Interactions Between the Argininyl Moieties of Neurotensin and the Catechol Protons of Dopamine

The interaction between dopamine and neurotensin as well as other Arg-containing peptides was studied to provide more chemical details of how dopamine binds to the neuropeptide neurotensin. The stoichiometry of 1:1, dopamine to neurotensin, was confirmed by additional electroanalytical and ultraviolet-visible spectroscopic studies. By analyses of the 205- to 340-nm difference spectra of fixed concentrations of dopamine in the presence of increasing amounts of neurotensin, the dissociation constant of the interaction was found to be 5.9 x 10(-8) mol/L. This finding confirmed (by a second physical method) the previously reported KD value obtained by electroanalytical techniques. The associations between dopamine and neurotensin as well as the neurotensin fragment Pro7-Arg8-Arg9-Pro10 were found to be pH dependent when the dissociation constant was measured as a function of pH (in 150 mmol/L NaCl). The results of studies of the formal potential of dopamine in the presence of Arg and Arg-containing peptides confirmed that catechol protons are directly involved in the association and that the chemical species of dopamine associated with neurotensin is a catecholate form. The (pseudo)-first-order rate constant of dissociation of the complex at pH 7.6, measured by the chronoamperometric and rotating disk electroanalytical techniques, was found to be approximately 10(5) s-1, indicating that the rate of formation of the complex is under diffusion control. A hypothetical chemical structure of the neurotensin-dopamine complex is suggested.     

Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D2 receptor

The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of [³H]-ATP to [³H]-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC₅₀ values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC₅₀ values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D₂ dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity.     

Striatal Transporter for Dopamine: Catechol Structure-Activity Studies and Susceptibility to Chemical Modification

Abstract: The apparent second-order association rate constant of dopamine binding to the striatal transporter (～1 ± 10⁶M^?1 s^?1) as well as the transporter turnover number (～1.5 s^?1) was estimated using rotating disk electrode voltammetry to monitor apparent zero trans entry of dopamine into striatal suspensions. The substrate specificity of the transporter was also assessed using catechol derivatives. Dopamine and norepinephrine were transported, whereas epinephrine and the acidic metabolites of dopamine were not transported. The metabolite, 3-meth-oxytyramine, was transported with a K_m seven times greater than and a V_max close to that for dopamine. 4-Methoxytyramine was transported more facilely than the 3-methoxy derivative. N-Alkylation of the amine side chain of dopamine reduced transport dramatically. 4-Ethylcatechol and 3,4-dihydroxybenzylamine were transported with velocities 79 and 91 % less than that for dopamine, respectively. The rigid analogue 6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene was transported with a greater velocity than the 5,7-dihydroxy derivative. Finally, the apparent K_mvalues for 4-ethylcatechol, 1-amino-2-phenylethane, tyramine, and m-tyramine as cosubstrates with dopamine were 1.1, 11, 17, and 2.6 μM, respectively. Pretreatments of striatal suspensions with chloroethylnorapomorphine, N-ethylmaleimide, Hg²⁺, 4,5-dihydroxy-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (a redox modulator of receptors in neuronal as well as other tissues), and neuraminidase reduced the velocity of transport of dopamine, whereas N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline had no effect. Thus, the dopamine transporter requires an intact catechol with a primary ethylamine side chain for optimal activity relative to shorter side chain derivatives (side chains longer than two carbons were not tested), the 3-hydroxyl group of dopamine is the more critical hydroxyl group, and the β rotamer of the extended conformation of dopamine is transported preferentially. The catechol appears to mediate the recognition of the substrate, whereas the amine side chain apparently facilitates the conformational change of the transporter that results in movement of dopamine into or across the membrane. The transporter distinguishes between agents known to block dopamine recognition sites on dopamine receptors? appears to possess a reduction/oxidation modulatory site, and requires sulfhydryl groups and external glycosylation for optimal function.     

Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro. II. The platelet-derived growth factor A-chain contains a sequence that specifically binds heparin

Summary Synthetic oligopeptides were used to study the specificity of the interaction between heparin and platelet-derived growth factor (PDGF) in competition experiments. DNA synthesis in PDGF-dependent human arterial smooth muscle cell (hASMC) cultures was used as a biological tracer of PDGF activity. Oligo-108-124 (corresponding to amino acid residues 108-124 of the long PDGF A-chain isoform) had no effect on DNA synthesis in itself but competed at 10⁻¹⁰ M concentration effectively with PDGF for binding to heparin and released the block on thymidine incorporation induced by heparin. Poly-lysine-serine (lysine:serine ratio 3:1) was also effective but at a considerably higher concentration (10⁻⁶ M). Poly-arginine-serine did not compete with PDGF for heparin as deduced from the cell assay. This suggested that among basic amino acids, lysine was more important than arginine for heparin binding. Deletion of lysine residues 115 and 116 in Oligo-108-124 abolished its effect on the interaction between PDGF and heparin in the cell assay. Likewise, Oligo-69-84 (corresponding to the PDGF A-chain residues 69–84), with three lysine residues interrupted by a proline, was ineffective. In Oligo-108-124, the lysine residues are interrupted by an arginine. Our results suggested that the binding between PDGF and heparin is specific and that the amino acid sequence [-Lys¹¹⁵-Lys¹¹⁶-Arg¹¹⁷-Lys¹¹⁸-Arg¹¹⁹-] is of major importance. They do not however, exclude other domains of the PDGF A or B chains as additional binding sites for heparin nor do they exclude the possibility that heparin and the PDGF receptor share a common binding site.     

Mapping of cytochrome P450 2B4 substrate binding sites by photolabile probe 3-azidiamantane: identification of putative substrate access regions

To investigate structure-function relationships of cytochromes P450 (CYP), 3-azidiamantane was employed for photoaffinity labeling of rabbit microsomal CYP2B4. Four diamantane labeled tryptic fragments were identified by mass spectrometry and sequencing: peptide I (Leu³⁵⁹-Lys³⁷³), peptide II (Leu³⁰-Arg⁴⁸), peptide III (Phe¹²⁷-Arg¹⁴⁰), and peptide IV (Arg⁴³⁴-Arg⁴⁴³). Their positions were projected into CYP2B4 model structures and compared with substrate binding sites, proposed by docking of diamantane. We identified novel binding regions outside the active site of CYP2B4. One of them, defined with diamantane modified Arg¹³³, marks a possible entrance to the active site from the heme proximal face. In addition to crystal structures of CYP2B4 chimeras and molecular dynamics simulations, our data of photoaffinity labeling of the full CYP2B4 molecule provide further insight into functional and structural aspects of substrate binding.     

Selective labeling of alpha-noradrenergic receptors in rat brain with [3H]dihydroergokryptine.

Using concentrations of [³H] dihydroergokryptine between 0.1 and 5 nM, saturable binding can be demonstrated in rat cerebral cortical membranes with a dissociation constant (K_D) of about 0.8 nM. α-Noradrenergic agonists and antagonists compete for the sites labeled by these low concentrations of [³H] dihydroergokryptine with relative potencies characteristics of classical α-noradrenergic receptors. The very low potency of serotonin in competing for these binding sites indicates that, in contrast to findings with higher concentrations of [³H] DHE, low concentrations do not label serotonin receptors. Moreover, the low potency of dopamine in competing for [³H] dihydroergokryptine binding in both striatal and cortical membranes indicates that no detectable portion of binding is associated with postsynaptic dopamine receptors.     

Chronic neuroleptic treatment specifically alters the number of dopamine receptors in rat brain

Trifluoperazine dihydrochloride (2.8–4.0 mg/kg/day) was administered continuously to rats in drinking water for six months. Animals killed at this time exhibited an increase in the number of dopamine receptors in the striatum and mesolimbic area, with a corresponding decrease in affinity (increase in the dissociation constant) for ³H-spiperone binding. In frontal cortex, ³H-spiperone binding to 5-HT receptors indicated no apparent change in numbers of receptors, but a slight increase in the dissociation constant. There was no obvious alteration in ³H-apomorphine binding in the striatum and mesolimbic area, but the individual results were very variable. The number and binding affinity of muscarinic receptors in striatum, mesolimbic area and cerebral cortex as identified by ³H-dexetemide were unchanged. Nor was there any alternation in the number or binding affinity of H-1 receptors identified by ³H-mepyramine, or of α-noradrenergic receptors identified by ³H-WB 4101, in cerebral cortex. The number and binding affinity of GABA receptors in the cerebellum identified by ³H-muscimol also was not altered.Chronic neuroleptic administration to rats appears to alter specifically the number of cerebral dopamine receptors.     

High-Affinity Receptor Sites and Rapid Proteolytic Inactivation of Neurotensin in Primary Cultured Neurons

The present article describes the interaction of neurotensin with specific receptors in pure primary cultured neurons and the mechanisms by which this peptide is inactivated by these cells. Neurotensin binding sites are not detectable in nondifferentiated neurons and appear during maturation. The binding at 37 degrees C of [monoiodo-Tyr3]neurotensin to monolayers of neurons 96 h after plating is saturable and characterized by a dissociation constant of 300 pM and a maximal binding capacity of 178 fmol/mg of protein. The binding parameters as well as the specificity of these receptors toward neurotensin analogues reveal close similarities between the binding sites present in primary cultured neurons and those described in other membrane preparations or cells. Neurotensin is rapidly degraded by primary cultured neurons. The sites of primary inactivating cleavages are the Pro7-Arg8, Arg8-Arg9, and Pro10-Tyr11 bonds. Proline endopeptidase is totally responsible for the cleavage at the Pro7-Arg8 bond and contributes to the hydrolysis mainly at the Pro10-Tyr11 site. However, the latter breakdown is also generated by a neurotensin-degrading neutral metallopeptidase. The cleavage at the Arg8-Arg9 bond is due to a peptidase that can be specifically inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanyl-p- aminobenzoate. The secondary processing occurring on neurotensin degradation products are: a bestatin-sensitive aminopeptidasic conversion of neurotensin11-13 to free Tyr11, and a rapid cleavage of neurotensin8-13 by proline endopeptidase. A model for the inactivation of neurotensin in primary cultured neurons is proposed and compared to that previously described for purified rat brain synaptic membranes.     

Affinity of arginase for ornithine carbamoyltransferase in Saccharomyces cerevisiae.

The association between the two trimeric enzymes ornithine carbamoyltransferase and arginase, which is under the control of arginine and ornithine, is endothermic (ΔH° = + 14.6 kcal mol^?1). The process is clearly entropy-driven (ΔS° = + 94.7 cal mol^?1 deg.^?1) allowing a dissociation constant of 0.1 nm for the complex at optimal pH 8, 30 °C and an ionic strength of 0.025. The stability of the complex is moderately sensitive to pH and ionic strength. The dissociation constant of the complex was measured in a medium at cellular pH and salt concentration and found to be close to the constant expected for operative inhibition of ornithine Carbamoyltransferase in the yeast cell. The importance of the presence of arginine as an effector for the formation of the complex under the above conditions is clearly demonstrated.     

[3H]Neurotensin(8–13) Binds in Human Brain to the Same Sites as Does [3H]Neurotensin but with Higher Affinity

The binding of [3H]neurotensin(8-13) to membranes from human frontal cortex at 0 degree C was time dependent, specific, saturable, and reversible. Saturation isotherms provided an equilibrium dissociation constant (KD) of 0.52 nM, and the maximal number of binding sites (Bmax) was 3.5 pmol/g original wet weight of tissue. Scatchard analysis yielded a straight line, and the Hill coefficient was equal to 1, a result indicating that [3H]neurotensin(8-13) bound to single, noncoopertive sites. The KD values of several analogs of neurotensin determined in competition with [3H]neurotensin(8-13) were similar to those previously determined in competition with [3H]neurotensin. The regional distribution of binding sites for [3H]neurotensin(8-13) was also similar to that for [3H]neurotensin. These results suggest that [3H]neurotensin(8-13) binds to the same sites as [3H]neurotensin and that [3H]neurotensin(8-13) has a higher affinity than [3H]neurotensin for these sites in human brain.     

Proton magnetic resonance studies of bradykinin antagonists

Bradykinin (BK) is a peptide hormone with sequence Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹ and has been implicated in a multitude of pathophysiological processes such as the ability to lower systemic blood pressure and stimulate pain. BK analogues having bulky, β-branched D -aliphatic residues at position 7 combined with bulky L -aliphatic residues at position 8 have now been observed to be strong antagonists. Conformational studies based on two-dimensional nmr experiments in methanol/water (80/20 v/v) were carried out on several such active antagonists in a polar solvent. Included in this study were the very active antagonists, [D -Arg⁰, Hyp³, Thi⁵, D -Cpg⁷, Cpg⁸]-BK [Cpg: α-cyclo-pentyl-glycine; Hyp: trans-4-hydroxy-L -proline; Thi: β-(2-thienyl)-L -alanine] ( I ), [D -Arg⁰, Hyp³, D -Cpg⁷, Cpg⁸] -BK ( II ), as well as its variant with D -Cpg⁷ replaced by Cpg⁷, namely [D -Arg⁰, Hyp³, Cpg⁷, Cpg⁸]-BK ( III ). A turn-like structure, which coexists with the extended conformation, was observed between residues 2 and 5 for the most active antagonists I and II , in direct correlation with the peptide activities. No turn-like structure was found for residues 6–9. In peptide III , a turn-like structure was not identified. The existence of a turn at the C-terminal end of bradykinin and its analogues has been predicted by empirical calculations and supported by nmr measurements. But the present nmr study on the most active antagonists ( I , II ) does not support this hypothesis. Instead, the data suggest that a turn-like structure between residues 2 and 5 could be important for antagonist activity. Finally, one weak inhibitor [D -Cpg⁷]-BK ( IV ) showed no defined secondary structure. © 1993 John Wiley & Sons, Inc.     

Preparation of a pure monoiodo derivative of the bee venom neurotoxin apamin and its binding properties to rat brain synaptosomes

The preparation and purification of an active monoiodo derivative of apamin is described. Radiolabeled monoiodoapamin (2000 Ci/mmol) binds specifically to rat brain synaptosomes at 0 degrees C and pH 7.5 with a second order rate constant of association (ka = 2.6 x 10(7) M-1 s-1) and a first order rate constant of dissociation (kd = 3.8 x 10(-4) s-1). The maximal binding capacity is 12.5 fmol/mg of protein and the dissociation constant is 15-25 pM for the monoiodo derivative and 10 pM for the native toxin. The apamin receptor is destroyed by proteases suggesting that it is of a proteic nature. Neurotensin and its COOH-terminal partial sequences are the only molecules unrelated to apamin that are able to displace monoiodoapamin from its receptor at low concentrations. Half-displacement occurs at 170 nM neurotensin. This property is due to the presence in the COOH-terminal sequence of neurotensin of two contiguous arginine residues, a structure analogous to that of the apamin active site. The binding of monoiodoapamin to its receptor is sensitive to cations. Increasing K+ or Rb+ concentrations from 10 microM to 5 mM selectively enhances the binding by a factor of 1.8. Increasing the concentration of any cation from 1 to 100 mM completely inhibits iodoapamin binding. Both effects are due to a cation-induced modulation of the affinity of monoidoapamin for its receptor without any change of the maximal toxin binding capacity of synaptosomes. Guanidinium and molecules containing a guanidinium group are better inhibitors of iodoapamin binding than other inorganic cations or positively charged organic molecules.     

Magnetic resonance studies of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

The binding of Mn²⁺ to the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium was examined by electron paramagnetic resonance studies. Two types of binding sites were observed: one to two tight sites with a dissociation constant of 3–5 μm and five to six weaker sites with a dissociation constant of 40–70 μm. The activator constant for Mn²⁺ was found to be 9 μm for the glutamine-linked anthranilate synthetase activity and 4 μm for the phosphoribosyltransferase activity. These values are both in the range of the dissociation constant for the tight sites. Water proton relaxation rate measurements showed that the binary enhancement values for both classes of sites were equivalent, ?_b = 10.7 ± 2.0. The addition of chorismate to the Mn²⁺-enzyme complexes when predominantly the tight Mn²⁺ sites were occupied resulted in a large decrease in the observed enhancement (?_T = 2.0). Addition of 5-phosphoribosyl-1-pyrophosphate to the enzyme-Mn²⁺ complexes caused large decreases in the water proton relaxation rate (?_T = 1.5) when tight or tight plus weaker Mn²⁺ sites were occupied. No changes in the water proton relaxation rate were observed when glutamine, pyruvate, or anthranilate were added; a small decrease was observed when enzyme-Mn²⁺ was titrated with tryptophan. Tryptophan significantly altered the effect of the binding of chorismate but not of 5-phosphoribosyl-1-pyrophosphate. The effect of tryptophan on the water proton relaxation rate of a Mn²⁺-enzyme-chorismate complex using a variant enzyme complex which is tryptophan hypersensitive (P. D. Robison, and H. R. Levy, 1976, Biochim. Biophys. Acta. 445, 475–485) occurred at lower concentrations than for the normal enzyme complex. The uncomplexed anthranilate synthetase subunit was titrated with Mn²⁺ and found to have one to two binding sites with a dissociation constant of 300 ± 100 μm. This dissociation constant is much larger than the activator constant for Mn²⁺ for uncomplexed anthranilate synthetase which was determined to be 4 μm. These results indicate that the Mn²⁺-binding sites on anthranilate synthetase are altered when the enzyme complex is formed and that both chorismate and 5-phosphoribosyl-1-pyrophosphate interact closely with enzyme-bound Mn²⁺ or cause a large effect upon its environment.     

Specific binding and pharmacological interactions of apamin,the neurotoxin from bee venom,with guinea pig colon

This paper describes the interaction of apamin, the bee venom neurotoxin, with its receptor in the guinea pig colon. The pharmacological activity of the toxin was assayed by measuring its contracting effect on guinea pig colon preparations that had been previously relaxed by neurotensin. The IC₅₀ value of apamin in this $\text{in vitro}$ bioassay is 7 nM. These pharmacological data are compared to the binding properties of apamin to smooth muscle membranes prepared from guinea pig colon. The highly radiolabeled monoiododerivative of apamin binds to its colon receptor with a dissociation constant $\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}^1\text{= 36}\text{pM}$. The maximal binding capacity of colonic membranes is 30^dfmol/mg of protein. The dissociation constant of the unmodified toxin is 23 pM. The difference between the toxin concentrations that produce half-maximal effects in the binding and pharmacological studies arises from the different experimental conditions used for the two assays.     

Ligand-stabilized conformational states of human beta(2) adrenergic receptor: insight into G-protein-coupled receptor activation

G-protein-coupled receptors (GPCRs) are known to exist in dynamic equilibrium between inactive- and several active-state conformations, even in the absence of a ligand. Recent experimental studies on the β₂ adrenergic receptor (β₂AR) indicate that structurally different ligands with varying efficacies trigger distinct conformational changes and stabilize different receptor conformations. We have developed a computational method to study the ligand-induced rotational orientation changes in the transmembrane helices of GPCRs. This method involves a systematic spanning of the rotational orientation of the transmembrane helices (TMs) that are in the vicinity of the ligand for predicting the helical rotations that occur on ligand binding. The predicted ligand-stabilized receptor conformations are characterized by a simultaneous lowering of the ligand binding energy and a significant gain in interhelical and receptor-ligand hydrogen bonds. Using the β₂AR as a model, we show that the receptor conformational state depends on the structure and efficacy of the ligand for a given signaling pathway. We have studied the ligand-stabilized receptor conformations of five different ligands, a full agonist, norepinephrine; a partial agonist, salbutamol; a weak partial agonist, dopamine; a very weak agonist, catechol; and an inverse agonist, ICI-115881. The predicted ligand-stabilized receptor models correlate well with the experimentally observed conformational switches in β₂AR, namely, the breaking of the ionic lock between R131^3.50 at the intracellular end of TM3 (part of the DRY motif) and E268^6.30 on TM6, and the rotamer toggle switch on W286^6.48 on TM6. In agreement with trp-bimane quenching experiments, we found that norepinephrine and dopamine break the ionic lock and engage the rotamer toggle switch, whereas salbutamol, a noncatechol partial agonist only breaks the ionic lock, and the weak agonist catechol only engages the rotamer toggle switch. Norepinephrine and dopamine occupy the same binding region, between TM3, TM5, and TM6, whereas the binding site of salbutamol is shifted toward TM4. Catechol binds deeper into the protein cavity compared to the other ligands, making contact with TM5 and TM6. A part of the catechol binding site overlaps with those of dopamine and norepinephrine but not with that of salbutamol. Virtual ligand screening on 10,060 ligands on the norepinephrine-stabilized receptor conformation shows an enrichment of 38% compared to ligand unbound receptor conformation. These results show that ligand-induced conformational changes are important for developing functionally specific drugs that will stabilize a particular receptor conformation. These studies represent the first step toward a more universally applicable computational method for studying ligand efficacy and GPCR activation.     

A proton magnetic resonance study of two synthetic agonist–antagonist pairs of bradykinin analogues

The conformation of two agonist–antagonist pairs of bradykinin (Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹) analogues were studied in CD₃OH/H₂O solution by ¹H-nmr techniques. The first agonist peptide studied, D -Arg⁰-Arg¹-Pro²-Hyp³-Gly⁴-Thi⁵-Ser⁶-Pro⁷-Thi⁸-Arg⁹, differs from the bradykinin sequence by the addition of D -Arg⁰, the replacement of the Phe moieties in positions 5 and 8 by Thi (Thi = β-(2-thienyl)-L -alanine), and Hyp³ (Hyp = L -4-hydroxy-L -proline) in position 3. In the corresponding antagonist sequence, Pro⁷ is replaced by D -Phe⁷. The second agonist–antagonist pair studied does not contain the D -Arg⁰ residue, which is present only to slow down the rate of metabolism. Based on complete resonance assignments from two-dimensional total correlation spectroscopy and rotating frame nuclear Overhauser effect spectroscopy spectra at 500 MHz, the peptides were analyzed in terms of intraresidue, sequential, and medium-range nuclear Overhauser effects, amide proton temperature coefficients, and vicinal coupling constants. Both agonist peptides show clear evidence for the existence of a type I β-turn comprising the C-terminal residues Ser⁶-Pro⁷-Thi⁸-Arg⁹ in fast conformational equilibrium with extended structures throughout. Although the conformational space is dominated by extended structures, the presence of the β-turn is spectroscopically clearly discernible. The two antagonist peptides, on the other hand, do not show evidence of turn formation but rather the presence of an extended conformation with some irregularities in the N-terminal region of the peptide. While the existence of a turn at the C-terminal end of bradykinin and its analogues with agonist activity has been predicted by empirical calculations and measurements in very apolar solvents, this study, for the first time, provides evidence based on physical data in a polar solvent environment that the turn is present, that it is type I and that it is essential for agonist activity. In the particular solvent used in these studies, the Pro⁷ to D -Phe⁷ substitution precluded the formation of the turn for the C-terminal residues of the antagonist. © 1993 John Wiley & Sons, Inc.     

Comparison of Neuroleptic Binding Characteristics in Rat Striatum and Renal Cortex

Abstract

The binding characteristics of the dopaminergic ligand, ³H- spiperone, were compared in renal cortical and striatal membrane homogenates of the rat. This ligand labelled a single class of high affinity binding sites in striatum with an apparent dissociation constant (Kd) of 0.13 nM and a maximal number of binding sites (Bmax) of 890 fmol/mg protein representing D-2 receptors. In the renal cortex, ³H-spiperone identified a population of binding sites with a Bmax and a Kd of 310 fmol/mg protein and 5.1 nM, respectively. The antagonist displacing profile suggests the dopaminergic nature of the renal binding site. The affinities of dopamine antagonists for the peripheral ³H-spiperone binding site were in general in the micromolar range while the affinities of D-2 or D-2/D-1 dopamine antagonists in striatum were in the nanomolar range. Moreover, these sites showed differential stereoselectivity for (+)- and (-)-isomers of sulpiride. In conclusion, the presence of a D-2/DA-2 dopamine receptor population in renal cortex could not be confirmed. The pharmacological properties of the peripheral ³H-spiperone binding site are also different from the DA-1 receptor but seem to resemble those previously reported for dopamine receptors in sympathetic ganglia and adrenal medulla.     

In vitro triiodothyronine binding to cytoplasmic proteins from human red blood cells.

In vitro incubations of cytosol proteins from human red blood cells with [¹²⁵I] labelled L-3,5,3′ triiodothyronine demonstrated the existence of high affinity and limited capacity binding sites for T₃. At 4°C, the rate constant of association was 3 × 10⁷ M^?1h^?1, and the rate constant of dissociation was 9.10^?3h^?1. The dissociation constant K_d was calculated from these data or measured by Scatchard analysis and found to be between 3 and 7.10^?10M. The maximum binding capacity was 1.4 f moles of L-3,5,3′ triiodothyronine per mg cytosol proteins. A close parallel between the biological pontency of the analogs of L-T₃ was observed.     

Verification of both the sequence and conformational specificity of neurotensin in binding to mast cells

Neurotensin (NT) analogs, modified at Arg⁸ and Arg⁹, were used to assess the role of Arg in NT binding to mast cells. [D-Arg⁸]- and [D-Arg⁹]-NT bound 4–5 times better than NT, whereas [D-Arg^8,9]-NT had the same binding affinity as NT. Binding of [Ala⁸]-NT was not parallel to NT and exhibited a dissociation constant 38-fold lower than NT while [Ala⁹]-NT had 32% binding. C-terminal peptides, NT_8–13 and NT_9–13, had about 65% binding. These data suggest that Arg⁸ plays a greater role than Arg⁹ in the binding to mast cell NT receptors. Reduction of the disulfide bond in [Cys^2,13]-NT produced an analog 4-times more potent than NT, while the cyclized form had only 3% binding. Thus, a linear peptide with a free C-terminus appears to be required for binding.