The ORL1 receptor, an orphan receptor whose human and murine complementary DNAs have recently been characterized, structurally resembles opioid receptors and is negatively coupled with adenylate cyclase. ORL1 transcripts are particularly abundant in the central nervous system. Here we report the isolation, on the basis of its ability to inhibit the cyclase in a stable recombinant CHO(ORL1+) cell line, of a neuropeptide that resembles dynorphin A9 and whose amino acid sequence is Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln. The rat-brain cDNA encodes the peptide flanked by Lys-Arg proteolytic cleavage motifs. The synthetic heptadecapeptide potently inhibits adenylate cyclase in CHO(ORL1+) cells in culture and induces hyperalgesia when administered intracerebroventricularly to mice. Taken together, these data indicate that the newly discovered heptadecapeptide is an endogenous agonist of the ORL1 receptor and that it may be endowed with pro-nociceptive properties.

Selective PCR amplification of human and mouse genomic DNAs with oligonucleotides encoding highly conserved regions of the S-opioid and somatostatin receptors generated a human DNA probe (hOPO1, 761 bp) and its murine counterpart (mOP86, 447 bp). hOPO1 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 ,u-, 6-and rc-opioid receptors and, in CHO-Kl cells stably transfected with a pRc/CMV:hORLl construct, ORLl 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 o (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.

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A new concept, that of a mnemonical transition, is proposed to explain the departure from Michaelian behavior of monomeric enzymes following ordered reaction mechanisms. The concept integrates three simple ideas : the free enzyme occurs under two conformational states in equilibrium ; the collision of any of these forms with the first substrate induces the same third new configuration required for proper substrate binding; the collision of only one of these enzyme forms with the last product stabilizes that form without any new conformational change. This whole set of definitions is equivalent to assuming that the free enzyme which is released after catalysis, is in a conformation different from the initial one. The enzyme can be said to "recall" for a while the configuration stabilized by the last product before relapsing to the initial conformation. The non-hyperbolic behavior is thus the consequence of the cooperation of two different conformations of the free enzyme to the overall reaction process.The reciprocal steady-state rate equations have been established and thoroughly discussed both for one-substrate, one-product and two-substrate, two-product mnemonical enzymes. The departure from Michaelian behavior does not appear as a consequence of a slow conformational transition, but is defined in a simpler way by the relative values of the activation free energies of conformation changes required for substrate binding on the two enzyme forms.A two-substrate, two-product enzyme following an ordered reaction mechanism and exhibiting the mnemonical transition has a very distinctive kinetic behavior. The curvature of the primary plots is observed with regard to the first substrate only, and is independant of the concentration of the second substrate as well as that of the first product. The enzyme is not inhibited by an excess of the substrate and the primary plots are either concave up or down. The slopes and the intercepts of the straight lines obtained in double reciprocal plots with the second substrate should give, when these are replotted against the reciprocal of the first substrate concentration, a straight line and a curve, respectively. The cooperation of the enzyme conformations to the overall reaction process can be either positive or negative. Since the reciprocal plots cannot exhibit an extreme, the extent of that cooperation can be measured by the numerical value of the second derivative of the reciprocal rate equation. The extent of cooperation between the free enzyme forms is highly controlled by the concentration of the last product. If the cooperation was already negative, the product strengthens that cooperation. If, on the other hand, the cooperation was positive, the product decreases or even reverses that cooperation.A very general property of one-sited mnemonical enzymes is that cooperation between enzyme forms is only kinetic and does not appear in the substrate binding isotherms.It is well known that the random binding of subof asymptotic or degeneracy conditions of the recipstrates on a...

Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p2l), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p2l region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig. Nociceptin (1) or orphanin FQ (2), a newly discovered neuropeptide of 17 amino acids, is the natural agonist of the opioid receptor-like (ORL1) receptor, a G protein-coupled receptor whose human (3) and murine (4-10) cDNAs had been identified previously.The ORL1 receptor is most closely related to opioid receptors in terms of primary structure ('60% homology), yet its in vitro pharmacological profile is clearly not opioid. When expressed in CHO cells, ORL1 responds to high (micromolar)

The irreversible thermal inactivation of Bacillus licheniformis alpha-amylase was studied. A two-step behaviour in the irreversible denaturation process was found. Our experimental results are consistent only with the two-step model and rule out the two-isoenzyme one. They suggest that the deactivation mechanism involves the existence of a temperature-dependent intermediate form. Therefore the enzyme could exist in a great number of active conformational states. We have shown that Ca2+ is necessary for the structural integrity of alpha-amylase. Indeed, dialysis against chelating agents leads to a reversible enzyme inactivation, though molecular sieving has no effect. Further, the key role of Ca2+ in the alpha-amylase thermostability is reported. The stabilizing effect of Ca2+ is reflected by the decrease of the denaturation constants of both the native and the intermediate forms. Below 75 degrees C, in the presence of 5 mM-CaCl2, alpha-amylase is completely thermostable. Neither other metal ions nor substrate have a positive effect on enzyme thermostability. The effect of temperature on the native enzyme and on one intermediate form was studied. Both forms exhibit the same optimum temperature. Identical activation parameters for the hydrolytic reaction catalysed by these two forms were found.

A site-directed mutagenesis approach has been used to gain insight into the molecular events whereby the heptadecapeptide nociceptin binds and activates the opioid receptor-like 1 (ORL1) receptor, a G protein-coupled receptor. Alanine mutation, in the human ORL1 receptor, of transmembrane amino acid residues that are conserved in opioid receptors, Asp(130) and Tyr(131) in transmembrane segment (TM) III, Phe(220) and Phe(224) in TM V, and Trp(276) in TM VI, yields mutant receptors with reduced affinity, and proportionally decreased reactivity, toward nociceptin. Least to most deleterious in this respect are Ala substitutions of Phe(220) approximately W276A < Tyr(131) << Phe(224) 10,000 nM compared with 0.8 nM at the wild-type receptor). In all respects, this mutant receptor appears to be functionally inactive, indicating that residue Gln(286) may play a pivotal role in ORL1 receptor-mediated transduction of the nociceptin signal.

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