Solubilization and reconstitution of dopamine D1 receptor from bovine striatal membranes: Effects of agonist and antagonist pretreatment

The bovine striatal dopamine D₁ receptor was solubilized with a combination of sodium cholate and NaCl in the presence of phospholipids, following treatment of membranes with a dopaminergic agonist (SKF-82526-J) or antagonist (SCH-23390). The solubilized receptors were subsequently reconstituted into lipid vesicles by gel-filtration. A comparison of ligand-binding properties shows that the solubilized and reconstituted receptors bound [³H]SCH-23390 to a homogeneous site in a saturable, stereospecific and reversible manner with a K_d of 0.95 and 1.1 nM and a B_max of 918 and 885 fmol/mg protein respectively for agonist- and antagonist-pretreated preparations. These values are very similar to those obtained for membrane-bound receptors. The competition of antagonists for [³H]SCH-23390 binding exhibited a clear D₁ dopaminergic order in the reconstituted preparation obtained from either agonist or antagonist-pretreated membranes, except that (+)butaclamol was about four-fold more potent thancis-flupentixol in displacing [³H]SCH-23390 binding in preparation obtained from agonist-pretreated membranes compared to antagonist-pretreated membranes. The agonist/[³H]SCH-23390 competition studies revealed the presence of a highaffinity component of agonist binding in both the reconstituted receptor preparations. The number of high-affinity agonist binding sites, however, is 40–80% higher in reconstituted preparation obtained from antagonist-treated membrane compared to that obrained from the agonist-treated membrane. In both the preparations, 100 M guanylylimidodiphosphate (Gpp(NH)p) completely abolished the high-affinity component of agonist binding compared to partial abolition in the native membranes, indicating a close association of a G-protein with the solubilized receptors. Whether the receptor was solubilized following agonist or antagonist preincubation of the membranes, the receptor-detergent complex eluted from a steric-exclusion HPLC column with an apparent molecular size of 360,000. Preincubation of the solubilized preparations with Gpp(NH)p had virtually no effect on the elution profile suggesting a lack of guanine nucleotide-dependent dissociation of G-protein receptor complex.     

Partial purification of dopamine D2 receptors using lectin affinity columns

Dopamine D₂ receptors , detected by [³H]spiperone Dopamine D₂ receptors , detected by [³H]spiperone binding, were solubilized from bovine caudate nucleus by cholate/sodium chloride and were found to bind to wheat germ agglutinin immobilized on agarose. Specific elution could be achieved with N-acetylglucosamine whereas other sugars tested were inactive in this regard . The eluted preparation was enriched in solubilized receptors about sevenfold. The pharmaco-logical properties of the preparation were essentially unchanged by the lectin affinity purification procedure. The D₂ dopamine receptor is therefore a glycoprotein. binding, were solubilized from bovine caudate nucJeus by cholate/sodium chloride and were found to bind to wheat germ agglutinin immobilized on agarose. Specific elution could be achieved with N-acetylglucosamine whereas other sugars tested were inactive in this regard . The eluted preparation was enriched in solubilized receptors about sevenfold. The pharmacological properties of the preparation were essentially unchanged by the lectin affinity purification procedure. The D₂ dopamine receptor is therefore a glycoprotein.     

Specific [3H]quinuclidinyl benzilate binding activity in Digitonin-solubilized preparations from bovine brain

Receptors for the specific muscarinic radioligand [³H]quinuclidinyl benzilate ([³H]QNB) were solubilized by digitonin from a particulate preparation of bovine brain without significant alteration in binding affinities for muscarinic antagonists. Electron microscopy and sucrose density gradient sedimentation analysis confirmed the solubility of these receptors in aqueous solutions of digitonin. Equilibrium and kinetic studies of [³H]QNB binding to solubilized receptors indicated that binding was stereoselective and was blocked by muscarinic compounds. These tests permit tentative identification of digitonin-solubilized [³H]QNB binding sites as muscarinic acetylcholine receptors. Digitonin-solubilized receptors were homogeneous with respect to sedimentation behavior and binding affinities for agonist and antagonist drugs, unlike membrane-bound receptors. Enzyme digestion studies and treatment with group-specific reagents indicated that muscarinic receptors are proteins whose binding activity could be disrupted by reduction with dithiothreitol or by modification of sulfhydryl residues.     

Partial Purification and Pharmacology of Peripheral-Type Benzodiazepine Receptors

Abstract

This report describes the results obtained with a new photo-affinity ligand for the “peripheral-type” benzodiazepine binding site (PBS), using a digitonin solubilized preparation from rat heart or adrenals.

The specific binding activity of the solubilized adrenal preparation is higher than 50 pmo1/mg protein, with binding proper-ties and pharmacological specificity identical to the membrane bound PBS. The apparent molecular weight of the solubilized PBS, determined by gel filtration is 215 KDa.

The photoaffinity ligand (PK 14105) is a nitrophenyl derivative of PK 11195, which attaches covalently and specifically to all the PBS when cardiac membranes are irradiated with this compound under ultraviolet light. After photolabelling with [³H]PK 14105 and solubilization in SDS of heart or adrenal membranes, gel electrophoresis indicates the existence of a single protein band whose molecular weight (18 KDa) is unaltered by incubation with sulphydryl-reducing or protein cross-linking agents. This molecule seems to be a low molecular weight, acidic protein.

Diethylpyrocarbonate decreases partially (60 %) the binding of [³H]PK 11195 without affecting [³H] RO5-4864 binding, which implies a vital histidine residue in the binding domain of [³H] -PK 11195. Treatment with phospholipase A₂ or mellitin, a stimulant of endogenous PLA₂, led to a selective, loss of [³H]RO5-4864 binding with no change in the binding of [³H]PK 11195.

Such differences between a benzodiazepine ligand and an isoquinoline ligand suggest that these compounds may induce.     

Different sedimentation properties of agonist- and antagonist-labelled platelet alpha2 adrenergic receptors

Alpha₂ adrenergic receptors were solubilized from human platelet particulate preparations with digitonin. The solubilized alpha₂ receptors retained the essential binding specificity characteristics of the membrane-bound receptors. The alpha₂ receptors could be labelled in platelet membranes with either agonist ([³H]epinephrine) or antagonist ([³H]yohimbine) radioligands. When these membranes were solubilized with digitonin and centrifuged on sucrose density gradients, the sedimentation coefficient of the agonist-labelled receptor (14.6S) was greater than that of the antagonist-labelled receptor (12.9S). This observation may provide insight into the mechanism of adenylate cyclase inhibition by alpha₂ adrenergic receptors.     

Solubilization of angiotensin II receptors in bovine adrenal cortex

Angiotensin II receptors in bovine adrenal cortex were solubilized with 1% digitonin solution. Binding of ³H-angiotensin II to the solubilized receptors could be assayed by gel filtration on Sephadex G-50 column. Scatchard analysis indicated two classes of binding sites with K_d of 15 and 170 nM. Maximal number of binding sites were estimated at approximately 120 and 470 fmole/mg protein for the high and low affinity binding sites respectively. Pharmacologically active angiotensin II analogues including angiotensin II, Sar¹-Ile⁸-angiotensin II, desAsp¹-angiotensin II, desAsp¹-Ile⁸-angiotensin II were all active in inhibiting the specific ³H-angiotensin II binding with relative affinities similar to those in membrane preparations. The inactive angiotensin II precursor, angiotensin I was much weaker in inhibiting the specific ³H-angiotensin II binding thus indicating the specificity of angiotensin II receptors in the solubilized state was maintained.     

Stabilization of Functional Recombinant Cannabinoid Receptor CB2 in Detergent Micelles and Lipid Bilayers

Elucidation of the molecular mechanisms of activation of G protein-coupled receptors (GPCRs) is among the most challenging tasks for modern membrane biology. For studies by high resolution analytical methods, these integral membrane receptors have to be expressed in large quantities, solubilized from cell membranes and purified in detergent micelles, which may result in a severe destabilization and a loss of function. Here, we report insights into differential effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB₂, and provide guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. While we previously described the expression in Escherichia coli, purification and liposome-reconstitution of multi-milligram quantities of CB₂, here we report an efficient stabilization of the recombinant receptor in micelles - crucial for functional and structural characterization. The effects of detergents, lipids and specific ligands on structural stability of CB₂ were assessed by studying activation of G proteins by the purified receptor reconstituted into liposomes. Functional structure of the ligand binding pocket of the receptor was confirmed by binding of ²H-labeled ligand measured by solid-state NMR. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB₂ in dodecyl maltoside (DDM)/CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB₂ reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions.     

Solubilization of High-Affinity, Guanine Nucjeotide-Sensitive μ-Opioid Receptors from 7315c Cell Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane-associated receptors with morphine before their solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [³H]-etorphine with high affinity (K_D= 0.304 ± 0.06 nM; B_max= 154 ± 33 fmol/mg of protein). Of the membrane-associated [³H]etorphine binding sites, 40 ± 5% were recovered in the solubilized fraction. Both μ-selective and non-selective enkephalins competed with [³H]etorphine for the solubilized binding sites; in contrast, 5- and K-opioid enkephalins failed to compete with [³H]etorphine for the solubilized binding sites at concentrations of <1 μM.The μ-selective ligand [³H][D-Ala²,A/-Me-Phe⁴,Gly⁵-ol]enkephalin also bound with high affinity (K_D= 0.79 rM; B_max= 108±17 fmol/mg of protein) to the solubilized material. Of the membrane-associated [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin binding sites, 43 ± 3% were recovered in the solubilized material. Guanosine 5′-O-(3-thiotriphosphate), GTP, and guanosine 5′-O-(2-thiodiphosphate), but not adenylylimidodiphosphate, diminished [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin binding in a concentration-dependent manner. Finally, μ-opioid receptors from rat brain membranes were also solubilized in a high-affinity, guanine nucleotide-sensitive state if membrane-associated receptors were occupied with morphine before and during their solubilization with the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate.     

Reconstitution of affinity-purified dopamine D2 receptor binding activities by specific lipids

The role of lipids in maintaining ligand binding properties of affinity-purified bovine striatal dopamine D₂ receptor was investigated in detail. The receptor, purified on a haloperidol-linked Sepharose CL6B affinity column, exhibited low [³H]spiroperidol binding unless reconstituted with soybean phospholipids. In order to understand the role of individual phospholipids in maintaining the receptor binding activity, the purified preparation was reconstituted separately with individual phospholipids and assayed for [³H]spiroperidol binding. Except for phosphatidylcholine and phosphatidylethanolamine, that respectively restored 30 and 20% binding as compared to that obtained with soybean lipids, reconstitution with other lipids had very little effect. When various combinations of phospholipids were used for reconstitution, a phosphatidylcholine and phosphatidylserine mixture seemed to almost fully restore the receptor binding. A mixture of phosphatidylcholine and phosphatidylethanolamine was as effective as phosphatidylcholine alone in reconstituting ligand binding; however, when phosphatidylserine was also included in the mixture, there was a pronounced increase in binding (about 2-fold compared to the soybean lipids and about 6-fold compared to the phosphatidylcholine-phosphatidylethanolamine mixture). Substitution of other phospholipids or cholesterol for phosphatidylserine in phosphatidylcholine and phosphatidylethanolamine mixture had little effect. Maximal reconstitution of [³H]spiroperidol binding was obtained with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine mixture (2:2:1, w/w) at a concentration of 0.5 mg/ml. The reconstituted receptor exhibited high affinity binding for [³H]spiroperidol which was comparable to that obtained with membrane or solubilized preparations. Various dopaminergic antagonists and agonists showed appropriate order of potency for the reconstituted receptor. The presently described reconstitution data suggest a role of specific phospholipids in preserving the binding properties of dopamine D₂ receptor and should prove useful in studies on functional reconstitution of the receptor.     

Solubilization and Characterization of the A2-Adenosine Receptor

Abstract

Binding of [³H]CGS 21680, an agonist radioligand selective for A₂-adenosine receptors (A₂AR), to membranes and solubilized preparations from bovine brain striatum revealed labelling of a single high affinity binding state. In membranes, guanine nucleotides per se were ineffective in modulating agonist binding whereas cations, Na⁺ and Mg⁺⁺, had distinct effects. The addition of NaCl (200 mM) as well as the Mg⁺⁺-free preparation of membranes led to a significant decrease in binding affinity and the number of binding sites. Moreover, the presence of Na⁺ was required for the demonstration of a guanine nucleotide effect, i.e. a decrease in maximal binding. Following solubilization, agonist-A AR interactions were sensitive to guanine nucleotides even in the absence of Na⁺²; guanine nucleotides and Na⁺ had additive effects in reducing the number of binding sites. Moreover, the effect of GTP was reversible, i.e. binding returned to control levels upon removal of the nucleotide. This suggests the A₂AR and its G protein (presumably G_S) are solubilized as a functional unit and may not dissociate even in the presence of GTP following solubilization. We, therefore, believe that a “tight” association exists between receptor and G protein (G_S), and that guanine nucleotides and sodium act at different sites on the R–G complex. Drawing an analogy with similar observations on the avian β-adrenergic receptor (Hertel et al, J.Biol.Chem. 265:17988–94, 1990; Parker & Ross, J.Biol.Chem. 266:9987–96, 1991) we postulate that the regulatory features of the A₂AR can be attributed to a distinct receptor domain that interacts with cellular regulatory elements.     

Solubilization,purification and reconstitution of Ca-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

Purification and reconstitution of the 32Pi-ATP exchange activity of bovine chromaffin granule membrane

Ghosts derived from bovine chromaffin granules have a ³²P_i-ATP exchange activity which is associated with the H⁺ pump of that membrane. This activity was low when compared to bacteria, chloroplasts or submitochondrial particles, but had similar properties (K_m for ATP and P_i, ATP/Mg²⁺ ratio, pH profile, inhibition by dicyclohexylcarbodiimide and tributyltin) to the ATPase from above membranes. The ³²P_i-ATP exchange activity was solubilized by cholate/octylglucoside mixtures. The soluble extract was lipid depleted by ammonium sulfate fractionation and partially purified by sucrose gradient centrifugation. The purified preparation was reconstituted with phospholipids by freeze-thawing. The reconstituted vesicles had a ³²P_i-ATP exchange sensitive to dicyclohexylcarbodiimide and trybutyltin and an ATPase with a sensitivity to the inhibitors which varied with the reconstitution conditions. The α- and β-subunits of F₁-ATPase were major components of the preparation.     

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2alpha receptors.

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [³H] prostaglandin (PG)F₂α binding compared to E₁ binding. Lubrol WX (LWX) tended to cause a greater loss of [³H]PGF₂α than E₁ binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [³H]PGE₁ binding was greater than for PGF₂α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF₂α binding without affecting PGE₁ binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE₁ receptors compared to 100% solubilization of F₂α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE₁ and F₂α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE₁ and F₂α receptors association with the membrane structure.     

Angiotensin-Induced Changes in the Apparent Size of Rat Liver Angiotensin Receptors

Abstract

Angiotensin receptors from rat liver were labeled using four different ligands : (Sar¹-(³H)Tyr⁴)-Angiotensin II ((³H)SarAII); (Sar¹-(³H)Tyr⁴-IIe⁸) -Angiotensin II ((³H)SarIIeAII); (Sar¹-(¹²⁵I)Tyr⁴-(4′-N₃)Phe⁸)-Anglotensin II (IN₃AII); (Sar¹-(¹²⁵I)Tyr⁴-(4′N₃D-Phe)⁸)-Angiotensin II (IN₃DPheAII) (³H) SarAII and IN₃AII behaved like agonists and (³H) SarlleAII and IN₃DPheAII like antagonists. All four ligands labeled the same population of sites. The azido derivatives allowed covalent labeling of receptors with a high yield (about 40%). Membranes were solubilized by Triton X-100 under experimental conditions which ensured complete solubilization of the liganded receptors in a stable form (less than 40% dissociation after 20 h). The apparent size of liganded angiotensin receptors was determined by gel filtration on Ultrogel ACA-34 columns and by SDS gel electrophoresis (in the case of covalent labeling). The apparent Stokes radius of solubilized angiotensin receptors was different wether the receptor was labeled with an agonist (Stokes radius = 6.2 ± 0. 1 nm (6) after labeling with (³H) SarAII) or with an antagonist (Stokes radii of 5. 5 ± 0. 1 (7), and 5.6 ± 0.1 nm (4) after labeling with (³H) SarIIeAII and IN₃DPheAII respectively). After covalent labeling with IN₃All anglotensin receptors were eluted as a mixture of light and heavy forms. SDS gel electrophoresis revealed only one molecular entity of M_r 64,000. It is concluded that binding of an agonist to liver angiotensin receptors triggers or stabilizes an interaction with another membrane component Involved in the coupling of the receptor to its primary effector.     

Solubilization of muscarinic acetylcholine receptor by zwitterionic detergent from rat brain cortex

The muscarinic acetylcholine receptor was solubilized from rat brain cortex by zwitterionic detergent 3-[(3-chloramidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). About 15% of the binding activity was solubilized and 40% of the activity was destroyed by the detergent. Binding of the muscarinic antagonist [³H]-N-methyl-4-piperidyl benzilate (4NMPB) was saturable. Scatchard analysis revealed a single population of binding sites with K_D value of 0.7 nM and a B_max value of 340 fmoles/mg protein. The homogenate and the CHAPS treated pellet and soluble receptors showed similar affinity for the agonists oxotremorine and carbamylcholine and for the antagonists QNB and atropine. The dissociation of 4NMPB from the soluble receptors appears slightly slower than from the membrane bound receptors.     

Effect of chromatin associated factors on the activity of thyroid hormone receptors in rat liver and brain

Receptors for thyroid hormones were extracted by 0.4 M KCl from the nuclei of rat liver and brain, and their binding properties compared to the properties of these receptors in unextracted nuclear suspensions. The inhibititory effect of a non-iodinated thyroid hormone analogue, 3,5,dimethyl-3′-isopropyl-l-thyronine (DIMIT) on [¹²⁵I]-T₃ binding was observed in the nuclear suspension of brain, but absent when the solubilized receptors of the same organ were tested. The initial properties of the receptor could be restored in a system containing the receptor and the extracted chromatin. Moreover, when the liver solubilized receptor was supplemented with the brain chromatin extract, the hepatic receptor acquired the binding ability of the brain receptors. The data suggest that chromatin associated components may confer organ specificity in thyroid hormone effects, and play a role in the selectivity of the recognition of thyroid hormone analogues by the receptor.     

Water Soluble Receptors for Human Growth Hormone from Rabbit Liver

Abstract

Soluble receptors that bind human growth hormone have been prepared by incubation of liver membranes from pregnant female rabbits in 1 mM Tris buffer (pH 7.5 or 9.0) at 4°C. Up to 29% of the growth hormone binding sites could be solubilized within 48 hours. The kinetics of binding of human growth hormone to the soluble receptor, the hormonal specificity and the binding parameters calculated by Scatchard analysis (K_a 2.2 × 10⁹ M^-1, capacity 409 fmole/mg) were essentially unchanged compared with those for the parent membrane-associated (particulate) receptor. Gel filtration on Ultrogel AcA22 indicated that the major binding peak eluted at a molecular weight of 300,000 daltons. Specificity studies showed that the soluble binding sites had a moderately high affinity for ovine prolactin (K_a ～1 × 10⁸ M^-1), but negligible affinity for insulin. Although aqueous extraction gives a lower yield of binding sites for human growth hormone than detergent extraction, it nevertheless avoids some of the problems associated with use of detergents and should facilitate the subsequent purification of the receptor in a relatively unaltered state. It may also have applicability for solubilization of other hormone receptor systems.     

Solubilization and Characterization of Striatal Dopamine Receptors

Abstract: Dopamine receptor binding proteins were sol-ubilized with the detergent 3–(3–cholamidopropyl) dimethylammonio - 2 - hydroxy - 1– propanesulfonate (CHAPSO) from bovine and rat striatal membranes. The binding of the dopamine antagonist [³H]spiroperidol ([³H]Spi) to the solubilized dopamine receptors was determined by the polyethyleneglycol method. The CHAPSO-solubilized dopamine receptor binding proteins remain in the supernatant fraction following centrifuga-tion at 100,000 ×g for 2 h. The CHAPSO-solubilized dopamine receptor proteins, as well as the prelabeled [³H]Spi-receptor protein complex, bind specifically to wheat germ agglutinin (WGA)-agarose columns, which is consistent with an identification as glycoproteins. HPLC analysis of the CHAPSO-solubilized, prelabeled [³H]Spi-receptor protein complex (CHAPSO preparation) reveals association with a high molecular weight form, indicating the formation of aggregates and/or micelles. Treatment of the WGA-agarose-bound [³H]Spi-receptor protein complex with digitonin (CHAPSO-digitonin preparation) results in dissociation of the high molecular weight form into lower molecular weight forms. The HPLC profile of the prelabeled [³H]Spi-receptor complex in the CHAPSO-digitonin preparation reveals two radioactive peaks. The major peak had a retention time of 16 min, corresponding to an apparent MW of 175,000, whereas the minor peak had a retention time of 21 min, corresponding to an apparent MW of 49,000. The CHAPSO-solubilized dopamine receptor binding proteins are sensitive to modulation by GTP, indicating that the association with the GTP binding component is preserved in the “soluble” state. The potencies of dopamine antagonists and agonists for inhibiting the binding of [³H]Spi to CHAPSO-solubilized dopamine receptor proteins are similar to those for membrane-bound proteins. Chronic treatment with haloperidol increases the B_max, and does not change the K_D for [³H]Spi in the CHAPSO-solubilized and in the membrane-bound preparations. Thus, the CHAPSO-solubilized dopamine receptor proteins retain the binding characteristics of the supersensitive membrane-bound dopamine receptors.     

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2α receptors

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [³H] prostaglandin (PG)F₂α binding compared to E₁ binding. Lubrol WX (LWX) tended to cause a greater loss of [³H]PGF₂α than E₁ binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [³H]PGE₁ binding was greater than for PGF₂α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF₂α binding without affecting PGE₁ binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE₁ receptors compared to 100% solubilization of F₂α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE₁ and F₂α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE₁ and F₂α receptors association with the membrane structure.