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Opioid and opioid-like receptors are activated by a variety of endogenous peptides including [Met]enkephalin (met), [Leu]enkephalin (leu), β-endorphin (β-end), α-neodynorphin, dynorphin A (dynA), dynorphin B (dynB), big dynorphin (Big dyn), nociceptin/orphanin FQ (N/OFQ); endomorphin-1 and endomorphin-2 are also potential endogenous peptides. The Greek letter nomenclature for the opioid receptors, μ, δ and κ, is well established, and NC-IUPHAR considers this nomenclature appropriate, along with the symbols spelled out (mu, delta, and kappa), and the acronyms, MOP, DOP, and KOP [124, 101, 92]. However the acronyms MOR, DOR and KOR are still widely used in the literature. The human N/OFQ receptor, NOP, is considered 'opioid-related' rather than opioid because, while it exhibits a high degree of structural homology with the conventional opioid receptors [304], it displays a distinct pharmacology. Currently there are numerous clinically used drugs, such as morphine and many other opioid analgesics, as well as antagonists such as naloxone. The majority of clinically used opiates are relatively selective μ agonists or partial agonists, though there are some μ/κ compounds, such as butorphanol, in clinical use. κ opioid agonists, such as the alkaloid nalfurafine and the peripherally acting peptide difelikefalin, are in clinical use for itch.

Opioid and opioid-like receptors are activated by a variety of endogenous peptides including [Met]enkephalin (met), [Leu]enkephalin (leu), β-endorphin (β-end), α-neodynorphin, dynorphin A (dynA), dynorphin B (dynB), big dynorphin (Big dyn), nociceptin/orphanin FQ (N/OFQ); endomorphin-1 and endomorphin-2 are also potential endogenous peptides. The Greek letter nomenclature for the opioid receptors, μ, δ and κ, is well established, and NC-IUPHAR considers this nomenclature appropriate, along with the symbols spelled out (mu, delta, and kappa), and the acronyms, MOP, DOP, and KOP. [121, 100, 91]. The human N/OFQ receptor, NOP, is considered 'opioid-related' rather than opioid because, while it exhibits a high degree of structural homology with the conventional opioid receptors [294], it displays a distinct pharmacology. Currently there are numerous clinically used drugs, such as morphine and many other opioid analgesics, as well as antagonists such as naloxone, however only for the μ receptor.

Opioid and opioid-like receptors are activated by a variety of endogenous peptides including [Met]enkephalin (met), [Leu]enkephalin (leu), β-endorphin (β-end), α-neodynorphin, dynorphin A (dynA), dynorphin B (dynB), big dynorphin (Big dyn), nociceptin/orphanin FQ (N/OFQ); endomorphin-1 and endomorphin-2 are also potential endogenous peptides. The Greek letter nomenclature for the opioid receptors, μ, δ and κ, is well established, and NC-IUPHAR considers this nomenclature appropriate, along with the symbols spelled out (mu, delta, and kappa), and the acronyms, MOP, DOP, and KOP. [116, 96, 88]. The human N/OFQ receptor, NOP, is considered 'opioid-related' rather than opioid because, while it exhibits a high degree of structural homology with the conventional opioid receptors [282], it displays a distinct pharmacology. Currently there are numerous clinically used drugs, such as morphine and many other opioid analgesics, as well as antagonists such as naloxone, however only for the μ receptor.

The second extracellular loop (ECL2) of the Noc receptor has been proposed to be involved in ligand binding and selectivity. The interaction of Noc with a constrained cyclic synthetic peptide, mimicking the ECL2, has been studied using fluorescence and NMR spectroscopies. Selective binding was shown with a dissociation constant of ∼10 µM (observed with the constrained cyclic loop and not with the open chain), and residues involved in ligand binding and selectivity have been identified. This bimolecular complex is stabilized by (i) ionic interactions between the two Noc basic motives and the ECL2 acidic residues; (ii) hydrophobic contacts involving Noc FGGF N-terminal sequence and an ECL2 tryptophane residue. Our data confirm that Noc receptor's ECL2 contributes actively to ligand binding and selectivity by providing the peptidic ligand with a low affinity-binding site. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.

A human neuroblastoma cell line, SK-N-BE, was shown to express a substantial amount of opioid receptors (200-300 fmol/mg of protein). A ligand binding profile of these receptors revealed that they could belong to two distinct subtypes of delta-opioid receptors. Results from sucrose-gradient sedimentation experiments were compared with similar data obtained with the mu-opioid receptor of the rabbit cerebellum and the delta-opioid receptor of the hybrid NG108-15 cell line and have shown that the opioid receptor of the SK-N-BE cell line behaved hydrodynamically as an intermediate between mu- and delta-opioid receptors. Taken together, pharmacological and hydrodynamic studies suggest that the opioid receptors present in the SK-N-BE cell membranes could belong to two delta-opioid receptor subtypes interacting allosterically. Functional experiments suggest that at least one of these subtypes of delta-opioid receptor is negatively coupled to the adenylate cyclase via a Gi protein and that the opiate receptors of the SK-N-BE neuroblastoma cell line undergo a rapid down-regulation when preincubated in the presence of the high-affinity opioid, etorphine.

The effects of the addition of clonidine to diuretics on the mobilization of ascites in the short term (diuretic response and requirement of diuretics) and the long term (readmissions for tense ascites and requirement of diuretics) were examined in patients with cirrhosis and with increased sympathetic nervous system (SNS) activity. We also studied neurohormonal, hemodynamic effects and side effects of clonidine and diuretics. Patients were randomized to receive placebo (group 1, n = 32) or clonidine (0.075 mg) twice daily (group 2, n = 32) for 3 months. After 8 days and for 10 days duration, spironolactone (200 mg/day) was added in both groups. After this period, the dosages of diuretics were individually increased until diuretic response. Responding patients were discharged and followed at the outpatient clinic. During the first hospitalization, the time needed for diuretic response was shorter in group 2 than in group 1. The mean requirement for diuretics was significantly higher in group 1 than in group 2, and the diuretic complications (hyperkalemia and renal impairment) were significantly lower in group 2. Clonidine induced a permanent decrease in SNS activity and delayed decrease in renin/aldosterone levels. During the follow-up, the time to the first readmission for tense ascites was shorter in group 1 than in group 2. Readmissions related to tense ascites or diuretic complications were significantly lower in group 2. The mean requirement for diuretics was significantly higher in group 1 than in group 2. In conclusion, the additional administration of clonidine to diuretics induced an earlier diuretic response associated with fewer diuretic requirements and complications.

In search for effective antagonist structures for the nociceptin (NOP) receptor, a number of N-acylated oligopeptides, including N-acyl tetra- and pentapeptides selective for the kappa-opioid receptor, as well as N-acyl hexapeptides bearing the Ac-Arg-Tyr-Tyr-Arg-Ile-Lys (Ac-RYYRIK) core sequence originally isolated from combinatorial chemical libraries, were synthesized and studied in radioreceptor binding assays, [(35)S]GTPgammaS functional tests and in mouse vas deferens (MVD) bioassays. The properties of the novel antagonist candidates were compared to known antagonists. A new antagonist structure with a reduced, primer alcohol C-terminus, Ac-Arg-Tyr-Tyr-Arg-Ile-lysinol (Ac-RYYRIK-ol) was described in the mouse vas deferens tests, showing an equilibrium inhibitory constant value (K(e)) of 2.44 nM, and no agonist effect at 10 microM ligand concentration. Schild-analysis indicated a clearly competitive interaction at the NOP receptor, whereas the peptide did not affect the action of the delta-opioid receptor agonist [D-Ala(2),D-Leu(5)]enkephalin. Ac-RYYRIK-ol also exhibited a high affinity in [(3)H]nociceptin-NH(2) binding competition assays using rat brain membranes. Agonist-induced G-protein activation via NOP receptors was studied in [(35)S]GTPgammaS binding stimulation assays by the use of both native brain tissue preparations and membranes from cultured CHO cells expressing recombinant nociceptin receptors. Ac-RYYRIK-ol displayed only weak intrinsic agonist activity, whereas it effectively inhibited the stimulation generated by nociceptin. The results support the high potency and antagonist nature of Ac-RYYRIK-ol and reveal important roles for both the N- and the C-terminal region of the molecule.

To better understand opioid receptor-like 1 (ORL1) internalization, we fused the C terminus of ORL1, the nociceptin (noc) receptor, to the N terminus of a green fluorescent protein and used the fusion protein to characterize receptor endocytosis in live human embryonic kidney cells. The fusion altered neither the affinity of the receptor for noc or other ORL1 receptor ligands nor the ability of the receptor to mediate agonist-induced binding of GTPgamma(35)S, i.e. coupling with heterotrimeric G protein. Confocal microscopy showed that the fluorescent receptor was mostly associated (75%) with the periplasmic membrane. In the presence of 0.1 microm noc, approximately 80% of receptors were internalized, and half-maximum internalization was reached in approximately 12 min at 22 C and approximately 6 min at 37 C. After washing, a normal receptor level was recovered within 70 min at 22 C. The lack of internalization in the presence of 0.45 m sucrose suggests that noc-induced receptor endocytosis mainly occurred via clathrin-coated pits. Coincubation of the recombinant cells with noc and tetramethylrhodamine-transferrin showed that ORL1 was mainly internalized through the endosome compartment. Lofentanil and Ro64-6198 ([(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one]) promoted endocytosis of the fluorescent receptor as efficiently as noc. Among the two ORL1 receptor antagonists, J-113397 (1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one), but not III-BTD, blocked the noc-induced internalization of the fluorescent receptor. Two partial agonists were dramatically less efficient than noc to promote ORL1 internalization. They recruited very little (the pseudopeptide [Phe(1)psi(CH(2)-NH)Gly(2)]-noc-(1-13)NH(2)) or no (the hexapeptide Ac-Arg-Tyr-Tyr-Lys-Trp-Arg-NH(2)) G protein receptor kinase type 2 coupled to red fluorescent protein 1 at the membrane, suggesting that subsequent receptor phosphorylation necessary for internalization via coated pits is altered. Thus, partial agonists that induce a prolonged cell response without causing substantial receptor internalization may be good tools for further clinical treatments.

The interaction of Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH(2) (HP1), a high-affinity partial agonist of the opioid receptor like (ORL1) receptor, has been investigated using the photo-labile analogue [p-benzoyl-L-Phe (Bpa)(2)]-HP1. In recombinant CHO cells expressing the human ORL1 receptor, [Bpa(2)]-HP1 binds the receptor with high affinity (K; approximately 3nM) and is as potent as HP1 in stimulating GTPgammaS binding (50-60% of nociceptin maximal effect). UV irradiation at 365nm of the complex formed by the ORL1 receptor and radio-iodinated [Bpa(2)]-HP1 results in the irreversible labelling of a glycoprotein of M(r) approximately 66kDa, as determined by SDS-PAGE. Cyanogen bromide (CNBr) and enzymatic footprints of the photo-labelled receptor and an engineered receptor mutant (L113M), containing an additional CNBR cleavage site, allowed the photoreactive region to be identified as ORL1[107-113] at the C-terminal of TM helix II. In addition the presence of a disulphide bridge between Cysl23 and Cys200 has been confirmed biochemically.

Nociceptin/orphanin FQ (noc/oFQ) is the first novel bioactive substance to have been discovered by the implementation of a functional genomics/reverse pharmacology approach. The neuropeptide was indeed identified in brain extracts as the natural ligand of a previously cloned orphan G protein-coupled receptor, the opioid receptor-like 1 (ORL1) receptor. Since its discovery in 1995, noc/oFQ has been the subject of intensive study to establish its role in normal brain function and its possible involvement in neurophysiopathology. Although the neuropeptide, an inhibitor of neuronal activity, has been found to have a wide spectrum of pharmacological effects in vivo, none has been as intensively investigated as its action on nociception and nociceptive processing. There is now substantial evidence that noc/oFQ has a modulatory role in nociception. However, dependent on the dose and site of injection, and possibly the animal's genetic background and even psychological status, the peptide has been variously reported to cause allodynia, hyperalgesia, analgesia, and even pain, in rodents. Overall, noc/oFQ tends to facilitate pain when administered supraspinally, and to inhibit it when administered spinally. These opposing effects beg the obvious, yet still unanswered, question as to what would be the net effect on nociception of an ORL1 receptor ligand, agonist or antagonist, able to target supraspinal and spinal sites simultaneously. Owing to the research effort of several drug companies, such ligands, i.e. nonpeptidic, brain-penetrating agonists and antagonists, have recently been produced whose systematic screening in animal models of acute and inflammatory pain may help validate the ORL1 receptor as the target for novel, non-opioid analgesics.

The first primary structure of a plant aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.19) is reported. The enzyme of pea (Pisum sativum) seedlings subjected to our study oxidises ω-aminoaldehydes to the corresponding ω-amino acids. Although pea does not accumulate betaine aldehyde as a compatible osmolyte, the N-terminal sequence of a purified pea AMADH resembles those of plant betaine aldehyde dehydrogenases (BADHs). On the basis of an anticipated pea AMADH homology to these enzymes, degenerated oligonucleotide primers were designed and used for PCR amplification. Two cDNA fragments were obtained in initial 5′ RACE experiments. Subsequent 5′and 3′ RACE performed with specific non-degenerated primers provided two putative cDNAs of the plant BADH family. Both encoded protein sequences (AMADH1 and AMADH2) are highly homologous to those of plant BADHs. They show 81% identity and 92% in mutual alignment. As a deduced product of the first cDNA, AMADH1 completely matches the N-terminal sequence of pea AMADH analysed previously by Edman degradation. AMADH 2 represents a putative AMADH or BADH that has not yet been isolated and characterised. We also tried to identify essential amino acid residues of a purified pea AMADH by both determination of its dissociation constants and evaluation of inhibition effects of specific modification reagents. From our results, it is clear that there are Cys (pK = 8.0) and Glu/Asp residues at the active site participating in the catalysis. This is in accordance with the presence of the conserved Glu and Cys active site regions of plant BADHs in both AMADH1 and AMADH2.

The therapeutic potential of selected peptides and proteins is enormous, with applications ranging from use as therapeutic vaccines, as modulators of intracellular signaling pathways and as highly selective agents capable of recognizing unique extracellular targets. We have been pursuing development of hybrid lipid-based carrier formulations designed to take advantage of the therapeutic benefits of peptides selected for their ability to act in a complementary fashion with the carrier system. In this regard, it is critical to have simple and versatile methods to promote and control the binding of diverse peptides to a broad range of carrier formulations. As demonstrated here, recombinant proteins and synthetic peptides containing poly-histidine residues (4 to 10) can be specifically bound to liposomes containing a metal-ion-chelating lipid, DOGS-NTA-Ni. The potential of this approach is demonstrated using two functional peptides, AntpHD-Cw3 (applications for vaccine production) and AHNP (specificity for Her-2 expressing cells).

The binding sites of nociceptin (also named orphanin FQ), the endogenous ligand of ORL1 (opiate receptor like 1), were localized in rat brain, using an autoradiographic procedure. High levels of binding were observed in the cingulate, retrosplenial, perirhinal, insular and occipital cortex, anterior and posteromedial cortical amygdaloid nuclei, basolateral amygdaloid nucleus, amygdaloid complex, posterior hippocampus, dorsal endopiriform, central medial thalamic, paraventricular, rhomboid thalamic, suprachiasmatic, ventromedial hypothalamic nuclei, mammillary complex, superficial gray layer of the superior colliculus, locus coeruleus, dorsal raphe nucleus. More moderate labelling was observed in the prefrontal, fronto-parietal, temporal, piriform cortex, dentate gyrus, anterior olfactory nucleus, olfactory tubercle, shell of nucleus accumbens, claustrum, lateral septum, laterodorsal thalamic, medial habenular, subthalamic, reuniens thalamic nuclei, subiculum, periaqueductal grey matter and pons. A lower binding site density was observed in the anterior and medial hippocampus, olfactory bulb, caudate putamen, the core of the nucleus accumbens, medial septum, ventrolateral, ventroposterolateral and mediodorsal thalamic nuclei, lateral and medial geniculate nuclei, hypothalamic area, substantia nigra, ventral tegmentum area and interpedoncular nucleus. A moderate and similar labelling was found in the dorsal and ventral horn of the spinal cord. No labelling was apparent in the corpus callosum. Thus, it appears that the ORL1 receptor is particularly abundant in the cerebral cortex, limbic system of the rat brain and some areas involved in pain perception.

Hydrolyse par la plasmine de la caseine β bovine dephosphorylee par une proteine phosphatase de type 2A purifiee chez Yarrowia lipolytica. La caseine β peut etre dephosphorylee par la sous-unite catalytique d'une proteine phosphatase de type 2A purifiee chez la levure Yarrowia lipolytica La dephosphorylation complete est obtenue en 24 a 30 h en tampon Tris (pH 7.5). A l'inverse, elle est largement inhibee par le citrate de sodium 13 mmol.L -1 a pH 6.8 (80 % d'inhibition). Apres dephosphorylation, la plasminolyse de la caseine β est augmentee puisque l'on observe une production supplementaire de 30 % de peptides solubles dans le TCA a 3 %. La dephosphorylation se traduit par la disparition du peptide f 29-105/107 et l'apparition d'un nouveau peptide non phosphoryle de 8 200 g.mol -1 . Il pourrait s'agir du peptide f 33-105/107 provenant de la partie centrale de la sequence proteique de la caseine β et obtenu par attaque de la liaison peptidique Lys 32 -Phe 33 .

Nociceptin and the ORL1 receptor share high sequence similarity with opioid peptides, particularly dynorphin A, and their receptors. However, nociceptin and dynorphin A may use distinct molecular pathways to bind and activate their cognate receptors. Activation of the kappa-opioid receptor by dynorphin A is thought to require interactions of its N-terminal hydrophobic domain (Y(1)GGF) with the receptor opioid binding pocket, located within the transmembrane helix bundle, while activation of the ORL1 receptor appears to require interactions of the positively charged core (R(8)KSARK) of nociceptin with the negatively charged second extracellular receptor loop.

Nociceptin (noc), a neuropeptide also known as orphanin FQ, is the endogenous ligand of the G protein-coupled receptor (GPCR) ORL1 (Opioid Receptor-Like1). Noc (FGGFTGARKSARKLANQ) bears a structura...

Prepronociceptin contains, in addition to nociceptin, other potentially excisable peptides which may have physiological significance. We have here considered NocII, a heptadecapeptide whose sequence lies immediately downstream of that of nociceptin in the precursor polypeptide, as well as NocIII which corresponds to NocII extended by a stretch of three arginine residues. When i.c.v.-administered in mice, NocII (10 – 10,000 ng) stimulated horizontal locomotor activity and decreased the latency to paw licking but neither to rearing nor escape jumping in the hot plate test (55 °C). When nociceptin (100 ng) and NocII (100 ng) were simultaneously intracerebroventricularly injected, each peptide produced its own effect without modifying the effect of the other. NocII was ineffective in the tail flick and writhing tests. NocIII (NocII-Arg-Arg-Arg) was inactive in all tests, even when assayed as long as 40 min following i.c.v. administration. The fact that NocII, but not its very close structural analogue NocIII, is biologically active indicates that their may exist a specific receptor to NocII.

The anatomical localization of nociceptin receptors was examined by in vitro quantitative autoradiography techniques in rat spinal cord sections by using [(125)I-Tyr(14)]nociceptin. [(125)I-Tyr(14)]nociceptin appeared to interact with a single class of binding sites (K(D)=0.1 nM) present in the grey matter in all laminae of the spinal cord from cervical to sacral levels. Pre-incubation of sections in the presence of 150 mM NaCl, did not modify the radioligand affinity but significantly augmented the number of accessible binding sites and increased specific binding of [(125)I-Tyr(14)]nociceptin differentially on each laminae. In particular, the superficial layers of the dorsal horn exhibited the highest density of sites after pre-wash. Continuous intrathecal infusion of morphine produced a tolerance accompanied by a significant increase in nociceptin site density in the superficial layers. Thus, nociceptin binding sites may have different properties dependent upon the layer and may be up-regulated during the process of opioid-induced tolerance.

A commercial soybean isolate was phosphorylated using casein kinase II purified from the yeast Yarrowia lipolytica. Both major reserve proteins, β-conglycinin and glycinin, were phosphorylated in a sequential way. The soybean isolate incorporated up to 0.7mol phosphate per mole in 2h. It was found that the phosphoester bonds were stable over time. The solubility of the phosphorylated isolate with respect to pH was not dramatically increased in comparison with the native one. However, counting the radioactivity of 32 P incorporated into the proteins (only the solubility of the phosphorylated proteins was measured in this case) showed that the solubility of the proteins was dramatically improved (up to 90% solubility for phosphorylated β-conglycinin at pH 4). β-Conglycinin became more soluble in the presence of CaCl 2 upon phosphorylation; this was not the case for the isolate. The iron-binding capacity of the soy isolate and β-conglycinin was significantly improved after phosphorylation (two and six times respectively).

The peptides formed upon action of purified cardoon rennet on the Ala189-Phe190-Leu191-Leu192-Tyr193 β-casein fragment were separated by capillary electrophoresis in an uncoated fused silica capillary. There was a linear correlation between electrophoretic mobility and Z/M2/3 (Z, net charge; M, molecular mass) under all experimental conditions tested; under the optimal condicitions, 25 kV and 40 °C, the correlation coefficient was 0.994. The reported method is fast (migration times less than 7 min) and may be used to study the action of aspartic proteinases on the C-terminal domain of β-casein and thus to help elucidate their effect on cheese quality.

We have discovered a multienzymatic complex in fresh young sugarcane leaves. This complex is constituted of three enzymes: PEPcase, NADP-MDH and malic enzyme. After successive molecular sieving chromatography, we have obtained a highly purified sample of the complex which has a molecular weight of 711 kDa. Its functional interest has been evaluated by comparing the kinetic properties of the enzymes in their free forms to those in their complexed form. We show that the association of the three enzymes leads to important changes in their respective kinetic properties.

Homology cloning and, more recently, the sequencing of whole genomes, have identified many open reading frames encoding proteins of unknown function, in particular putative G protein-coupled membrane receptors. Identification of orphan receptors in this way has marked the advent of 'reverse pharmacology' to identify the corresponding physiological ligands. This approach has led to the discovery of the ORL1 (Opioid Receptor-Like 1) receptor, and of its natural ligand, nociceptin/orphanin FQ (noc/oFQ), the basic components of a new peptide-based signalling pathway in the nervous system. Based on genetic criteria, the ORL1 and opioid receptors belong to the same family, as do noc/oFQ and opioid peptides. The marked structural analogy between the ORLI and opioid receptors, especially the kappa-opioid receptor, and the noc/oFQ and opioid peptides, particularly dynorphin A, is not reflected anatomically since noc/oFQ and opioid peptides appear to be located in separate neuronal circuits. Noc/oFQ triggers the same G protein-mediated signalling pathways as do opioids, however, to produce pharmacological effects that sometimes differ from, and even oppose, those of opioids. Noc/oFQ stimulates an outward K+ current and/or inhibits voltage-gated Ca2+ channels, thereby reducing synaptic efficacy, i.e. neuronal activity. In the rat, noc/oFQ is endowed with supraspinal pronociceptive/anti-opioid properties (it suppresses opioid-mediated analgesia), while convergent electrophysiological and behavioural data indicate that the peptide is a spinal analgesic. Noc/oFQ has not yet been found to precipitate withdrawal in morphine-tolerant rats. Nor does it elicit motivational effects, suggesting it lacks abuse liability. Also, by acting supraspinally, noc/oFQ impairs motor performance, suppresses spatial learning, induces feeding, and regulates basal and stress-induced release of pituitary hormones. Noc/oFQ is also active when administered intravenously, exhibiting potent smooth muscle relaxant, diuretic, and antinatriuretic properties. Last but not least, noc/oFQ appears to regulate stimulated immune function, and to be involved in neuronal differentiation. The discovery of noc/oFQ, a neuropeptide with multiple functions, will certainly improve our knowledge of brain physiology, and may find therapeutic applications, for example in the management of pain or hyponatremic and water-retaining diseases. However, given the wide distribution of noc/oFQ and its receptor, the pharmacological profile of noc/oFQ is likely to be incomplete, and other as yet unknown functions of the peptide remain to be discovered. Most helpful in this respect will be the identification of new ligands of the ORL1 receptor, particularly antagonists. If research on noc/oFQ carries on unabated at the present pace, potentially clinically interesting new compounds could become available in the not too distant future.

The catalytic subunit of a protein phosphatase 2A (PP2Ac) purified from Yarrowia lipolytica was immobilized by covalent coupling on CNBr-Sepharose 4B with a fixation yield of about 70−85%. The specific activity of free PP2Ac for substrate phosvitin was almost totally preserved by the immobilization process. The immobilized enzyme exhibits strongly improved thermostability. Phosvitin dephosphorylation by immobilized PP2Ac attained 14% and 35% yield after 3 and 17 h incubation, respectively, and resulted in modified phosvitin properties, e.g., improved solubility and alteration of ultraviolet absorption spectrum. Electrophoretic data indicated that β-phosvitin was preferentially dephosphorylated by the immobilized enzyme. Presence of a reductant such as DTT in the reaction medium improved dephosphorylation efficiency by inducing formation of a phosphate complex which would prevent enzyme inhibition by the released phosphate. This work indicates that immobilization of protein phosphatases is a performant too...

The binding sites of [3H]nociceptin (also named Orphanin FQ), the endogenous ligand of the ORL1 (opiate receptor like 1) receptor, were localized in the central nervous system of the mouse using an autoradiographic procedure. A high density of binding sites was seen in the cerebral cortex, paraventricular nucleus of the thalamus, amygdaloid complex, suprachiasmatic nucleus, medial thalamus and medial geniculate nucleus. Moderate binding was observed in the nucleus accumbens, lateral septum, lateral thalamus, hippocampus, periaqueductal grey matter and pons. Finally, low levels of binding were seen in the striatum, olfactory tubercle, hypothalamus and substantia nigra. Thus, it appears that the ORL1 receptor is particularly abundant in the cerebral cortex and limbic system of the mouse brain.

Resume Une activite proteine phosphatase intracellulaire a ete mise en evidence chez la levure Yarrowia lipolytica . Cette activite est maximale en debut de la phase exponentielle de croissance et augmentee largement lorsque le milieu de culture est appauvri en phosphate. La proteine phosphatase majeure retrouvee sur milieu appauvri en phosphate a ete purifiee. Cette enzyme est dissociee par un traitement ethanolique et son activite est augmentee legerement (30%) par l'heparine et fortement (150 a 300%) par les polycations. Il s'agirait donc d'une proteine phosphatase de type 2A, composee d'une sous-unite catalytique et d'autres sous-unites. Son pH optimum est 7,2, son Km apparent pour la caseine 37 μM et la vitesse maximale apparente 3,6 pmol de 32 P libere min −1 pmol −1 enzyme.

Nociceptin, also known as orphanin FQ, was recently identified as the naturally occurring agonist of orphan opioid receptor-like ORL1 receptor (Meunier et al., 1995, Nature 377, 532; Reinscheid et al., 1995, Science 270, 792). Nociceptin is a heptadecapeptide which, although it resembles dynorphin A, the endogenous agonist of the kappa-opioid receptor, displays very low potency in competing with binding of [3H]diprenorphine to or inhibiting adenylate cyclase via mu-, delta- and kappa-opioid receptors. Tritium-labeled nociceptin ([3H]nociceptin) was used here to establish a pharmacological profile in vitro of the ORL 1 receptor. In membranes from recombinant Chinese hamster ovary (CHO) cells expressing the ORL 1 receptor, equilibrium binding of [3H]nociceptin is highly specific, saturable (Bmax in the range 1.3-1.8 pmol/mg protein) and of high affinity (Kd approximately equal to 0.1 nM). It is selectively decreased in the presence of Na+ ions and/or of the GTP analog 5'-guanylylimido-diphosphate, an allosteric regulation that is analogous to that of opiate binding to opioid receptors. A few opiates, namely lofentanil, a 4-anilinopiperidine derivative and etorphine, a 6,14-endo-ethenotetrahydrothebaine derivative, were found to be quite potent not only in competing with binding of [3H]nociceptin at the ORL 1 receptor but also in inhibiting forskolin-induced accumulation of cyclic AMP in intact recombinant CHO cells. In a preliminary attempt to delineate active parts of the neuropeptide, nociceptin analogs were also tested, including N- and C-terminal truncation products. Our results suggest that the highly basic, internal core of nociceptin might be essential in conferring on the peptide both affinity for and activity at the ORL 1 receptor. In this respect, the message and address division of dynorphin A, nociceptin's closest structural analog, do not seem to apply to nociceptin.

The recently characterized heptadecapeptide nociceptin, the endogenous agonist of the orphan opioid receptor-like 1 (ORL 1 receptor), has been tested for its effects on locomotion and exploratory behaviour in mice. I.c.v. administration of as little as 10 ng of nociceptin/animal stimulated locomotor activity. This effect was dose-dependent, increasing in intensity up to 100 ng and in duration for doses in the range of 1000–10 000 ng. The stimulation of horizontal locomotion elicited by 100 ng nociceptin was accompanied by a stimulation of the vertical component of locomotion. These effects were not reversed by high doses (1.5 and 4.5 mg/kg s.c.) of the opioid receptor antagonist naloxone. Increasing doses of the dopamine D2 receptor antagonist haloperidol (0.1–0.5 mg/kg i.p.) as well as of the dopamine D1 receptor antagonist SCH 23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride] (0.0075–0.03 mg/kg s.c.) reversed this effect, suggesting that nociceptin exerts its motor-stimulant actions by increasing central dopaminergic transmission. Nociceptin was also found to increase the number of head dips in the hole-board test, indicating that the peptide stimulates exploratory behaviour.

A casein kinase II purified from the lipolytic yeast Yarrowia lipolytica has been used to phosphorylate purified soy storage proteins and their isolated constitutive subunits. β-Conglycinin appeared to be a good substrate for the enzyme, glycinin being much poorer. Phosphate was incorporated specifically in the α and α‘ subunits of β-conglycinin and in the acidic subunits of glycinin. Phosphorylation stochiometries of 0.73 mol of P/mol for β-conglycinin and 0.20 mol of P/mol for glycinin were determined. To improve the accessibility of the phosphorylation sites of glycinin and β-conglycinin for the enzyme, these proteins were succinylated at different rates. Both glycinin and β-conglycinin became better substrates after extensive succinylation. Phosphate incorporation after succinylation was still occurring in the α and α‘ subunits of β-conglycinin but also in an unknown low molecular weight fraction. The acidic subunits of succinylated glycinin were still mainly phosphorylated, but the basic subunits bec...

The present paper provides evidence that spinach chloroplasts possess a sulphite oxidase activity coupled with oxygen consumption and reduction of ferricyanide. This activity is associated with thylakoids and solubilized by non-ionic biological detergents. The pH and temperature dependencies of sulphite oxidase activity solubilized by Triton X-100 from spinach thylakoids were consistent with those of an intrinsic membrane protein. This isolated activity was insensitive towards radical scavengers (mannitol, mannose and fructose) and catalase, and was inhibited only with very high concentrations of superoxide dismutase. Thus, observed sulphite oxidation was not induced through the photosynthetic electron transport system, but achieved via a thylakoid membrane enzymic system showing a sulphite oxidase activity. Kinetic parameters of thylakoid sulphite oxidase were measured and compared with those of other sulphite oxidases.

Enzymes extracted from 10-d-old Lupinus albus seedlings were able to (1) polymerize casein and (2) incorporate [ 14 C]putrescine into dimethylcasein (modified casein). High molecular weight polymers formed were visualized by SDS-polyacrylamide gel electrophoresis. [ 14 C]Putrescine incorporation was not only due to transglutaminase activity but also due to diamine oxidase. The use of diamine oxidase inhibitor allowed us to localize a transglutaminase activity in the pellet after centrifugation at 41400g of the filtered homogenate of seedlings and a diamine oxidase activity mainly in the supernatant. Covalent conjugation of monodansylcadaverine and [ 14 C]putrescine to dimethylcasein by enzymes contained in the 41400g pellet was vizualized by fluorescent detection or by autoradiography in SDS-polyacrylamide gel electrophoresis. The enzyme contained in the pellet fraction was able to polymerize not only casein but also spinach ribulose-1,5-bisphosphate carboxylase/oxygenase and 7S soybean globulins. As a control we used purified diamine oxidase from porcine kidney to check the inability of this enzyme to catalyze casein polymerization. Moreover, 54% of transglutaminase activity contained in the pellet has been solubilized by 5% (v/v) detergent (Triton X-100) contrary to high concentrated salts. Transglutaminase was recovered in the 108000g supernatant. This suggests that this enzyme was an integral membrane protein.

Calcium-activated human placental factor XIII was assayed for polymerization on some food proteins. High molecular weight polymers were visualized on SDS-PAGE. Polymerization of soybean proteins, β-conglycin, a trimer [α,α',β], and glycinin, a hexamer of A-B dimer, depends on protein concentration, time, and the presence of DTT, although no disulfide bridge is known for β-conglycin. FXIIIa can also polymerize spinach RuBisCO, especially the L subunit. These proteins, β-conglycin, glycinin, and RuBisCO, were then assayed for incorporation of monodansylcadaverine to identify the subunit(s) containing reactive glutaminyl residues. Although all subunits can be polymerized, when purified, only the α of β-conglycin, A of glycinin, and S of RuBisCO subunits were labeled on the whole proteins. These findings could be explained by the better accessibility of the reactive glutaminyl residues from the isolated subunits to FXIIIa

Summary A reconstituted thylakoid system including isolated spinach thylakoids, ferredoxin, thioredoxin andferredoxin-thioredoxin reductase (FTR) was shown to provide sufficient electron transfer flow to mediate photoactivation of sedoheptulose-1,7-bisphosphatase (SBPase). Progress curves were linear under these conditions, in contrast to the lags observed after reductive activation of SBPase by DTT. In this case the lag was suppressed in the presence of ferredoxin + thioredoxin, and addition of FTR to this incubation medium resulted in a substantial increase of SBPase activity. Evidence obtained from gel filtration suggests that during the activation process, incubation of SBPase with ferredoxin, thioredoxin and FTR resulted in enzyme-protein or enzyme-enzyme associations and/or conformational changes.

Wheat or spinach chloroplasts were fed with 35 S-labelled elemental sulphur (S 0 ). 35 S-labelled compounds appearing thereafter were analysed. Five 35 S-compounds were recovered, thiosulphate, sulphite, sulphate, cysteine and glutathione. The effect of environmental conditions was also studied. The measurement of S 0 metabolism rate was compared to the recovery of the introduced S 0 into other S compounds. The specific radioactivity of thiosulphate and sulphate was determined. The results obtained have permitted the elucidation of the precise biochemical pathway of S 0 oxidation. The oxidation of sulphite in wheat and spinach chloroplasts occurs via both non-enzymatic (in particular the operation of the photosynthetic electron transport chain) and enzymatic processes (sulphite oxidase activity).

1. 1. A protein kinase from the lipolytic yeast Yarrowia lipolytica has been purified to nearby homogeneity. 2. 2. The enzyme is able to phosphorylate casein and soybean purified storage proteins (β-conglycinin and glycinin) in the absence of cAMP. 3. 3. Its activity and autophosphorylation are inhibited by very low concentrations of heparin. 4. 4. Its native structure which is a tetramer of 170 kDa composed of two kinds of subunits, α and β (42 and 35 kDa, respectively), β being autophosphorylated. Through ordered aggregation phenomena 1800 and 580 kDa species can also be observed. 5. 5. This enzyme belongs to the casein kinase II family. 6. 6. We describe the use of a protein kinase to phosphorylate easily available reserve proteins. This system is a promising tool to study the specificity of the enzyme, and to find out the link between the phosphorylation, conformational changes, and functional properties.

Selective PCR amplification of human and mouse genomic DNAs with oligonucleotides encoding highly conserved regions of the delta-opioid and somatostatin receptors generated a human DNA probe (hOP01, 761 bp) and its murine counterpart (mOP86, 447 bp). hOP01 was used to screen a cDNA library from human brainstem. A clone (named hORL1) was isolated, sequenced and found to encode a protein of 370 amino acids whose primary structure displays the seven putative membrane-spanning domains of a G protein-coupled membrane receptor. The hORL1 receptor is most closely related to opioid receptors not only on structural (sequence) but also on functional grounds: hORL1 is 49-50% identical to the murine mu-, delta- and kappa-opioid receptors and, in CHO-K1 cells stably transfected with a pRc/CMV:hORL1 construct, ORL1 mediates inhibition of adenylyl cyclase by etorphine, a 'universal' (nonselective) opiate agonist. Yet, hORL1 appears not to be a typical opioid receptor. Neither is it a somatostatin or sigma (N-allylnormetazocine) receptor. mRNAs hybridizing with synthetic oligonucleotides complementary to mOP86 are present in many regions of the mouse brain and spinal cord, particularly in limbic (amygdala, hippocampus, septum, habenula, ...) and hypothalamic structures. We conclude that the hORL1 receptor is a new member of the opioid receptor family with a potential role in modulating a number of brain functions, including instinctive behaviours and emotions.

35 S 0 was offered to wheat intact chloroplasts and 35 S products analysed. Radioactivity in SO 4 2− was determined after precipitation by BaCl 2 . Bimane derivatives of S compounds were separated by HPLC and detected by fluorimetry and radioactivity monitoring. It has been observed that fresh chloroplasts were able to oxidize 35 S 0 to 35 SO 4 2− via the production of 35 S 2 O 3 2− and 35 SO 3 2− . From the characteristics of this oxidation, the operation of chloroplastic enzymatic mechanisms is proposed.

Placental FXIII a catalyzes the formation of e-(γ-glutamyl)-lysine bonds within and between protein molecules. Therefore, this enzyme, activated by calcium, was used to cross-link myosin, actin and 7S globulin. High-concentration myosin and globulin solutions formed gels when they were treated by FXIII a . The breaking strength of globulin gel is greater than that of myosin gel

1. Phanerochaete chrysosporium INA-12 secretes several lignin peroxidase isoenzymes. This paper reports an improved procedure for the purification of the different isoforms compared to those previously described. 2. Lignin peroxidases are first concentrated and prefractionated on fast-flow ion-exchangers which avoid concentration by ultrafiltration and dialysis. 3. Further purification is achieved by hydrophobic interaction chromatography and anion-exchange FPLC. 4. Two major forms were purified to homogeneity. Kinetic measurements and protein characterization (isoelectric points, phosphate content) suggest that they are similar to those produced by P. chrysosporium BKM strain.

1. 1. FXIII was isolated and purified over 4000 fold from human placenta to apparent electrophoretic homogeneity by a new procedure inclucling ethanol precipitation. DEAE-Cellulose, molecular sieving on Sephacryl S-300 and Phenyl-Sepharose chromatography. 2. 2. Its pl was about 5.1. Under appropriate conditions, the incubation of FXIII in the presence of thrombin did not lead to inactivation cut in the polypeptidic chain. 3. 3. FXIII was also activated by CaCI2 and, in a lesser extent, by other divalent cations like SrCl2, Bad; or MgCl2. 4. 4. The bincling of calcium to FXIII exhibited a negative cooperativity. 5. 5. The activity-pH curve of the calcium-activated enzyme did not appear very different from that of the thrombin-activated enzyme.

A method to the study of β-casein proteolysis by aspartic proteinases is developed. The 3% trichloroacetic acid-soluble peptides of β-casein digested with cardosin A were separated by capillary zone electrophoresis under different experimental conditions in an uncoated fused silica capillary. The best separation was at pH 3.01, 30 kV and 30 °C.

Digitonin treatment of frog brain membranes in 50 mM Tris-HCl yields a soluble extract that contains nearly equal amounts of free and G protein-bound opioid receptor molecules (Mollereau et al., 1988, J. Biol. Chem. 263, 18003). We report here that the balance of the two forms of the opioid receptor in digitonin solution is dependent on the environment of the membrane suspension at the time of solubilization with the detergent. Preincubating the membrane suspension with 50 microM GppNHp or with 120 mM NaCl results, in the two cases, in a digitonin extract that no longer displays the G protein-bound form of the receptor, i.e., the form of the receptor which exhibits high affinity for the opiate agonist etorphine in binding studies, as well as large apparent molecular size in sucrose gradients. Assuming that the situation in soluble extracts faithfully reflects the one in the membrane, these results would exclude the possibility that in a physiological environment the opioid receptor is in part precoupled with a G protein in the absence of an opioid agonist.

The influence of various additives such as polyhydric alcohols, sugars, polyethylene glycol, and substrates on the thermostability of Bacillus licheniformis α-amylase has been studied. Results obtained showed a thermostabilizing effect of most of the additives tested (ethylene glycol, glycerol, sorbitol, glucose, maltose, maltotriose, and polyethylene glycol). The stabilizing efficiency appears to be strongly dependent on additive concentration. α-Amylase thermostability has also been assayed in organic solvents. In these media, particularly in octane, α-amylase acquired a remarkable stability since no deactivation occurred at 75°C for at least 90 min .

The synthesis of aryldiazonium and arylazido derivatives of carfentanil, etonitazene, and naltrexone and of a triazaspirodecane derivative is described. The chemical stability and the spectral characteristics of these compounds were verified, and their binding affinity constants for the different opioid receptor classes were determined, in the absence of light, from competition experiments. With the exception of the naltrexyl derivatives, which remained nonselective, all compounds tested displayed a pronounced mu-binding selectivity with mu/delta and mu/kappa ratios ranging from 12 to 1000. After irradiation, only the arylazido probes led to an irreversible mu-binding-site inactivation. This inactivation fulfilled the criteria for photoaffinity labeling such as protection against inactivation by other opiate ligands and absence of an effect of scavengers on the extent of the inactivation. Most of the photoactivatable probes formed long-lasting reversible complexes with the opioid binding sites: an efficient dissociation procedure was thus required to discriminate between pseudoirreversible and covalent complexes. The marked differences in labeling efficacy between aryldiazonium salts and their corresponding arylazido derivatives are discussed.

Rabbit and guinea-pig cerebellum membranes contain a very high (greater than 80%) proportion of mu- and kappa-opioid receptors, respectively. Rabbit (mu) and guinea-pig (kappa) cerebellum membranes were (i) labeled either with the opiate agonist, [3H]etorphine (Kd = 0.1-0.2 nM), or with the opiate antagonist, [3H]diprenorphine (Kd = 0.1 nM), in the absence or presence of Na+ and/or 5'-guanylylimidodiphosphate (GppNHp), (ii) solubilized with digitonin (1%, w:v) and (iii) the radioactivity in the soluble extracts analyzed by ultracentrifugation in sucrose gradients. In the soluble extracts from rabbit cerebellum (mu) membranes, bound [3H]etorphine sedimented faster (S20,w congruent to 12S) than bound [3H]diprenorphine (10S), while in those from guinea-pig cerebellum (kappa) membranes, bound [3H]etorphine and bound [3H]diprenorphine sedimented at the same position (12S). Na+ selectively decreased recovery of the bound tritiated agonist in the two soluble preparations. When they had been generated in the presence of GppNHp but in the absence of Na+, the [3H]etorphine complexes of the mu- and kappa-opioid receptors as well as the [3H]diprenorphine complex of the kappa-opioid receptor were all recovered at position 10S, indicating that GppNHp had induced a decrease of the apparent molecular size of the two types of opioid receptors. These data are interpreted in terms of mu- and kappa-opioid receptors being capable of physically interacting with a G protein (GTP binding regulatory protein) yet, unlike the mu-opioid receptor which does so only in the presence of an agonist, the kappa-opioid receptor appears to be precoupled with a G protein.

The putative regulatory effect of opioids on adenylate cyclase was investigated in two different preparations containing, respectively, two different populations of opioid receptors: the rabbit cerebellum (greater than 75% mu-opioid receptors) and the guinea pig cerebellum (greater than 80% kappa-opioid receptors). In the mu-preparation, but not in the kappa-preparation, opioids inhibited the basal and the forskolin-stimulated adenylate cyclase activity in a dose-dependent manner and stereospecifically. The inhibition was in the 20-30% range, required the presence in the assay medium of Mg2+ and of GTP, but was independent of the presence of Na+. Pharmacological characterization of the inhibitory response in the rabbit cerebellum clearly showed that it was under the control of a mu-opioid binding site, with the effect being elicited by non-selective (etorphine and morphine) and mu-selective (Tyr-D-Ala-Gly-Me-Phe-Gly-ol) agonists, whereas delta- and kappa-selective agonists were almost totally ineffective. ADP ribosylation of inhibitory GTP-binding protein by pertussis toxin failed to block the inhibitory effect of opioids, and data presented suggest that this failure is likely to be the consequence of a limited access of the toxin to its substrate in rabbit cerebellum membranes.

The oxidized form of spinach chloroplast fructose-1,6-bisphosphatase is devoid of activity under the H+ and Mg2+ concentrations which prevail in the illuminated chloroplast stroma. In contrast, the reductive activation of this enzyme form results in activity under these conditions. In this paper we demonstrate that preincubation of the oxidized enzyme in the presence of Ca2+/fructose-1,6-bisphosphate gives rise to an active enzyme under these same conditions. The activation appears to be fast at pH 8 and the final level of activity is roughly of the same order of magnitude as that of the enzyme reductively activated by thioredoxin. The ph/activity profile of the activated oxidized enzyme also resembles that of the reduced enzyme.

Background Opiate addiction reflects plastic changes that endurably alter synaptic transmission within relevant neuronal circuits. The biochemical mechanisms of these adaptations remain largely unknown and proteomics-based approaches could lead to a broad characterization of the molecular events underlying adaptations to chronic drug exposure. Results Thus, we have started proteomic analyses of the effects of chronic morphine exposure in a recombinant human neuroblastoma SH-SY5Y clone that stably overexpresses the μ-opioid receptor. Cells were treated with morphine for 6, 24 and 72 hours, the proteins were separated by 2-D gel electrophoresis and stained with Coomassie blue, and the protein map was compared with that obtained from untreated cells. Spots showing a statistically significant variation were selected for identification using mass spectrometric analyses. Conclusion A total of 45 proteins were identified, including proteins involved in cellular metabolism, cytoskeleton organization, vesicular trafficking, transcriptional and translational regulation, and cell signaling.

The heptadecapeptide nociceptin (NOP; FGGFT-GARKSARKLANQ) was discovered as the natural ligand of the NOP receptor, the fourth cloned member of the opioid receptor family. NOP is derived from a larger precursor polypeptide (prepro-NOP) which is widely distributed in the nervous system. NOP is primarily an inhibitory neuropeptide that acts on neurons to depress synaptic transmission. NOP is reported to modulate pain, stress and anxiety, feeding, learning and memory, reward and addiction, and the functioning of the heart and vessels, airways, kidney, urinary bladder, intestine, and immune cells. The broad spectrum of pharmacological effects of NOP suggests potential therapeutic applications for NOP receptor agonists and/or antagonists.