Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes

High-affinity binding sites for mono[125I]iodoapamin were detected in membranes (Kd = 59 pM, Bmax = 24 fmol/mg protein) and cultured cells (Kd = 69 pM, Bmax = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (Kd = 67 pM, Bmax 42 fmol/mg protein) and liver (Kd = 15 pM, Bmax = 43 fmol/mg protein). Binding was stimulated by K+ ions (K0.5 = 0.3-0.5 mM). Covalent labeling with arylazide [125I]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85-87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.     

The relationship between high-affinity noncatalytic binding of snake venom phospholipases A2 to brain synaptic plasma membranes and their central lethal potencies

The basic phospholipase A2 from Naja nigricollis (African spitting cobra) snake venom is enzymatically less active but more toxic than the acidic phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom, following injection into the right lateral ventricle of the brain of rats. When radiolabeled with 125I, these phospholipases A2 retained enzymatic activities and lethal potencies. Both enzymes bound with high affinity and specificity to brain synaptic plasma membrane preparations in vitro even in the absence of calcium, suggesting a non-catalytic binding. The acidic enzyme, in a calcium-free medium, had two binding components with Kd values of 1 X 10(-10) and 2.75 X 10(-8) M and Bmax values of 6 X 10(-13) and 3.4 X 10(-11) mol/mg, respectively. Multiple specific and nonspecific binding components were observed for each phospholipase A2; saturability for all of the binding sites was conclusively demonstrated only for the N. naja atra phospholipase A2 in a calcium-free medium (Bmax = 3.4 X 10(-11) mol/mg). The levels of specific and total binding were 150 pmol/mg and 450 pmol/mg, respectively, for the comparatively toxic enzyme and 15 pmol/mg and 35 pmol/mg, respectively, for the comparatively nontoxic enzyme at a concentration of 2.5 X 10(-8) M. These levels of binding (both total and specific) were directly correlated with the intraventricular lethal potencies of the phospholipases A2 (0.5 and 5.0 micrograms/rat for the N. nigricollis and N. naja atra phospholipases A2, respectively), suggesting a possible relationship between binding and lethal potency. Carbamylation of lysines reduced the levels of binding and the lethal potencies of both enzymes to a greater extent than their enzymatic activities. Pretreatment with high temperature, proteinases, phospholipases A2 or C suggested that radiolabeled phospholipase A2 binds to phospholipids rather than proteins. However, only the N. naja atra phospholipase A2 manifested a strict dependence on a divalent cation (Ca2+ or Sr2+) for most of its binding. The N. nigricollis enzyme demonstrated a much lower rate of dissociation from synaptic plasma membranes than did N. naja atra phospholipase A2, suggesting that hydrophobic interactions are more important in the binding of the more toxic enzyme as compared to the less toxic enzyme. It is proposed that differences in the extent of high-affinity noncatalytic binding to membrane phospholipids may be at least partly responsible for the marked difference in central toxicities of these two phospholipases A2.     

Cholecystokinin-8 suppressed 3H-etorphine binding to rat brain opiate receptors

Radio receptor assay (RRA) was adopted to analyse the influence of CCK-8 on 3H-etorphine binding to opiate receptors in rat brain synaptosomal membranes (P2). In the competition experiment CCK-8 (1pM to 1 microM) suppressed the binding of 3H-etorphine. This effect was completely reversed by proglumide at 1 microM. Rosenthal analysis for saturation revealed two populations of 3H-etorphine binding sites. CCK-8 (1pM to 1 microM) inhibited 3H-etorphine binding to the high affinity sites by an increase in Kd (up to +235%) and decrease in Bmax (up to -80%) without significant changes in the Kd and Bmax of the low affinity sites. This effect of CCK-8 (10nM) was also completely reversed by proglumide at 1 microM. Unsulfated CCK-8 (100pM to 1 microM) produced only a slight increase in Kd of the high affinity sites (+64%) without affecting Bmax. The results suggest that CCK-8 might be capable of suppressing the high affinity opioid binding sites via the activation of CCK receptor.     

Melatonin binding sites in the brain of European sea bass (Dicentrarchus labrax)

Characteristics, day-night changes, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) modulation, and localization of melatonin binding sites in the brain of a marine teleost, European sea bass Dicentrarchus labrax, were studied by radioreceptor assay using 2-[(125)I]iodomelatonin as a radioligand. The specific binding to the sea bass brain membranes was rapid, stable, saturable and reversible. The radioligand binds to a single class of receptor site with the affinity (Kd) of 9.3 +/-0.6 pM and total binding capacity (Bmax) of 39.08 +/-0.86 fmol/mg protein (mean+/-SEM, n=4) at mid-light under light-dark (LD) cycles of 12:12. Day-night changes were observed neither in the Kd nor in the Bmax under LD 12:12. Treatment with GTPgammaS significantly increased the Kd and decreased the Bmax both at mid-light and mid-dark. The binding sites were highly specific for 2-phenylmelatonin, 2-iodomelatonin, melatonin, and 6-chloromelatonin. Distribution of melatonin binding sites in the sea bass brain was uneven: The Bmax was determined to be highest in mesencephalic optic tectum-tegmentum and hypothalamus, intermediate in telencephalon, cerebellum-vestibulolateral lobe and medulla oblongata-spinal cord, and lowest in olfactory bulbs with the Kd in the low picomolar range. These results indicate that melatonin released from the pineal organ and/or retina plays neuromodulatory roles in the sea bass brain via G protein-coupled melatonin receptors.     

Distinct platelet-activating factor binding sites in synaptic endings and in intracellular membranes of rat cerebral cortex

The binding of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC or PAF, platelet-activating factor) to synaptic plasma membranes, microsomal membranes, and other rat cerebral cortex subcellular fractions was studied. Using several PAF-binding antagonists, three distinct sites were identified. Two of them were in intracellular membranes (microsomes) and one in synaptic plasma membranes. Microsomal membranes were prepared after obtaining a 43,500 x g pellet from the postmitochondrial supernatant and subsequent centrifugation at 105,000 x g of the resulting supernatant. Most plasma membrane markers were retained in the 43,500 x g pellet (Sun, G.Y., Huang, H.-M., Kelleher, J.A., Stubbs, E.B., Sun, A. Y. (1988) Neurochem. Int. 12, 69-77). Microsomes were purified by density-gradient centrifugation and marker enzymes showed relatively very low contamination by plasma membrane markers. Myelin and mitochondria were devoid of specific PAF binding. A site displaying the highest PAF-binding affinity reported to date in all cells and membranes (KD = 22.5 +/- 1.7 pM and Bmax 8.75 = fmol/mg protein), was found in the microsomal fraction. There was a second binding site in microsomal fractions (KD = 25.0 +/- 0.8 nM and Bmax = 0.96 pmol/mg protein. Ca2+ decreases PAF affinity for the microsomal binding sites. The third binding site displays relatively low specific PAF binding and is present in synaptosomal plasma membranes. Moreover, displacement curves by a wide variety of PAF antagonists indicated different affinities for each of the binding sites described here. These results indicate that PAF-binding sites are heterogeneous in rat cerebral cortex, and they imply that the microsomal membrane sites may be involved, at least in part, in intracellular events such as gene expression.     

Identification and affinity labeling of very high affinity binding sites for the phenylalkylamine series of Ca+ channel blockers in the Drosophila nervous system

The interaction of putative Ca2+ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-[3H]D888 and (+/-)-[3H]verapamil. These ligands recognize a single class (Kd = 0.1-0.4 nM; Bmax = 1600-1800 fmol/mg of protein) of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a Kd value as exceptionally low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca2+ channel blockers as well as bepridil inhibited (-)-[3H]D888 binding in a competitive way with Kd values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor Kd = 0.24 nM), was used in photoaffinity experiments. A protein of Mr 135,000 +/- 5,000 was specifically labeled after ultraviolet irradiation.     

Cyclic oxidation-reduction reactions regulate thromboxane A2/prostaglandin H2 receptor number and affinity in human platelet membranes

The radiolabeled thromboxane A2/prostaglandin H2 (TXA2/PGH2) agonist 125I-BOP bound to the TXA2/PGH2 receptor on human platelet membranes. Scatchard analysis showed that pretreatment of platelet membranes with the reducing agent dithiothreitol (DTT) (10 mM) for 10 min decreased maximal 125I-BOP binding (Bmax) from 1.51 +/- 0.11 pmol/mg to 0.51 +/- 0.05 pmol/mg (p = 0.001) and increased the affinity of the remaining binding sites (Kd = 647 +/- 64 pM (untreated), 363 +/- 46 pM (treated), p = 0.006). Prolonged incubation of membranes with DTT (10 mM) for 40 min further reduced the Bmax to 0.23 +/- 0.08 pmol/mg (p = 0.001 from untreated), and the binding affinity remained elevated (Kd = 334 +/- 117 pM, p = 0.035 from untreated). Kinetic analysis of 125I-BOP binding indicated that the apparent increase in binding affinity after DTT treatment was due exclusively to an increase in the rate of ligand-receptor association with no change in dissociation rate. The effects of DTT on 125I-BOP binding were dose-dependent with an EC50 of 8.1 +/- 0.2 mM. DTT inactivation of TXA2/PGH2 receptors was time-dependent with a second order rate constant (k2) of 0.123 M-1 s-1 at 20 degrees C. The platelet membrane 125I-BOP binding site was partially protected from DTT inactivation by prior occupation with the ligand. TXA2/PGH2 receptor protection by I-BOP was dose-dependent and linearly related (r = 0.97, p = 0.002) to the proportion of receptors occupied, but was incomplete since agonist occupation of 89% of the total number of receptors resulted in only a 38% protective effect. Inhibition of 125I-BOP binding after reduction with DTT could be made permanent by addition of the sulfhydryl alkylating agent N-ethylmaleimide (25 mM), but was completely reversed by reoxidation with dithionitrobenzoic acid (DTNB) (5 mM). Oxidation of untreated receptors with DTNB resulted in a 64% increase in 125I-BOP binding sites from 1.65 +/- 0.12 pmol/mg to 2.70 +/- 0.08 pmol/mg (p = 0.013) without affecting binding affinity. DTNB-induced increases in 125I-BOP binding were concentration-dependent with an EC50 of 668 +/- 106 microM and occurred in less than 1 min at 37 degrees C. In the absence of DTT, alkylation of free sulfhydryl groups with N-ethylmaleimide reduced 125I-BOP Bmax in platelet membranes to 0.85 +/- 0.08 pmol/mg (p = 0.003), but did not change the affinity of the remaining receptors. The EC50 for N-ethylmaleimide inactivation of TXA2/PGH2 receptors was 139 +/- 8 mM, and the k2 in time course experiments was 0.067 M-1 s-1 at 20 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

beta-Adrenergic receptors of brain cells. Membrane integrity implies apparent positive cooperativity and higher affinity

Beta-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [3H]dihydro-L-alprenolol ([3H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [3H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, beta-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of Bmax = 12.01 fmol/10(6) cells (7200 sites/cell), Kd = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with L-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k+ = 8.31 X 10(7) M-1 X min-1 and k- = 0.28 min-1 from the association results, and k- = 0.24 min-1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; Kd, as (k-/k+)1/n was found to be 101 pM. Analysis of the equilibrium binding of [3H]DHA to rat and mouse living brain cells resulted in values of Bmax = 13.04 fmol/10(6) cells (7800 sites/cell), Kd = 43.85 pM and n = 2.52, and Bmax = 8.08 fmol/10(6) cells (4800 sites/cell), Kd = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the beta-receptor in mammalian objects, too. The [3H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, Kd in the range 1-2 nM, and a Bmax of 10(3) - 10(4) sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the beta-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.     

Guanine Nucleotides Regulate 2-[125I]Iodomelatonin Binding Sites in Chick Retinal Pigment Epithelium but Not in Neuronal Retina

The characteristics of the binding sites labeled by the radioligand 2-[125I]iodomelatonin were compared in chicken neuronal retina and retinal pigment epithelium (RPE). Specific binding of 2-[125I]iodomelatonin in both sites was stable, saturable, reversible, and of high affinity. Scatchard analysis revealed an affinity constant (KD) of 446 +/- 55 pM and a total number of binding sites (Bmax) of 25.4 +/- 2.2 fmol/mg of protein for neuronal retina. For RPE the KD was 34.1 +/- 2.2 pM and the Bmax 59.5 +/- 5.2 fmol/mg of protein. Competition experiments with various melatonin analogues gave the following order of affinities: 2-iodomelatonin greater than 2-chloromelatonin greater than melatonin greater than 6-chloromelatonin greater than 6-hydroxymelatonin greater than N-acetylserotonin greater than 6-methoxyharmalan greater than 5-hydroxytryptamine. Linear regression of log Ki values from neuronal retina and RPE gave a highly significant correlation (r = 0.994, n = 8; p less than 0.001). GTP inhibited specific binding to RPE membranes in a concentration-dependent manner, but not in neuronal retinal membranes. The present results strongly suggest that a single type of melatonin receptor is found in neuronal retina and RPE, and that the site in RPE is coupled to a guanine nucleotide-binding regulatory protein (G protein), but that in neuronal retina is not.     

Identification and characterization of melatonin binding sites in the gastrointestinal tract of ducks.

Utilizing 2-[125I]iodomelatonin as the radioligand, melatonin binding sites were identified and characterized in the jejunum of ducks. These sites were found to be reversible, saturable, specific and exhibited high affinity for melatonin. Scatchard analyses have established the equilibrium dissociation constant (Kd) for tissues collected during mid-photophase to be 40.9 +/- 7 pM and the maximum quantity of binding sites (Bmax) to be 2.0 +/- 0.4 fmol/mg protein while Kd of samples collected during mid-scotophase was found to be 54.1 +/- 10 pM with a corresponding Bmax of 1.5 +/- 0.3 fmol/mg protein. These Kd values are in good proximity to the kinetically derived equilibrium dissociation constant of 47.3 +/- 20 pM. No significant difference in Kd or Bmax was detected between the mid-light and mid-dark samples. Pharmacological profile of these binding sites, developed by their interactions with other indoles and compounds, indicated that these binding sites are highly specific for melatonin. Subcellularly, different densities of binding sites were localized to various fractions in the following order: nuclear greater than microsomal greater than mitochondrial greater than cytosolic. These binding sites in the jejunum might be the receptors accountable for promoting paracrine activities for the locally synthesized gastrointestinal melatonin and/or responsible for eliciting hormonal actions via interactions with melatonin of pineal origin.     

Identification of specific vasopressin binding sites in cell membranes of the adenohypophysis, liver, kidney and adrenal glands of the rat

3H vasopressin specifically binds to the binding sites in liver, kidney and adenohypophysis with Bmax = 11.8 +/- 5.6, 1.7 +/- 0.5 and 4.9 +/- 1.2 pmol/g tissue and Kd = 1.5 +/- 0.5, 0.66 +/- 0.21 and 0.84 +/- 0.21 nM, correspondingly. Specific binding increases in the presence of Mg2+ and Ni2+ and decreases at high temperature (37 degrees). The presence of high affinity binding sites for 3H vasopressin was shown in the rat adrenals, binding was saturable and reversible. Concentration of vasopressin binding sites in adrenals is 6-8 fold less than in adenohypophysis. It is supposed that vasopressin receptors in adrenals may participate in the regulation of corticosteroid secretion.     

Regulation of 1,4-dihydropyridine and beta-adrenergic receptor sites in coronary artery smooth muscle membranes.

The receptor sites for 1,4-dihydropyridine (DHP) calcium channel ligands were identified and pharmacologically characterized in partially purified canine coronary artery smooth muscle (CSM) membranes (purification factor for 1,4-DHPs 2.8 and 2.2 respectively) using Ca2+ channel agonist (-)-S-[3H]BAYK 8644 and antagonist (+)-[3H]PN 200-110 as radioligands. The beta-adrenergic receptors were identified with (-)-3-[125I]iodocyanopindolol (ICYP). Specific binding of 1,4-DHPs and ICYP to membrane fraction was saturable, reversible and of both high and low affinity. The Kd for 1,4-DHP Ca2+ channel agonist was 0.59 +/- 0.05 and for antagonist 0.35 +/- 0.06 nmol/l and for low affinity binding sites Kd = 9.0 +/- 0.18 and 18.0 +/- 1.1 nmol/l. The high affinity 1,4-DHP binding (Bmax = 265 +/- 21 and 492 +/- 12 fmol/mg protein), showed stereoselectivity, temperature-dependence as well as pharmacological specificity: isoprenaline- and GTP-sensitivity, positive modulation with dilthiazem and negative modulation with verapamil, that is, properties characteristic of 1,4-DHP receptor sites on L-type Ca2+ channels. The low affinity binding sites were characterized as nonselective, temperature independent, dipyridamol-sensitive and represented a nucleoside transporter. The proportion of high affinity binding sites identified in the CSM membranes was 1.85 : 1.0 in favour of the antagonist. Results obtained with [125I]omega Conotoxin GVI A demonstrated that CSM membrane fractions isolated from median layers of coronary artery were devoid of substantial contamination with fragments of neuronal cells.     

Investigation of the binding of Ca2+, Mg2+, Mn2+ and K+ to the vitamin D-dependent Ca2+-binding protein from pig duodenum.

The cation-binding properties of the vitamin D-dependent Ca2+-binding protein from pig duodenum were investigated, mainly by flow dialysis. The protein bound two Ca2+ ions with high affinity, and Mg2+, Mn2+ and K+ were all bound competitively with Ca2+ at both sites. The sites were distinguished by their different affinities for Mn2+, the one with the higher affinity being designated A (Kd 0.61 +/- 0.02 microM) and the other B (Kd 50 +/- 6 microM). Competitive binding studies allied to fluorimetric titration with Mg2+ showed that site A bound Ca2+, Mg2+ and K+ with Kd values of 4.7 +/- 0.8 nM, 94 +/- 18 microM and 1.6 +/- 0.3 mM respectively, and site B bound the same three cations with Kd values of 6.3 +/- 1.8 nM, 127 +/- 38 microM and 2.1 +/- 0.6 mM. For the binding of these cations, therefore, there was no significant difference between the two sites. In the presence of 1 mM-Mg2+ and 150 mM-K+, both sites bound Ca2+ with an apparent Kd of 0.5 microM. The cation-binding properties were discussed relative to those of parvalbumin, troponin C and the vitamin D-dependent Ca2+-binding protein from chick duodenum.     

Thyrotropin releasing hormone (TRH) binding sites in the adult human brain: localization and characterization

In the current study, we found evidence for the existence of binding sites for TRH in synaptic membrane preparations of several regions of the postmortem adult human brain. High levels of specific binding (fmol [3H]Me-TRH/mg protein/2 hr) were found in limbic structures: amygdala (7.1 +/- 0.6, Mean +/- SE), hippocampus (2.8 +/- 0.3), and temporal cortex (2.4 +/- 0.8). Intermediate levels of binding were found in the hypothalamus and nucleus accumbens whereas binding was low to undetectable in frontal and occipital cortex, cerebellum, pons, medulla and corpus striatum. Binding of the radioligand was linear over protein concentrations of 0.05-1.5 mg, and greater than 6 hr of incubation was required to achieve maximal binding. In the amygdala, binding was inhibited in the presence of TRH and Me-TRH but not in the presence of up to 1 microM concentrations of cyclo (His-Pro), TRH-OH, pGlu-His or peptides unrelated to TRH. Pretreatment of amygdala synaptic membranes with detergents, proteases or phospholipases disrupted [3H]Me-TRH binding; pretreatment with DNase or collagenase had no effect on binding. Saturation and association/dissociation analyses of the binding of [3H]Me-TRH to purified amygdala synaptic membranes revealed the presence of a high affinity (KD = 2.0 nM), low capacity (Bmax = 180 +/- 16 fmoles/mg protein) binding site. These results demonstrate that a highly specific membrane associated receptor for TRH is present in the adult human brain. The specific role that this receptor plays in brain function remains to be elucidated.     

High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor. G protein interactions

The binding of the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions, [3H]QNB bound to a homogenous population of muscarinic receptors (Kd, 118 +/- 19 pM; Bmax, 572 +/- 42 fmol/mg membrane protein, n = 12). The addition of G protein activators AlF4- or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) + Mg2+ increased the Kd by 77 +/- 7% (n = 4, P less than 0.05) and 83 +/- 27% (n = 7, P less than 0.05), respectively, without a change in the Bmax or homogeneity of the binding site. GTP gamma S added without exogenous Mg2+ did not affect [3H]QNB binding. Thus, optimal QNB binding requires a muscarinic receptor/G protein interaction.     

Effects of N-phthalamoyl-L-glutaminic acid, a selective agonist of NMDA receptor, on binding of 3H-L-glutamate with synaptic membranes of the hippocampus]

In the present investigation the interaction of a novel selective NMDA receptors agonist, N-phthalamoyl-L-glutamic acid (PhGA), with the synaptic membranes preparation of human hippocampus was examined against NMDA. It was established that there are two binding sites of 3H-L-Glu, Kd1 = 0.35 +/- 0.11 nM, Bmax1 = 6.5 +/- 2.3 pmol/mg and Kd2 = 51 +/- 12 nM, Bmax2 = 98 +/- 17 pmol/mg. The inhibition constants (Ki) were calculated for NMDA and PhGA and were equal: Ki(NMDA) = 19 microM, Ki (PhGA) = 13 microM, respectively. It was concluded that PhGA is the partial agonist of the NMDA receptors.     

Substituted diphenylbutylpiperidines bind to a unique high affinity site on the L-type calcium channel. Evidence for a fourth site in the cardiac calcium entry blocker receptor complex

Fluspirilene binds with high affinity to a single class of sites in purified porcine cardiac sarcolemmal membrane vesicles at a Kd of 0.6 nM and a Bmax that is in approximately 1:1 stoichiometry with other Ca2+ entry blocker receptors. Fluspirilene binding is modulated by various classes of L-type Ca2+ channel effectors. Metal ion channel inhibitors (e.g. Cd2+) stimulate binding primarily by increasing ligand affinity, whereas channel substrates (e.g. Ca2+) inhibit binding. Dihydropyridine, aralkylamine, and benzothiazepine Ca2+ entry blockers partially inhibit binding with Ki values equivalent to their respective Kd values, indicating close coupling between binding sites for the former agents and the diphenylbutylpiperidine site. All of these agents function as mixed inhibitors and affect both Kd and Bmax of fluspirilene binding. Only other substituted diphenylbutylpiperidines (e.g. pimozide) inhibit binding competitively. Diphenylbutylpiperidines, on the other hand, block nitrendipine, D-600, and diltiazem binding through a noncompetitive mechanism with Ki values much reduced from their measured Kd values, suggesting that coupling between the diphenylbutylpiperidine site and receptors for diverse Ca2+ entry blockers is more indirect. In addition, high affinity sites have been detected for fluspirilene in bovine aortic sarcolemmal vesicles, rat brain synaptic membranes, and GH3 rat anterior pituitary cell plasma membranes. Fluspirilene also effectively blocks Ca2+ flux through L-type Ca2+ channels in GH3 cells. Together, these results suggest that fluspirilene binds with high affinity to a unique fourth site in the Ca2+ entry blocker receptor complex and that substituted diphenylbutylpiperidines represent a new structural class of potent L-type Ca2+ channel inhibitors.     

ACTH receptors in nervous tissue. High affinity binding-sequestration of [125I]Phe2,Nle4]ACTH 1-24 in homogenates and slices from rat brain

We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adrenergic receptors in the liver parenchyma in children with chronic hepatitis]

In the present study adrenergic receptors have been investigated in liver parenchyma, obtained at the resection of extrahepatic portal hypertension children without parenchymal affection (control group, n-7) and the resection of children in parenchymal affection (group of chronic hepatitis children, n-6). It has been shown, that the binding of beta-adrenergic radioligand 3H-dihydroalprenolol (3H-DHA) in liver parenchyma membranes of both control and chronic hepatitis groups was saturable and showed high affinity. The Scatchard analysis of the binding data indicated that the binding site was characterized by Kd and Bmax of 1.2 +/- 0.5 nM, 261.2 +/- 50 fmol/mg, respectively, for the control group; and 0.9 +/- 0.15 nM, 68.5 +/- 18.8 fmol/mg, respectively, for the group of chronic hepatitis patients; (mean+SEM). The binding of alpha 1-adrenergic antagonist 3H-prazosin (3H-PRZ) in liver parenchyma was also saturable and showed high affinity. The binding site is characterized by Kd = 0.6 +/- 0.12 nM, Bmax = 92.8 +/- 8.0 fmol/mg, for the control group; and Kd = 0.8 +/- 0.15 nM, Bmax = 195.0 +/- 22.0 fmol/mg, for the group of chronic hepatitis. It has been found that the number of binding sites of 3H-DHA significantly decreased and the number of binding sites of 3H-PRZ did not change in chronic hepatitis liver parenchyma in comparison with the control group. The results obtained suggest the important role of beta-adrenergic receptors in the pathogenesis of chronic hepatitis and in liver regeneration in children.     

Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy.

Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H2O and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 +/- 0.5 and 4.6 +/- 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 +/- 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 A. Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site n1 was observed to decrease from Kd = 0.35 +/- 0.09 microM for GS0 to Kd = 0.06 +/- 0.02 microM for GS12. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 +/- 0.9 microM and Kd = 2.6 +/- 0.7 microM for GS0 and GS12, respectively. Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 +/- 0.02 microM for GS0 to Kd = 0.35 +/- 0.09 microM for GS12. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 +/- 1.3 microM for GS0 and Kd = 4.0 +/- 1.0 microM for GS12). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991, Biochemistry 30:1413-1418), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities.