrenergic agonist isoproterenol. This effect was entirely blocked by the beta-antagonist propranolol. The catecholamine-sensitive adenylate cyclase activity established in the vesicle-Xenopus hybrids showed the characteristic agonist potency series of the donor frog erythrocyte beta 2-adrenergic receptor. Fusion of vesicles from desensitized frog erythrocytes in which the beta-adrenergic receptors had been inactivated with the group specific reagent dicyclohexylcarbodiimide, or of vesicles derived from control frog erythrocytes, which contain low amounts of beta-adrenergic receptor, did not establish catecholamine-sensitive adenylate cyclase activity in the hybrids. These data demonstrate that beta-adrenergic receptors internalized during desensitization retain their functionality when recoupled to an adenylate cyclase system from a different source. The functional uncoupling of these receptors during desensitization is thus more likely due to their sequestration away from the other components of the adenylate cyclase than to any alterations in the receptors themselves.

Adenylyl Cyclases↗
PubMed1983

Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase is associated with phosphorylation of the beta-adrenergic receptor.

Preincubation of turkey erythrocytes with catecholamines desensitizes the beta-adrenergic receptor-adenylate cyclase complex in the plasma membranes of these cells. Photoaffinity labeling of the beta-adrenergic receptors with 125I-labeled p-azidobenzylcarazolol (125I-pABC) and subsequent analysis by NaDodSO4/polyacrylamide gel electrophoresis demonstrates an altered mobility of receptor peptides from desensitized cells compared to controls [Stadel, J.M., Nambi, P., Lavin, T.N., Heald, S.L., Caron, M.G. & Lefkowitz, R.J. (1982) J. Biol. Chem. 257, 9242-9245]. The time course of alteration in beta-adrenergic receptor mobility correlates with that for desensitization of isoproterenol-stimulated adenylate cyclase activity. The altered mobility of the receptor peptides from desensitized cells is also observed if the receptors are first purified and then photoaffinity labeled with 125I-pABC. The cyclic nucleotide analog 8-bromoadenosine 3',5'-cyclic monophosphate partially mimics catecholamines in promoting desensitization of the adenylate cyclase and modification of the receptor. Phosphorylation of the beta-adrenergic receptor in intact turkey erythrocytes was assessed by preincubating the cells with [32P]orthophosphate, desensitizing them with catecholamine, purifying the receptors, and then subjecting them to NaDodSO4/polyacrylamide gel electrophoresis. Desensitization is associated with a 2- to 3-fold increase in 32P incorporation into the receptor, which also demonstrates the characteristic alterations in mobility. These effects are blocked by the beta-adrenergic antagonist propranolol. Purified turkey erythrocyte beta-adrenergic receptors could be phosphorylated by incubation with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase. The mobility of the phosphorylated receptor peptides on NaDodSO4/polyacrylamide gel electrophoresis appears to correspond to that of the desensitized receptors. These data show that catecholamine-induced desensitization of adenylate cyclase in turkey erythrocytes correlates with a stable modification of the beta-adrenergic receptor and is associated with agonist-promoted phosphorylation of beta-receptor peptides.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1982

Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase. Structural alterations in the beta-adrenergic receptor revealed by photoaffinity labeling.

Preincubation of turkey erythrocytes with isoproterenol results in an impaired ability of beta-adrenergic agonists to stimulate adenylate cyclase in membranes prepared from these cells. The biochemical basis for this agonist-induced desensitization was investigated using the new beta-adrenergic antagonist photoaffinity label [125I]p-azidobenzylcarazolol ([125I]PABC). Exposure of [125I]PABC-labeled turkey erythrocyte membranes to high intensity light leads to specific covalent incorporation of the labeled compound into two polypeptides, Mr approximately equal to 38,000 and 50,000, as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. Incorporation of [125I]PABC into these two polypeptides is completely blocked by a beta-adrenergic agonist and antagonist consistent with covalent labeling of the beta-adrenergic receptor. After desensitization of the turkey erythrocyte by preincubation with 10(-5) M isoproterenol, the beta-adrenergic receptor polypeptides specifically labeled by [125I]PABC in membranes prepared from desensitized erythrocytes were of larger apparent molecular weight (Mr approximately equal to 42,000 versus 38,000, and 53,000 versus 50,000) compared to controls. When included during the preincubation of the erythrocytes with isoproterenol, the antagonist propranolol (10(-5) M) inhibited both agonist-promoted desensitization of the adenylate cyclase and the altered mobility of the [125I]PABC-labeled receptor polypeptides. These data indicate that structural alterations in the beta-adrenergic receptor accompany the desensitization process in turkey erythrocytes.

Adenylyl Cyclase Inhibitors↗
PubMed1982

Mechanisms of hormone receptor-effector coupling: the beta-adrenergic receptor and adenylate cyclase.

The beta-adrenergic receptors that are coupled to adenylate cyclase have provided a model system for studying the mechanisms by which a plasma membrane receptor is coupled to a well-defined biochemical effector. The beta 2-adrenergic receptors from frog erythrocyte membranes have been purified to homogeneity and the ligand-binding subunit has been identified as a glycoprotein with an approximate molecular weight of 58,000. This subunit has also been identified with the use of newly developed photoaffinity reagents. Under the influence of agonist hormones (H), the receptors (R) form transient complexes with another component of this system, termed the nucleotide regulatory protein (N). Formation of this ternary complex, HRN, leads to the dissociation of GDP from N and the interaction of stimulatory GTP with N. N charged with GTP appears to activate the catalytic moiety of the adenylate cyclase enzyme. Although some striking analogies have been found for the mechanisms by which inhibitory receptors interact with adenylate cyclase, much less is known about the molecular properties of the components involved and the ways in which they interact to dampen adenylate cyclase activity in the plasma membrane.

Adenylyl Cyclases↗