An electron spin resonance study of synaptosome opiate receptors. The preparation and use of a spin labeled morphine

Copeland, E.S.; Boykin, Michael E.; Kelley, James A.; Kullberg, Max

doi:10.1016/s0006-3495(75)85889-9

Cited by 9 publications

(4 citation statements)

References 19 publications

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“…1 were observed for samples containing 1 mM dextrorphan, levorphanol, or nalorphine in 0.32 M sucrose (pH 7.5) with synap-2Reduction in line amplitude and binding are used interchangeably according to hypotheses developed in Ref. 1. Sample calculations SSB = k$[h(-I)f]ev -[h( l)]f dex = 9.68(0.6) = 5.8 pmol/mg SSBR = BRdeX -BR1e, = 36.5 -27.4 = 9.1 pmol/mg/min * Data taken from the experiment described in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The slope could thus be validly determined without synaptosomes. A calibration factor, k, was then derived from the slope and the synaptosomal protein concentration, k = [(cm/pmol/ml) (mg/ml)]' = pmol/mg/cm, (1) for use in expressing line amplitude decreases in terms of picomoles SLM bound per milligram of synaptosomal protein (vide infra). The calibration factor was dependent on observation temperature and on the nature of the spin label moiety on morphine.…”

Section: Methodsmentioning

confidence: 99%

“…We have recently reported certain benefits to be gained from the use of a spin labeled opiate probe in studies of the opiate receptor (1). Thus, the ability of the technique to differentiate bound and free probe simultaneously, without sample destruction or modification, seems to circumvent one great limitation of conventional radiolabel probe studies of the opiate receptor.…”

Section: Introductionmentioning

confidence: 99%

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Electron spin resonance study of the synaptosome opiate receptor: kinetics of stereospecific binding of spin labeled morphine

Copeland¹,

deBaare²

1976

Biophysical Journal

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Morphine, spin labeled on the 3- or 6-position has been used as the opiate ligand in a study of the time course of stereospecific opiate binding to intact synaptosomes isolated from non-cerebellar rat brain. The broadening of electron spin resonance lines induced by immobilization of the ligand on binding has been used to determine the concentration of bound opiate. The stereospecificity of the reaction was measured by comparing ligand binding in the presence of thousand-fold molar excesses of dextrorphan or levorphanol. Using both static and flow techniques, the binding process has been continuously monitored at times greater than 4.8 s after mixing spin labeled morphine with synaptosomes. It is shown that for this ligand and receptor preparation, binding takes place primarily during a delayed, abrupt process whose rate and time of onset are temperature dependent and reflect the presence of added opiate agonist or antagonist.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Electron spin resonance study of the synaptosome opiate receptor: kinetics of stereospecific binding of spin labeled morphine

Copeland¹,

deBaare²

1976

Biophysical Journal

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“…A synthetic morphine analogue was the first active G protein-coupled receptor ligand to contain a nitroxyl moiety (38). Later, a spin-labeled peptide hormone, ␣-melanocyte stimulating hormone, was synthesized and shown to be fully active (39).…”

Section: Fig 6 Epr Scans Of the Different Binding Conditions Of Spimentioning

confidence: 99%

Probing the Binding Pocket and Endocytosis of a G Protein-coupled Receptor in Live Cells Reported by a Spin-labeled Substance P Agonist

Shafer

Bennett

Kim³

et al. 2003

Journal of Biological Chemistry

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To probe the molecular nature of the binding pocket of a G protein-coupled receptor and the events immediately following the binding and activation, we have modified the substance P peptide, a potent agonist for the neurokinin-1 receptor, with a nitroxide spin probe specifically attached at Lys-3. The agonist properties and binding affinity of the spin-labeled substance P are similar to the native peptide. Using electron paramagnetic resonance (EPR) spectroscopy, the substance P analogue is capable of reporting the microenvironment found in the binding pocket of the receptor. The EPR spectrum of bound peptide indicates that the Lys-3 portion of the agonist is highly flexible. In addition, we detect a slight increase in the mobility of the bound peptide in the presence of a non-hydrolyzable analogue of GTP, indicative of the alternate conformational states described for this class of receptor. The down-regulation of neurokinin-tachykinin receptors is accomplished by a rapid internalization of the activated protein. Thus, it was also of interest to establish whether spin-labeled substance P could serve as a real time reporter for endocytosis. Our findings show the receptor agonist is efficiently endocytosed and the loss of EPR signal upon internalization provides a real time monitor of endocytosis. The rapid loss of signal suggests that endosomal trafficking vesicles maintain a reductive environment. Whereas the reductive capacity of the lysosome has been established, our findings indicate this capacity in early endosomes as well.The neurokinin-1 receptor (Nk1r) 1 is a neuropeptide receptor belonging to the G protein-coupled receptor superfamily (1). The receptor, also designated the substance P or tachykinin 1 receptor, has been implicated in numerous physiological functions, which include modulatory roles in the central nervous system and proinflammatory action (for review see Refs. 2 and 3). Although much debate still exists as to which neurokinin represents the physiological ligand for the receptor, the substance P neurokinin has been identified as the primary ligand (4).Because of the significant pathophysiological role of Nk1r, extensive pharmacological research has been carried out to characterize both agonist and antagonist binding properties of the receptor. Previously, extensive mutagenesis of the receptor identified separate binding sites for substance P and the antagonist CP-96,345 (5-9). These studies indicated that substance P interacts almost exclusively with the NH 2 -terminal extracellular residues, with little involvement by residues located in the membrane-spanning domain. The same studies revealed that the antagonist appeared to bind deeply into the protein, within a pocket formed by the transmembrane helices.Although site-directed mutagenesis identified residues important for ligand binding, it cannot differentiate side chains that have a direct coordination of the ligands from residues involved indirectly, such as through allosteric communication. For example, a Y216A substitution located nea...

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Preparation of Spin-Labeled Opiates; Morphine and Codeine

Cella

Kelley

1977

Journal of Pharmaceutical Sciences

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An electron spin resonance study of synaptosome opiate receptors. The preparation and use of a spin labeled morphine

Cited by 9 publications

References 19 publications

Electron spin resonance study of the synaptosome opiate receptor: kinetics of stereospecific binding of spin labeled morphine

Electron spin resonance study of the synaptosome opiate receptor: kinetics of stereospecific binding of spin labeled morphine

Probing the Binding Pocket and Endocytosis of a G Protein-coupled Receptor in Live Cells Reported by a Spin-labeled Substance P Agonist

Preparation of Spin-Labeled Opiates; Morphine and Codeine

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