Metkephamid (Tyr-D-Ala-Gly-Phe-N(Me)Met-NH2), a potent opioid peptide: Receptor binding and analgesic properties

The interaction of metkephamid (Tyr-D-Ala-Gly-Phe-N(Me)Met-NH₂) with ³H-dihydromorphine and ³H-D-Ala²-D-Leu⁵-enkephalin binding has been examined in rat brain homogenates. Displacements of both ³H-ligands by metkephamid indicate that metkephamid interacts competitively with greatest potency to the high affinity binding component for both ligands (mu₁ site). Unlike most enkephalins and opiates, metkephamid binds equipotently to both morphine-selective (mu₂) and enkephalin-selective (delta) binding sites. Metkephamid is differentiated from morphine by its better than 12-fold higher affinity for the delta receptor. Blockade of the high affinity (mu₁) binding in vivo with high doses of naloxazone dramatically reduces metkephamid's analgesic potency.     

Etonitazene: An opioid selective for the mu receptor types

Specific radioligand binding protocols were utilized to compare the affinity of morphine and the high-potency opioid etonitazene at mu₁, mu₂, delta, kappa₁ and sigma receptors. Both etonitazene and morphine displayed a mu₁-selective binding profile; however, etonitazene had a 2500-fold higher affinity at this receptor type. The latter result is consistent with the relative potencies of morphine and etonitazene in various behavioral tests.     

Characterization of a low affinity binding site for N6-substituted adenosine derivatives in rat testis membranes

Abstract

The binding characteristics of radiolabeled N⁶-(cyclohexyl)adenosine ([³H]CHA), N⁶-(R-phenylisopropyl)adenosine ([³H]R-PIA), 5′-N-ethylcarboxamidoadenosine ([³H]NECA), and 2-[4-(2-carboxyethyl)phenyl]ethyl-amino-5′-N-ethylcarboxamidoadenosine ([³H]CGS 21680), to rat testis membranes were investigated. Specific binding of [³H]CGS 21680, a selective agonist for the A_2a adenosine receptor, was very modest whilst the nonselective agonist [³H]NECA bound to rat testis membranes showing high binding capacity. At least two types of binding sites for [³H]NECA could be identified in rat testis membranes: high affinity sites and high capacity sites. Selective agonists for the At adenosine receptor, [³H]CHA and [³H]R-PIA bound with high affinity to a single class of binding sites. This high affinity binding site showed the typical pharmacological specificity of the A₁ adenosine receptor with a potency order for agonists of CHA R-PIA > NECA > N⁶-(S-phenylisopropyl)adenosine (S-PIA). In order to detect the presence of the A₃ adenosine receptor in these membranes we selectively blocked the A₁ receptor with a large molar excess of a xanthine antagonist, either 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or xanthine amine congener (XAC). In the presence of an antagonist a low affinity binding site for [³H]CHA and [³H]R-PIA was detected. This low affinity binding site showed a different pharmacological specificity than the high affinity binding site. In fact the potency order for agonists was CHA NECA = R-PIA > S-PIA. This finding suggests that the low affinity binding site represents the A₃ adenosine receptor.     

Hormone action at the membrane level VI. Binding of (—)-[3H]dihydroalprenolol and (±)-[3H]isoproterenol to turkey erythrocytes and correlation with adenylate cyclase activity

The binding of (±)-[³H]isoproterenol and (—)-[³H]dihydroalprenolol to intact turkey erythrocytes was studied using a rapid centrifugation technique. The binding of both ligands is rapid, dissociable, stereospecific and inhibited by (—)-propranolol. The total number of isoproterenol binding sites is 2800 sites/ cell. This consists of a low and high affinity site both of which show stereospecific binding. The high affinity isoproterenol site has a K_d of 15.5—19.5 nM and has 600 sites/cell. The low affinity isoproterenol site has a K_d of 195 nM and has 2200 sites/cell. The binding of (—)-[³H]dihydroalprenolol shows one type of site with a K_d of 7.8 nM and has 2500 sites/cell. The agonists epinephrine, norepinephrine, soterenol and p-hydroxyphenylisoproterenol which were tested by competition for binding showed a 6—25-fold greater affinity for the high affinity site determined by (±)-[³H]isoproterenol as compared to the (—)-[³H]dihydroalprenolol binding site. However, the antagonists propranolol, practolol and metrapolol showed similar affinities for the binding sites as determined by competition of binding of either labeled isoproterenol or dihydroalprenolol. These studies indicate that isoproterenol binding can recognize two independent stereospecific β-adrenergic receptors or can recognize two different conformational states of a single receptor. Provisional calculations are made on the turnover number of adenylate cyclase under physiological conditions using intact erythrocytes. The turnover number is 4000 molecules of cyclic AMP/10 min per high affinity receptor.     

A Study of the Cerebral Cortex Cholecystokinin Receptor Using Two Radiolabelled Probes: Evidence for a Common CCK 8 and CCK 4 Cholecystokinin Receptor Binding Site

Abstract: This study was directed at the issue of whether or not subpopulations of cholecystokinin (CCK) receptors exist within the CNS. This was achieved through the use of two radiolabelled probes, namely [¹²⁵I] Bolton-Hunter (BH) CCK 8 and [³H]pentagastrin (Boc-β-Ala CCK 4), in comparative studies under identical conditions. Both probes bound with high affinity to the mouse cerebral cortical CCK receptor binding site with apparent equilibrium dissociation constants (K_D) of 1.9 nM and 1.4 nM for [³H]pentagastrin and [¹²⁵I]BH CCK 8, respectively. The maximal binding capacity was 1.05 and 1.15 pmol/g weight for the tritium and iodinated probes, respectively. Hill analysis yielded Hill numbers close to unity, suggesting the absence of more than one binding site and the lack of cooperativity of CCK receptor binding. Kinetic studies revealed binding site homogeneity in that no evidence of multiphasic dissociation curves was seen. Computerised analysis of displacement binding data using LIGAND established that both radiolabelled probes bound to a single site, with the one-site model providing the best fit of the data. Similar rank orders of potency were obtained for various fragments of CCK 8 in competing for the CCK receptor, labelled with either probe. Both CCK 8 and CCK 4 bound with roughly equinanomolar affinity. These studies demonstrate that both CCK 8 and its shorter C-terminal fragment CCK 4 bind to a single class of high-affinity binding site, with as yet no evidence of CNS CCK receptor multiplicity.     

[3H]adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cell line and rat brain membranes

Adenosine binding sites on 108CC15 neuroblastoma × glioma hybrid cells and rat brain membranes were investigated using [³H]adenosine as labelled ligand. Both the hybrid cells and brain membranes were found to have a high affinity binding site, K_d 0.8 and 3 nM respectively. The same ligand was used to demonstrate two lower affinity binding sites on brain membranes, K_ds 1.4 and 29.1 μM and a single low affinity site on the hybrid cells, K_d 2.6 μM. Structure activity studies of the low affinity binding site on hybrid cells showed this to be an ‘R’ adenosine receptor of the A₂ subtype. It is concluded that [³H]adenosine can be used to demonstrate both high and low affinity binding sites and that 108CC15 hybrid cells provide a valuable system for studying adenosine receptors.     

Triiodothyronine nuclear receptor: Role of histones and DNA in hormone binding

The triiodothyronine (T₃) nuclear receptor was previously shown to lose rapidly its high affinity hormone-binding property after a partial purification from the nuclear extract. It was then found that histones + DNA added to the incubation medium with labeled T₃ could restore, at least in part, the high affinity T₃ binding. We now demonstrate that DNA alone increases the high affinity T₃ binding site concentration moderately, and only at low ionic strength where it can bind to the receptor. Total histones and all histone fractions studied (total core histones, F2a, H2B, H3, H4, H1) specifically increase, at low concentrations, the level of T₃ binding; but higher concentrations of some individualized histones, particularly arginine-rich histones, have an inhibitory effect. DNA, or several other polynucleotides, in the presence of histones increase the stimulating histone effect and reverse the inhibitory effect into a true activation. Histones increase the number of T₃ binding sites but decrease the affinity for T₃; addition of DNA restores the high affinity for T₃ and stabilizes the T₃-receptor complexes. Thus, some of the histone molecules could play a role in the maintenance of the T₃ binding site, but multiple interactions between histones or with DNA seem necessary to impair the negative effect exerted by other parts of the histone molecules. Whether these positive and negative effects of histones on the T₃ binding site are of biological relevance in the regulation of T₃ binding to its receptor remains to be determined.     

HIGH AFFINITY AMINO ACID TRANSPORT BY FROG SPINAL CORD SLICES

The characteristics of amino acid uptake by frog spinal cord slices was studied by in vitro incubations in appropriate media. The uptake mechanisms exhibited saturation; kinetic analysis demonstrated 2 distinct systems for the influx of the possible neurotransmitters: GABA, glycine, L-glutamic acid and L-aspartic acid. One system showed a comparatively high substrate affinity (K_m values, 10-26 μM) while the other system had a lower affinity (K_m, 0.4-1.6 mM).-Leucine, an amino acid presumably not a transmitter, was accumulated only by a low affinity mechanism (K_m 1.6 mM). The process responsible for high affinity uptake had many of the properties of an active transport mechanism. These included temperature sensitivity, energy dependence, requirement for Na⁺ ions and inhibition by ouabain. GABA and glycine uptake was inhibited only by closely related amino acids or structural analogues. The influx of L-glutamic acid was competitively inhibited by the presence of L-aspartic acid in the medium; the converse was also demonstrated. Thus, the high affinity uptake system for possible transmitter amino acids in the frog spinal cord closely resembles that described for mammalian CNS tissue. These results are compatible with the assumption that GABA, glycine, L-glutamic acid and L-aspartic acid are neurotransmitters in the amphibian spinal cord.     

Determination of binding affinity of metal cofactor to the active site of methionine aminopeptidase based on quantitation of functional enzyme

Determination of metal affinity to the active site of metalloenzymes constitutes an integral part in the understanding of enzyme catalysis and regulation. Nonlinear curve fitting of metal titration curves using the multiple independent binding sites (MIBS) model was adapted to determine K_D values based on functional enzyme concentrations. This approach provides a more accurate evaluation of K_D compared with existing methods that are based on total protein concentrations. We applied this concept to methionine aminopeptidase from Mycobacterium tuberculosis and showed that it is a monometalated enzyme with a K_D of 0.13 μM for Co²⁺.     

Benzodiazepine receptor: Heterogeneity in rabbit brain

Binding characteristics of benzodiazepine receptors were studied with synaptosomal and microsomal membranes from rabbit brain $\text{in}$$\text{vitro}$ utilizing [methyl-³H]diazepam. In synaptosomal membranes, both high and low affinity binding sites were identified with the dissociation constants (K_d) of 4.92 nM and 83.8 nM, respectively. However, only the high affinity site was identified with K_d of 3.96 nM with microsomal membranes. Benzodiazepine binding sites appear to include at least two subpopulations of receptors, one with high affinity and another with low affinity binding site.     

Field growth characteristics of two aquatic carnivorous plants,Aldrovanda vesiculosa andUtricularia australis

Basic growth characteristics of two species of free-floating submerged carnivorous plants, the very rare and stenotopicAldrovanda vesiculosa and the very common and eurytopicUtricularia australis, were investigated in a 10/11-day field growth experiment within three nylon enclosures at two artificialAldrovanda sites in the T?eboň region, S Bohemia, Czech Republic, at the peak of a growing season. Growth ofAldrovanda was best at a meso-eutrophic site (biomass doubling time,T ₂, 8.4–10.7 days, mean growth of new leaf whorls 0.96 whorls days^?1, 1.6 developed branches per shoot) and slower at an oligo-mesotrophic site (T ₂ 17.2–21.5 days, growth of whorls 1.01 whorls days^?1, 0.1–0.5 branches per shoot). Growth ofUtricularia was similar at both sites (T ₂ 19.8–33.2 days or 9.1–16.8 days, growth of whorls 3.1 or 2.7 whorls days^?1, 1.5–2.1 or 0.8–1.4 developed branches per shoot at the former or latter site, respectively). Throughout the experiment, both species at the meso-eutrophic site allocated relatively more biomass to the production and growth of branches, than to that of new whorls. The results show thatAldrovanda, although usually considered as competitively weaker, can grow faster during the growing season peak thanUtricularia due to frequent branching and the subsequent rapid growth and separation of daughter shoots. Very rapid growth of rootless aquatic carnivorous plants in nutrient-poor habitats allows the consideration of ecophysiological adaptations that enable the plants to gain limiting mineral nutrients. These adaptations include carnivory, efficient nutrient reutilization from senescent shoots, and very high affinity for mineral nutrient uptake from water. Comparison of growth rates of rare and stenotopicA. vesiculosa and very common and eurytopicU. australis shows that differences in their rarity do not seem to be based on differences of growth rate.     

Identification of conventional and novel endothelin receptors in sheep choroid plexus cells

Previous studies have demonstrated the presence of super-high affinity endothelin receptors with apparent K_d's on the order of pM in different brain tissues. This study was designed to characterize, in detail, the receptors present in SCP cells, a non-transformed sheep choroid plexus cell line. Competitive binding assays with receptor-selective ligands indicated the presence of at least three classes of binding sites: a conventional receptor of the ET_A subtype with a K_d = 0.4 nM that mediates an increase in intracellular levels of inositol 1,4,5-trisphosphate (IP₃) in response to ET-1 and two additional sites with much higher binding affinities. The latter two sites are not coupled to the common signal transduction pathways of IP₃, cAMP and cGMP. Northern blot analysis confirmed the presence of only the ET_A subtype mRNA in SCP cells. It remains to determined if the multiple binding sites are distinct gene products, multiple affinity states of a single receptor molecule or a result of cooperative association of one site with either the ligand or with other proteins.     

[3H]2-nitroimipramine: A selective “slowly-dissociating” probe of the imipramine binding site (“serotonin transporter”) in platelets and brain

Previous studies have demonstrated a close functional and structural relationship between the “high affinity” binding site for [³H]imipramine and the presynaptic and platelet uptake site(s) for serotonin. Recently we have synthesized several nitro derivatives of imipramine which have a very high affinity for the imipramine binding site and which dissociate very slowly when incubations are performed at 0–4°C. In this report, we describe the characteristics of [³H]2-nitroimipramine binding to platelet and brain membranes. Our results support the relative utility of this ligand for studying the impramine binding site (serotonin transporter) since this analogue has both a higher affinity and specific activity than [³H]imipramine. [³H]2-Nitroimipramine by virtue of its extremely slow dissociation rate should be a valuable tool in subsequent characterization and purification of the serotonin uptake or transport site.     

O2 Reactivity of Flavoproteins: DYNAMIC ACCESS OF DIOXYGEN TO THE ACTIVE SITE AND ROLE OF A H+ RELAY SYSTEM IN d-AMINO ACID OXIDASE*

Molecular dynamics simulations and implicit ligand sampling methods have identified trajectories and sites of high affinity for O₂ in the protein framework of the flavoprotein d-amino-acid oxidase (DAAO). A specific dynamic channel for the diffusion of O₂ leads from solvent to the flavin Si-side (amino acid substrate and product bind on the Re-side). Based on this, amino acids that flank the putative O₂ high affinity sites have been exchanged with bulky residues to introduce steric constraints. In G52V DAAO, the valine side chain occupies the site that in wild-type DAAO has the highest O₂ affinity. In this variant, the reactivity of the reduced enzyme with O₂ is decreased ≥100-fold and the turnover number ≈1000-fold thus verifying the concept. In addition, the simulations have identified a chain of three water molecules that might serve in relaying a H⁺ from the product imino acid =NH₂⁺ group bound on the flavin Re-side to the developing peroxide on the Si-side. This function would be comparable with that of a similarly located histidine in the flavoprotein glucose oxidase.     

Corticosteroid modulation of muscarinic receptors in rat myocardial membranes

Rat ventricular myocardial membanes contain muscarinic acetylcholine receptors which can be identified by binding of the muscarinic antagonist (-)-[³H]quinuclidinyl benzilate. Scatchard analysis of saturation binding data revealed binding to a single class of non-cooperative sites (0.693 pmol/mg protein) with high affinity (i.e. with an equilibrium dissociation constant of 0.24 nM). Competition binding curves of the agonist carbamylholine were shallow (with a Hill coefficient, n_H of 0.71) for membranes of untreated rats, suggesting the presence of two receptor subpopulations with different agonist affinity. These curves were steeper (n_H = 0.86) for adrenalectomized animals and more shallow (n_H = 0.62) for hydrocortisone-treated animals. In contrast, both treatments did not affect the total receptor number. This suggests that corticosteroids are required for the myocardial muscarinic receptors to adopt high agonist affinity. However, the inhibition of adenylate cyclase by muscarinic agonists disappeared after both corticosteroid treatment and adrenalectomy. But agonist receptor binding could still be modulated by guanine nucleotides. This indicates that both high and low affinity froms of muscarinic receptors induced by altered corticosteroid states retain functional coupling with the inhibitory nucleotide binding site, but are uncoupled from the adenylate cyclase catalytic subunit, C.     

The enzymatic binding of aminoacyl-tRNA to reticulocyte ribosomes: The stimulatory effect of donor site bound peptidyl-tRNA

The affinity of ribosomes for the elongation factors EF₁ and EF₂ changes while the ribosome is going through the different steps of the elongation cycle. In this communication we provide evidence that the affinity of the EF₁-aa-tRNA-GTP complex for the ribosomal acceptor site differs for ribosomes having their donor site either vacant or occupied by peptidyl-tRNA or by uncharged tRNA. Ribosomes having peptidyl-tRNA at their donor site bind the EF₁ complex with the highest affinity.Results are discussed in light of recent findings that the two elongation factors are not bound to the ribosome simultaneously.     

Taurine uptake in synaptosomal fractions of rat cerebral cortex.

Abstract— [³⁵S]Taurine was found to be accumulated in synaptosomal fractions of rat cerebral cortex. Kinetic analysis in the range of 1–800 μm -[³⁵S]taurine revealed at least two different uptake processes. A high affinity uptake with a K_m of 20 μM and a low affinity uptake with a K_m of about 450 μM. The high affinity component was dependent on temperature and energy, and virtually abolished in the absence of sodium. Examination of the influence of structural analogues and putative transmitter substances indicates that the high affinity uptake of taurine into synaptosomal fractions of rat cerebral cortex is unique and highly specific. No specific actions of several centrally acting drugs on taurine uptake could be observed.     

RNA Affinity for Molecular L-Histidine; Genetic Code Origins

Selection for affinity for free histidine yields a single RNA aptamer, which was isolated 54 times independently. This RNA is highly specific for the side chain and binds protonated L-histidine with 10²−10³-fold stereoselectivity and a dissociation constant (K_D) of 8–54 μM in different isolates. These histidine-binding RNAs have a common internal loop–hairpin loop structure, based on a conserved RAAGUGGGKKN_0–36 AUGUN_0–2AGKAACAG sequence. Notably, the repetitively isolated sequence contains two histidine anticodons, both implicated by conservation and chemical data in amino acid affinity. This site is probably the simplest structure that can meet our histidine affinity selection, which strengthens experimental support for a “stereochemical” origin of the genetic code.[Reviewing Editor: Niles Lehman]     

The actions of naloxazone on the binding and analgesic properties of morphiceptin (NH2Tyr-Pro-Phe-Pro-CONH2), a selective mu-receptor ligand

Active in both binding and biological assays, morphiceptin (NH₂ Tyr-Pro-Phe-Pro-CONH₂), a potent opioid peptide derivative of β-casamorphine, binds specifically and selectively to mu or morphine-type receptors with little affinity for delta sites. Displacement studies of a variety of ³H-labeled opiates and enkephalins show biphasic curves. Naloxazone, which blocks irreversibly and selectively high affinity opiate and enkephalin binding, abolishes morphiceptin's inhibition of binding at low concentrations, suggesting that the high affinity binding of enkephalins and opiates represents a mu or morphine-type receptor. Unlike the reversible antagonist naloxone, naloxazone treatment $\text{in}$$\text{vivo}$ inhibits for over 24 hours the analgesic activity of morphiceptin like it inhibits morphine, β-endorphin and enkephalin analgesia. Together, these studies imply that opiates and enkephalins bind with highest affinity to a mu receptor which mediates their analgesic activity. The ³H-D-ala²-D-leu⁵-enkephalin binding remaining after naloxazone treatment, representing a lower affinity site (K_D 4 nM), is quite insensitive to morphiceptin inhibition and has the characteristics of a delta receptor. However, the ³H-dihydromorphine binding present after naloxazone treatment, which also represents a lower affinity site (K_D 6 nM), is far more sensitive to both morphine and morphiceptin and may represent a second morphine-like, or mu, receptor.