Solubilization and Characterization of a [3H]Hemicholinium-3 Binding Site in Rat Brain

A sodium-dependent high-affinity [3H]-hemicholinium-3 ([3H]HCh-3) binding site was solubilized from rat striatal synaptic plasma membranes by 0.2% deoxycholate. Deoxycholate solubilization of the [3H]HCh-3 binding site was dependent upon both detergent concentration and ionic strength of the solubilization medium. Specific [3H]HCh-3 binding to the solubilized preparation was both sodium- and chloride-dependent and saturable, exhibiting an affinity of 14.2 nM and a capacity (Bmax) of 695 fmol/mg protein. Choline and other analogs inhibited specific [3H]HCh-3 binding to the solubilized preparation in a concentration-dependent manner with the similar rank order of potency observed in crude synaptic membranes. Treatments known to disrupt both protein and lipid moieties resulted in diminished specific [3H]HCh-3 binding. These results suggest that the characteristics of the solubilized [3H]HCh-3 binding site are similar to those of the membrane-bound site.     

Solubilization and characterization of kainate receptors from goldfish brain

The binding of [3H]kainate to goldfish brain membrane fragments was investigated. Scatchard analysis revealed a single class of binding sites in Tris-HCl buffer with a Kd of 352 nM and a Bmax of 3.1 pmol/mg wet weight. In Ringer's saline, [3H]kainate bound with a Bmax of 1.8 pmol/mg wet weight and a Kd of 214 nM. Binding in Ringer's saline, but not Tris-HCl buffer, displayed positive cooperativity with a Hill coefficient of 1.15. The [3H]kainate binding sites were solubilized in Ringer's saline using the nonionic detergent n-octyl-beta-D-glucopyranoside. Approximately 30-50% of the total number of membrane-bound binding sites were recovered on solubilization. The Kd of [3H]kainate for solubilized binding sites was approximately 200 nM. The rank order of potency for glutamatergic ligands at inhibiting [3H]kainate binding was identical and the competitive ligands had similar Ki values in both membranes and solubilized extracts. In membrane preparations, [3H]kainate displayed a two component off-rate with koff values of 0.97 min-1 and 0.07 min-1; in solubilized extracts, however, only a single off-rate (koff = 0.52 min-1) was observed. The hydrodynamic properties of n-octyl-beta-D-glucopyranoside solubilized [3H]kainate binding sites was investigated by sucrose density centrifugation. A single well defined peak was detected which yielded a sedimentation coefficient of 8.3 S. The results presented in this report suggest that goldfish brain may provide an ideal system in which to study kainate receptor biochemistry.     

N-methyl-D-aspartate/phencyclidine receptor complex of rat forebrain: purification and biochemical characterization

The N-methyl-D-aspartate (NMDA)/phencyclidine (PCP) receptor from rat forebrain was solubilized with sodium cholate and purified by affinity chromatography on amino-PCP-agarose. A 3700-fold purification was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol revealed four major bands of Mr 67,000, 57,000, 46,000, and 33,000. [3H]Azido-PCP was irreversibly incorporated into each of these bands after UV irradiation. The dissociation constant (Kd) of [1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to the purified NMDA/PCP receptor was 120 nM. The maximum specific binding (Bmax) for [3H]TCP binding was 3.3 nmol/mg of protein. The pharmacological profile of the purified receptor complex was similar to that of the membranal and soluble receptors. The binding of [3H]TCP to the purified receptor was modulated by the NMDA receptor ligands glutamate, glycine, and NMDA.     

Solubilization of Rat Brain Phencyclidine Receptors in an Active Binding Form That Is Sensitive to N-Methyl-d-Aspartate Receptor Ligands

Phencyclidine (PCP) receptors were successfully solubilized from rat forebrain membranes with 1% sodium cholate. Approximately 58% of the initial protein and 20-30% of the high-affinity PCP binding sites were solubilized. The high affinity toward PCP-like drugs, the stereo-selectivity of the sites, and the sensitivity to N-methyl-D-aspartate (NMDA) receptor ligands were preserved. Binding of the potent PCP receptor ligand N-[3H][1-(2-thienyl)cyclohexyl] piperidine ([3H]TCP) to the soluble receptors was saturable (KD = 35 nM), and PCP-like drugs inhibited [3H]TCP binding in a rank order of potency close to that observed for the membrane-bound receptors; the most potent inhibitors were TCP (Ki = 31 nM) and the anticonvulsant MK-801 (Ki = 50 nM). The NMDA receptor antagonist 2-amino-5-phosphonovaleric acid inhibited binding of [3H]TCP to the soluble receptors; glutamate or NMDA diminished this inhibition in a dose-dependent manner. Taken together, the results indicate that the soluble PCP receptor preparation contains the glutamate recognition sites and may represent a single receptor complex for PCP and NMDA, as suggested by electrophysiological data. The successful solubilization of the PCP receptors in an active binding form should now facilitate their purification.     

Molecular size of the 5-HT3 receptor solubilized from NCB 20 cells.

The 5-HT3 hydroxytryptamine receptor from NCB 20 cells was solubilized and the molecular and hydrodynamic properties of the receptor were investigated. The receptor was identified by binding of the radioligand 3-NN'-[3H]dimethyl-8-azabicyclo[3.2.1]octanyl indol-3-yl carboxylate ester [( 3H]Q ICS 205-930) to NCB 20 membranes (Bmax = 1.19 +/- 0.31 pmol/mg of protein; Kd = 0.43 +/- 0.076 nM) and was optimally solubilized with 0.5% deoxycholate. [3H]Q ICS 205-930 labelled one population of sites in solution (Bmax = 1.11 +/- 0.4 pmol/mg of protein; Kd = 0.48 +/- 0.06 nM; n = 4). The characteristics of [3H]Q ICS 205-930 binding were essentially unchanged by solubilization, and competition for [3H]Q ICS 205-930 binding by a series of 5-HT3 agonists and antagonists was consistent with binding to a 5-HT3 receptor site and was similar to that observed for 5-HT3 receptors solubilized from rat brain [McKernan, Quirk, Jackson & Ragan (1990) J. Neurochem. 54, 924-930]. Some physical properties of the solubilized receptor were investigated. The molecular size (Stokes radius) of the [3H]Q ICS 205-930-binding site was measured by gel-exclusion chromatography in a buffer containing 0.2% Lubrol and 0.5 M-NaCl and was determined as 4.81 +/- 0.15 nm (mean +/- S.E.M.; n = 6). Sucrose-density-gradient centrifugation was also performed under the same detergent and salt conditions to determine the partial specific volume (v) of the detergent-receptor site complex. This was found to be 0.794 ml.g-1. Sucrose-density-gradient centrifugation was carried out in both 1H2O and 2H2O to allow correction for detergent binding to the receptor. The Mr of the 5-HT3 receptor under these conditions was calculated as 249,000 +/- 18,000 (n = 3). The size and physical properties of the 5-HT3 receptor are similar to those observed for members of the family of ligand-gated ion channels.     

Solubilization and partial purification of somatostatin-28 preferring receptors from hamster pancreatic beta cells.

Somatostatin-28 (SRIF-28) preferring receptors were solubilized from hamster beta cell insulinoma using the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate. The binding of the iodinated [Leu8-D-TRP22-Tyr25]SRIF-28 analog (referred to as 125I[LWY] SRIF-28) to the solubilized fraction was time-dependent, saturable, and reversible. Scatchard analysis of equilibrium binding data indicated that the solubilized extract contained two classes of SRIF-28-binding sites: a high affinity site (Kd = 0.3 nM and Bmax = 1 pmol/mg protein) and a low affinity site (Kd = 13 nM and Bmax = 4.7 pmol/mg protein). The binding of 125I[LWY]SRIF-28 to solubilized SRIF-28 receptors was sensitive to the GTP analog guanosine-5'-O-thiotriphosphate, suggesting that receptors are functionally linked to a G-protein. By anion-exchange chromatography of the solubilized extract followed by chromatography on wheat germ agglutinin, a 46-fold purification of SRIF-28 receptors was obtained. At this stage of purification, only high affinity sites were found (Kd = 1 nM) and the GTP effect was not maintained. A specific protein of 37 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling. We suggest that this protein is the putative SRIF-28 receptor or a subunit thereof.     

Solubilization of the D-1 dopamine receptor from rat striatum

The D-1 dopamine receptor was extracted from rat striatal membranes with 0.7% sodium cholate and 1 M NaCl. Pretreatment of the membranes with a D-1 specific agonist, inclusion of crude phospholipids in the solubilization buffer, and subsequent removal of the detergent led to a maximal extraction of 48% of the receptor binding sites. The D-1 antagonist, [125I]SCH 23982, bound to single class of sites with a Kd of 1.8 nM and a Bmax of 1.65 pmol/mg protein. The solubilized receptors retained the ability to discriminate between active and inactive enantiomers of agonists and antagonists selective for the D-1 receptor.     

Solubilization and molecular size determination of the P2x purinoceptor from rat vas deferens.

Membranes of the rat vas deferens were shown to contain a high density of binding sites for [3H] alpha, beta-methylene ATP ([3H] alpha, beta-MeATP), a ligand selective for the P2X purinoceptor. Analysis demonstrated two classes, of high affinity (Kd = 1.8 nM, Bmax (maximum density) = 9.3 pmol/mg of protein) and of low affinity (Kd = 34 nM, Bmax = 29 pmol/mg of protein). The high affinity [3H] alpha, beta-MeATP binding sites were successfully solubilized with 2% digitonin: the Kd was then 1.6 nM. Both the association and dissociation of the receptor-ligand complex were rapid (half-time for association = 6.5 min). The rank order of potency of purinergic ligands in displacing [3H] alpha, beta-MeATP binding from the solubilized preparation was in accord with the pharmacological criteria for P2X purinoceptors. The receptor-detergent complex was separated by sucrose gradient ultracentrifugation from the ATPase enzymes also present in the preparation. The sedimentation coefficient of the receptor-detergent complex was 12.1 S. It was shown that [3H] alpha, beta-MeATP can function as a photoaffinity labeling reagent upon exposure to ultraviolet light; in the rat vas deferens membranes, it thus became cross-linked in a specific manner to a polypeptide of apparent molecular mass = 62,000 daltons, proposed to be the ligand-binding subunit of the functional P2X purinoceptor.     

Affinity chromatography of the beta-adrenergic receptor from turkey erythrocytes.

The beta 1-adrenergic receptors of turkey erythrocyte membranes have been identified by binding of the radioactively labeled antagonist (--)-[3H]dihydroalprenolol, solubilized by treatment of the membranes with the detergent digitonin, and purified by affinity chromatography. Binding of (--)-[3H]dihydroalprenolol to the membranes occurred to a single class of non-cooperative binding sites (0.2--0.3 pmol/mg protein) with a equilibrium dissociation constant (Kd) of 8 (+/- 2) nM. These sites were identified as the functional, adenylate-cyclase-linked beta 1-adrenergic receptors on the basis of: firstly, the fast association and dissociation binding kinetics at 30 degrees C; secondly, the stereospecific displacement of bound (--)-[3H]dihydroalprenolol by beta-adrenergic agonists and antagonists; and thirdly, the order of potencies for agonists to displace bound tracer (isoproterenol congruent to protokylol greater than norepinephrine congruent to epinephrine) similar to the one found for adenylate cyclase activation, and typical for beta 1-adrenergic receptors. Treatment of the membranes with the detergent digitonin solubilized 30% of the receptors in an active form. Digitonin solubilized also adenylate cyclase activity with a yield of 20 to 30%, provided the membranes were first treated with an effector known to produce a persistent active state of the enzyme: e.g. sodium fluoride. Binding sites for guanine nucleotides ([3H]p[NH]ppG) were solubilized as well. Their concentration (24 pmol/mg protein) was in large excess over the concentration of solubilized receptors (0.30--0.45 pmol/mg protein). Solubilized receptors were purified 500--2000-fold by affinity chromatography with a 25 to 35% yield, using an alprenolol-agarose affinity matrix. Affinity purified receptors were devoid of measurable adenylate cyclase activity and guanine nucleotide binding sites, thus showing that receptors and adenylate cyclase are distinct membrane constituents, and that guanine nucleotides apparently do not bind directly to the receptor molecules. Membrane-bound, solubilized and purified receptors were sensitive to inactivation by dithiothreitol, but not by N-ethylmaleimide, suggesting that receptors are at least partly constituted of protein molecules, with essential disulfide bonds.     

Binding of [3H]forskolin to solubilized preparations of adenylate cyclase

The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS).     

Solubilization of the N-Methyl-d-Aspartate Receptor Channel Complex from Rat and Porcine Brain

The N-methyl-D-aspartate (NMDA) receptor complex as defined by the binding of [3H]MK-801 has been solubilized from membranes prepared from both rat and porcine brain using the anionic detergent deoxycholate (DOC). Of the detergents tested DOC extracted the most receptors (21% for rat, 34% for pig), and the soluble complex, stabilized by the presence of MK-801, could be stored for up to 1 week at 4 degrees C with less than 25% loss in activity. Receptor preparations from both species exhibited [3H]MK-801 binding properties in solution very similar to those observed in membranes (Bmax = 485 +/- 67 fmol/mg of protein, KD = 11.5 +/- 2.9 nM in rat; Bmax = 728 +/- 108 fmol/mg of protein, KD = 7.1 +/- 1.6 nM in pig, n = 3). The pharmacological profile of the solubilized [3H]MK-801 binding site was virtually identical to that observed in membranes. The rank order of potency of: MK-801 greater than (-)-MK-801 = thienylcyclohexylpiperidine greater than dexoxadrol greater than SKF 10,047 greater than ketamine, for inhibition of [3H]MK-801 binding, was observed in all preparations. The receptor complex in solution exhibited many of the characteristic modulations observed in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilized Rat Brain Adenosine Receptors Have Two High-Affinity Binding Sites for l, 3-Dipropyl-8-Cyclopentylxanthine

The specific binding of L-N6-[3H]phenylisopropyladenosine (L-[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L-[3H]PIA (0.5-12.0 nM) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 +/- 0.03 and 0.66 +/- 0.05 pmol/mg of protein. In the presence of GTP, the binding of L-[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 +/- 0.09 and 0.93 +/- 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L-[3H]PIA binding by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1-300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 +/- 0.02 and 0.74 +/- 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.     

Two types of inositol trisphosphate binding in cardiac microsomes.

Two distinct types of [3H]IP3 binding were found in canine cardiac microsomes with high (Kd = 21 nM, Bmax = 0.66 pmol/mg) and low affinity (Kd = 230 nM, Bmax = 2.9 pmol/mg). Also found were low affinity [3H]IP4 binding (Kd = 190 nM, Bmax = 4.5 pmol/mg) and high affinity [3H]IP6 binding (Kd = 10 nM, Bmax = 4.9 pmol/mg). The rank order of potency to displace these radioligands indicates that binding of IP3 and IP6 is ligand-specific. Sucrose gradient centrifugation of the detergent-solubilized cardiac microsomes indicates that the molecular size of the cardiac high affinity IP3 receptor is similar to that of the aortic smooth muscle IP3 receptor and smaller than that of the ryanodine receptor which migrates more rapidly. The IP4 and IP6 binding migrates more slowly than the IP3 receptor.     

Solubilisation and Characterisation of a Putative Quisqualate-Type Glutamate Receptor from Chick Brain

The brains of 1-day-old chicks were shown to be a rich source of binding sites with the pharmacological characteristics expected of a quisqualate-type glutamate receptor. alpha-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) bound with KD and Bmax values, measured at 0 degree C in the presence of the chaotrope potassium thiocyanate, of 55 nM and 2.6 pmol/mg protein. The regional localisations of [3H]AMPA and [3H]kainate binding sites were manifestly different. The membrane-bound [3H]AMPA binding sites were efficiently solubilised by N-octyl-beta-D-glucopyranoside (1%) in the presence of 0.2 M thiocyanate. In the detergent extract the affinity was 69 nM and there was an apparent increase in the number of sites (Bmax, 4.6 pmol/mg protein). The rank order of potency for competitive ligands in displacing [3H]AMPA binding was quisqualate approximately AMPA greater than 6-cyano-7-nitroquinoxaline-2,3-dione greater than L-glutamate greater than kainate and was identical for the membrane-bound and solubilised sites. Dissociation was biphasic with rate constants of 0.117 min-1 and 0.015 min-1. The association rate constants for [3H]AMPA at the solubilised sites were 1.45 x 10(6) M-1 min-1 and 6.55 x 10(6) M-1 min-1. The kinetically derived KD values were 80.7 nM and 2.3 nM. The detection of higher affinity binding sites by kinetic analysis but not by equilibrium binding may be explained by the greater sensitivity of dissociation data to small populations of high-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulatory and protective effects of benzodiazepines on GABA receptors labeled with [3H]muscimol

We investigated the effects of benzodiazepines on [³H]muscimol binding to rat brain membranes and on heat inactivation of GABA receptors. Scatchard analysis of [³H]muscimol binding to frozen and 0.05% Triton X-100 treated membranes revealed two components; a higher affinity (Kd=2.2 nM, Bmax=1.2 pmol/mg protein) and a lower affinity component (Kd=15.9 nM, Bmax=4.4 pmol/mg protein). Diazepam and flurazepam (3 μM) increased significantly the specific binding of 40 nM but not of 2 nM [³H]muscimol. This stimulation was attributed to an increase in the affinity of the lower affinity component for GABA receptors. The time course of heat inactivation of GABA receptors revealed rapidly and then slowly denaturating Phases. These observations would suggest that there are multiple GABA receptors with different sensitivities to the heat treatment. Diazepam depressed remarkably the slowly denaturating phase(s). After heat treatment for 50 min, the single component of GABA receptors with Kd of 14.3 nM and Bmax of 0.6 pmol/mg protein survived, whereas in the membranes preincubated with 3 μM diazepam, the Kd and Bmax of the still viable GABA receptors were 14.8 nM and 1.14 pmol/mg protein, respectively. In light of these findings, the stimulation of the lower affinity component of GABA receptors may be related to the protective effect of these drugs against heat inactivation.     

Reconstitution of beta-adrenergic receptors into phospholipid vesicles: restoration of [125I]iodohydroxybenzylpindolol binding to digitonin-solubilized receptors

beta-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 A in diameter, and had a lipid content of 6 mumol phospholipid/mg protein. Specific binding of the beta-adrenergic ligand [3H]dihydroalprenolol ([3H]DNA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [Kd = 84 pM] and competed appropriately with (+) and (-) isomers of beta-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of beta-adrenergic receptor function in a defined lipid milieu.     

Solubilization and purification of the prostaglandin E2 receptor from cardiac sarcolemma.

A prostaglandin E2 (PGE2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [3H]PGE2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg2+, Ca2+ or of changes in buffer pH were observed on [3H]PGE2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE1 displaced over 90% of [3H]PGE2 from the CHAPS-solubilized receptor. PGD2, PGI2, PGF2 alpha and 6-keto-PGF1 alpha were not effective in displacing [3H]PGE2 from the receptor. Scatchard analysis of [3H]PGE2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE2 binding sites with Kd of 0.33 +/- 0.05 nM and 3.00 +/- 0.27 nM and Bmax of 0.5 +/- 0.04 and 2.0 +/- 0.1 pmol/mg of protein. The functional PGE2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [3H]PGE2. Unlabelled PGE1 and PGE2 displaced [3H]PGE2 from the purified receptor. Scatchard analysis of [3H]PGE2 binding to purified receptor revealed the presence of the two binding sites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M(r) of approx. 100,000. This 100-kDa was photolabelled with [3H]azido-PGE2, a photoactive derivative of PGE2. We propose that this 100-kDa protein is a cardiac PGE2 receptor.     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.     

Differential Solubilization of γ-Aminobutyric Acid Receptive Sites from Membranes of Mammalian Brain

Sodium-dependent (+Na) and sodium-independent (-Na) receptive sites for gamma-aminobutyric acid (GABA) residing in or on frozen synaptic plasma membranes (SPM) of bovine cerebral cortex were characterized as to binding constants, pharmacologic specificities, and sodium dependence. The SPM fraction was then treated with various concentrations of Triton X-100 resulting in the loss of pharmacologic specificity, binding characteristics, and sodium dependence associated with +Na GABA receptive sites in SPM. The resulting junctional complex preparation (JC), i.e., a fraction enriched in junctional complexes, possessed only the pharmacologic specificity and binding constants associated with -Na receptive sites whether assayed in the presence or absence of 100 mM-NaCl. This is probably due to the detergent dispersal or solubilization of the +Na GABA receptive site. The binding constants, KD and Bmax, for -Na GABA binding in SPM were 170 nM and 4.4 pmol/mg protein, while in JC they were 186 nM and 3.7 pmol/mg protein. Under repeated washing the KD was reduced to 60 +/- 6.9 nM and the Bmax was reduced to 2.5 +/- 0.5 pmol/mg protein in JC, probably owing to the removal of endogenous ligand or inhibitor, and not to inhibition by residual Triton X-100. Multiple extraction with 0.1% or 0.5% Triton X-100 did not alter the KD or Bmax values for the binding of [3H]GABA to JC. Sodium-independent GABA binding was lost from JC membranes with the use of sodium deoxycholate, probably through solubilization.     

Characterization of gingival epithelium epidermal growth factor receptor

The binding characteristics of gingival epithelium epidermal growth factor (EGF) receptor were investigated using epithelial cell membranes from bovine gingiva. The binding of [125I]EGF was found to be time and protein concentration dependent, reversible, and specific. Unlabeled EGF competed for [125I]EGF binding with IC50 of 0.25nM and maximum displacement of 93% at 0.81nM. Scatchard analysis of the binding data inferred the presence of two binding sites, one of high affinity (Kd = 3.3 nM and Bmax = 47.3fmol/mg protein) and the other of a low affinity (Kd = 1.6 microM and Bmax = 1.9pmol/mg protein). Crosslinking of [125I]EGF to gingival membranes followed by polyacrylamide gel electrophoresis and autoradiography revealed a receptor protein of 170kDa.