Physical characterization of native opiate receptors. Additional information from detailed binding analysis of a radiation-inactivated receptor.

Target size analyses of the etorphine receptor were performed on frozen rat brain P2 homogenates using the radiation inactivation technique. Multi-point saturation curves at each radiation dose revealed that the apparent dissociation constant for the binding of this ligand to its receptor is a function of the dose. Analysis of the results shows clearly that the ligand-binding macromolecule is functionally coupled to at least one other macromolecule. When the coupling is destroyed the ligand dissociation constant becomes larger by over an order of magnitude. Thus, the variation of the dissociation constant with dose yields interesting new information on the nature of the native receptor which has implications with respect to the conformation of the binding site and to solubilization and cloning methods directed towards sequencing the ligand-binding component of opiate receptors.

Nuclear magnetic resonance studies of flexible opiate conformations at monoclonal antibody binding sites. Quantitative interproton distances obtained from comparing theoretical and experimental transferred nuclear Overhauser enhancement: correlation with antibody sequence.

A previous publication described the use of qualitative intramolecular 1H-transferred nuclear Overhauser effect measurements to determine the conformations of flexible ligands at monoclonal anti-opiate antibody binding sites. This paper concentrates on the quantitative interpretation of experiments of this type using the ligand nalorphine (N-allyl morphine) and a single anti-opiate monoclonal antibody. I compare the experimental unidimensional driven nuclear Overhauser effect buildup curves to theoretical curves derived with a knowledge of the fixed interproton distances in the ligand. The discussion covers the potential accuracies of derived distances and concentrates on two problem areas associated with determining structures from this type of experiment. The most serious one is the case where, because of particular multiproton spatial distributions, spin diffusion is so rapid that it cannot be determined experimentally and where numerical fits of theoretical calculations are misleading. The results show that, while intraligand spin diffusion complicates the interpretation for some proton pairs, with many others accuracies within about 0.3 A for interproton distances from 2 to 4 A are attainable. The results confirm the earlier report that the conformation of nalorphine in this antibody binding site differs from the major one present in solution or in the crystal. An important aspect of the work is that theoretical prediction of nuclear Overhauser effect time-dependence is an important practical tool for recognizing cases where interpretation of experiments will be difficult. Initial data on protein-to-ligand transferred nuclear Overhauser effect are presented, which show that at least one aromatic amino acid residue is closely involved in the binding of the ligand. The companion paper presents the primary sequences of the variable regions of the antibodies being used in our studies. In this paper, these and associated immunochemical studies are correlated with the nuclear magnetic resonance results. The combination of data presented in the two papers provides a basis for future work on protein-ligand interproton distances in the range 1 to 5 A using both transferred nuclear Overhauser effect (for rapidly exchanging ligands) and isotope-edited, indirectly detected nuclear Overhauser effect (for tightly bound ligands).

Comparative sequence and immunochemical analyses of murine monoclonal anti-morphine antibodies.

A more complete characterization is given for four previously reported anti-morphine monoclonal antibodies that bind the hapten with high affinity and to which anti-idiotypic antibodies have been raised that mimic opiates at receptor binding sites. The variable (V) region nucleotide sequences of the heavy (H) and light (L) chains of these murine antibodies have been determined by direct sequencing of the poly(A)+ mRNAs using specific oligonucleotide primers and dideoxynucleotide chain-termination, and the deduced amino acid sequences are compared. The primary sequences predicted for the VH segments of 10C3 and 11C7 antibodies are closely associated with the VHIIIB subgroup of mouse H-chain (80 to 82% homology), while those for the V-regions of 3B9 and 12D4 H-chains correlate well with the VHIIC subgroup (64 to 67% homology). The 11C7, 10C3, 3B9 and 12D4 hybridoma cell lines use JH1, JH2, JH3 and JH4 DNA segments, respectively. Since considerable variations in length and primary sequence in the CDR3 (complementarity determining region) peptides of all the H-chains are evident, conservation of the D-region structure does not appear to be necessary for effective hapten binding. However, sequence homologies of the CDR2 regions of all the antibodies indicate that residues Glu H-50, Ile H-51, Pro H-52a and Tyr H-59 are conserved, and that these segments may be more critically involved in binding than the other H and L-chain hypervariable regions. The marked VL sequence homology, greater than 93%, among the L-chains and consensus lambda sequence, suggests derivation from a similar or identical VL germ-line gene. The L-chain J-region peptides for all the antibodies are classified JL1 and no VL-JL junctional diversity was apparent. The antimorphine antibody L-chains are apparently generated by the joining of a specific J-gene segment to a single germ-line V-gene segment, and minor sequence variations are the result of somatic mutations within the coding region. The leader sequence for one of the H-chains was determined. The inhibition of morphine binding by phenoxybenzylation or iodination of the affinity-purified immunoglobulins indicates the involvement of a single tyrosyl residue within or close to the antibody-combining site for the opiate. This conclusion is supported by the sequence data and nuclear magnetic resonance measurements reported in the accompanying paper, in which the results are used to interpret nuclear magnetic resonance measurements on one of the ligand-antibody systems. The possible importance of additional contact amino acids, tryptophan, aspartic and/or glutamic acids, is discussed.

Direct production of Fv-fragments from a family of monoclonal IgGs papain digestion.

Fv fragments of four monoclonal antibodies specific for morphine binding have been produced from their divalent IgG forms by papain digestion using the classic procedure for Fab formation. The binding characteristics of one of the Fv fragments have been determined relative to the intact antibody by equilibrium dialysis. Its dissociation constant is a factor of five lower than the IgG. Previous work had resulted in the sequences of each the chains for the four Fv fragments. The light chains are all from the highly homologous lambda subclass while the gamma heavy chains are closely related except for their CDR regions. In this work optical molar extinction coefficients are predicted from amino acid sequences for each of the fragments. It is found that they differ significantly from each other and from the commonly used value for intact IgG. Detailed comparisons between our results and those reported previously on the molecular masses of Fv-derived light and heavy chains and hapten-Fv dissociation constants are given based on analytical gel electrophoresis and electroblotting experiments using dye and immunovisualization techniques. Isoelectric focusing experiments have been performed and the pIs obtained are compared to those predicted theoretically from the chain sequences. Gel filtration column chromatography, acrylamide gel electrophoresis and equilibrium dialysis experiments are consistent with significant aggregation of the Fv fragments in neutral solution with accompanying inactivation of the binding site. Comparison of sequences for the Fv light and heavy chains are made with those which have been proposed to be important for chain dimer association and for canonical hypervariable regions. This methods of Fv production is not regarded as a general one. However, it may be an approach which is general to lambda chain containing antibodies.

Properties of murine anti-morphine antibodies.

Four lines of high affinity monoclonal antibodies directed against morphine have been isolated and affinity purified. Some of their properties, including cross-reactivities to a large set of selected opiate agonists and antagonists are described. Importantly, none of the immunoglobulins cross-react with D-Ala2-D-Leu5-enkephalin.

Differential effects of opioid and adrenergic agonists on proliferation in a cultured cell line.

A novel clonal cell line transfected with the delta-opioid receptor (delta-OR) encoding gene was used to study agonist-activated regulation of cell proliferation. In this cell line, endogenous beta2-adrenergic receptors (beta2-ARs) are coexpressed with the exogenous delta-ORs. Upon individual acute treatments with morphine and procaterol (a selective beta2-AR agonist), both the delta-OR and beta2-AR are coupled to differential modulation of cyclic AMP (cAMP) levels in accord with the classical second messenger response patterns to these agonists in the normal cellular settings of the receptors. But chronic morphine activation of the delta-OR inhibits cellular proliferation, while chronic procaterol activation of the beta2-AR stimulates it. Chronic treatment with the individual agonists is accompanied by differential activation of the mitogen-activated protein kinase (MAPK) isozymes, extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). The findings suggest that chronic beta2-AR activation stimulates proliferation by interacting with the ERK signalling cascade independent of a cAMP-mediated pathway. In contrast to treatment with individual agonists, chronic dual agonist treatment suppresses procaterol-induced stimulation of ERK activity and stimulation of proliferation indicating that a cross-regulatory interaction occurs between the delta-OR and beta2-AR signalling systems in the cells under these conditions.

A glass-fiber filtration assay for solution phase hapten-antibody binding. Effect of surface treatment with a polycation.

Radioligands bound to murine monoclonal and polyclonal antibodies in solution are rapidly and effectively separated from free ligand by filtration through either untreated or polyethylenimine (PEI)-pretreated glass-fiber filters. Ligand binding to selected anti-morphine monoclonal antibodies, determined by filtration through untreated filters, was significantly greater (1.5-3.0-fold) than the binding activities obtained by gel filtration. However, radioactivity retained by filters that were pretreated with PEI (pH 4) was essentially the same as that obtained using the Sephadex G-25 short-column assay. The pH dependence of the retention on the coated fibers suggests that the mechanism of binding of antibodies to glass is different from that to the PEI-treated surfaces. The quantitative aspects of the assay are reported. The method should prove useful where quantitative, rapid and inexpensive binding radioassays need to be performed.

A rapid binding assay for immunoglobulins which may be used as an alternative to ammonium sulfate precipitation methods.

This paper describes the conditions under which a short column packed with Sephadex G-25 may be eluted centrifugally to separate labeled ligands from binding immunoglobulins in a manner which can yield quantitative binding information. This method may be used as an alternative to the classical binding assay involving ammonium sulfate precipitation. The advantage is a large saving in total assay time over that procedure. The column assay is applicable to antisera and purified polyclonal and monoclonal immunoglobulins. The restrictions are that the labeled ligands must be of molecular weight less than approximately 5000 Da.

Two- and three-dimensional distributions of opioid receptors on NG108-15 cells visualized with the aid of fluorescence confocal microscopy and anti-idiotypic antibodies.

Polyclonal anti-idiotypic antibodies previously shown to be specific for mu- and delta-opioid receptor subclasses on rat brain membrane preparations using radioreceptor assays have been labeled with fluorescent conjugates of avidin. The resulting complexes have been used to study the distribution and properties of opioid receptors on living and fixed NG108-15 cells using normal fluorescence, and confocal fluorescence, microscopy. From the confocal data three-dimensional views of immunofluorescent distributions have been obtained using a computer transformation of the data. From experiments involving co-incubation with media containing naloxone, selective opioid peptides and stereoisomeric synthetic opioid analogs as blocking agents, the specificity and stereospecificity of immunofluorescent binding is reported. The effects on immunofluorescent binding by co-incubation with media containing a sulfhydryl reagent, 100 mM-NaCl and sodium azide are also reported. The results have implications concerning the use of immunoprecipitation in the biochemical isolation of the receptors. Finally, the confocal two-dimensional sections and two-dimensional reconstructions obtained from fixed cells show internal immunofluorescence in the form of cytoplasmic clusters. The reagents and methods should be applicable to visualizing native opioid receptor distributions on neurons.

The effects of morphine on cell proliferation.

There is increasing evidence that endogenous opioid peptides ("enkephalins") and other neurotransmitters have widespread, receptor-mediated roles as growth regulators in non-neuronal cells and tissues. For example, it is now believed that enkephalins produced in placental trophoblast giant cells have multiple roles in supporting embryo growth, and in maternal adaptation to pregnancy. Since plant and synthetic narcotics (e.g., morphine) bind to the same receptors, the questions immediately arise: Do narcotics also have actions as growth regulators? If so, do these actions have physiological significance in addicts? Recent work on the first of these questions is covered in this review. While the greatest volume of research has been focused on the proliferative effects of narcotics for cells of the immune system, the roles of opioid peptides and narcotics on the growth of a variety of other cells has come under study recently.

From outer to inner space: traveling along a scientific career from astrochemistry to drug research.

This professional history describes my journey as a research scientist after my early training and experiences in the pre- and early post-World War II United States. My graduate training concentrated on a problem in astrochemistry: phenomena on comets. As my career developed, I felt confident enough in myself as an experimentalist to enter, and make contributions to, several different fields: structural biochemistry (via nuclear magnetic resonance spectroscopy), molecular immunology, pharmacology, neurochemistry, and cell biology. One emphasis is on the nature and quality of my scientific training that permitted me to do cross-disciplinary work. A second emphasis is on the technical and intellectual developments in late twentieth-century science and how, along with the changes in American society as it passed through three major wars, they influenced my life and thought.

Subclass specificity of anti-idiotypic anti-opiate receptor antibodies in rat brain, guinea pig cerebellum, & neuroblastoma x glioma (NG 108-15).

Receptor subclass recognition properties of affinity-purified rabbit polyclonal anti-idiotypic anti-opiate receptor antibodies in various membrane preparations have been determined. The anti-receptor immunoglobulins significantly decrease binding of 3H-[D-Ala2,-MePhe4,Gly-ol5]enkephalin, a highly selective mu agonist, in rat neural membrane. In the presence of a concentration of the unlabeled ligand sufficient to block existing mu sites, the antibodies compete, to a lesser degree with 3H-[D-Ala2,D-Leu5]enkephalin for delta site occupancy in both rat neural membrane, and neuroblastoma x glioma membrane preparations. The antibodies do not displace 3H-ethylketocyclazocine from rat brain or guinea pig cerebellum.

Co-localization of mu and delta opioid receptors on SK-N-SH cells detected by fluorescence microscopy using labeled anti-idiotypic antibodies.

Selective fluorescence labeling of opioid receptor subclasses on SK-N-SH cultured cells has been accomplished using labeled polyclonal anti-idiotypic antibodies along with subclass-selective opioid agonists (DPDPE, delta-selective; DAMGO, mu-selective) as blocking reagents. Labeling of the cells was examined using conventional fluorescence microscopy. Co-localization of mu- and delta-opioid receptors on SK-N-SH cells has been studied by double labeling fluorescence experiments. In agreement with our own, and other workers', previous observations on NG108-15 cells, a subpopulation of viable cells in asynchronous cultures are labeled. Among those SK-N-SH cells that are labeled, both subclasses of receptors are seen. On the basis of sequential blocking experiments we interpret our combined results to be consistent with a model where mu- and delta- binding sites reside on different subunits of a multimeric complex.

Theoretical analysis of a morphine withdrawal phenotype in a cultured cell line.

We have previously described a delta-opioid receptor-expressing cultured cell line that proliferates in a defined medium and responds to chronic morphine treatment with an inhibition of its rate of proliferation. To help provide an explanation for this behavior, we have used computer simulation of cell cycle kinetics to analyze the observed rates of proliferation of these cells in the presence and absence of morphine, and after withdrawal of morphine treatment. We questioned whether the difference in cell kinetics observed for the cell populations under the different treatments could be due to changes in the length of the cell cycle, withdrawal of cells from the cycle into a quiescent state, or differences in cell renewal. This was investigated by comparing observed cell numbers as a function of time with the results of different computer simulations using different values for these parameters. We found that we can provide a satisfactory explanation of the experimental observations on the basis of changes in a small set of parameters: Untreated cells experience a slowdown of cell proliferation at about the culture density where multiple cell-cell contacts are made and, beginning then, a large fraction are shunted from G1 into a quiescent state. Chronic morphine treatment inhibits proliferation by slowing passage through G1, but the cells remain as sensitive to cell-cell contacts as the untreated cells. After drug withdrawal following a 6 day treatment with morphine, the cells exhibit a large temporary increase in their rate of proliferation compared with control or chronically treated cells but about 48 hours after withdrawal, when cell-cell contacts just begin to be made, the cells return to almost their pre-treatment total cell cycle time and, as before, a large fraction are shunted into a quiescent state. Taken in conjunction with previously published results, the present ones indicate a possible interaction between morphine-induced and insulin-induced nuclear signaling pathways to the nucleus.

Rabbit anti-idiotypic antibodies raised against monoclonal anti-morphine IgG block mu & delta opiate receptor sites.

Rabbit antibodies have been raised against murine monoclonal anti-morphine Fab fragments. Following affinity purification, these antibodies competitively inhibit morphine binding to anti-morphine monoclonal antibodies, yet do not recognize normal mouse IgGs, suggesting that the antibodies produced are anti-idiotypic with respect to the anti-morphine IgG. More importantly, the purified antibodies competitively inhibit binding of morphine, naloxone, and D-ala-2-D-leu-5-enkephalin to rat brain opiate receptors.

Opiate binding to subcellular fractions from guinea pig ileum.

Binding of 3H-etorphine and 3H-D-Ala2-D-Leu5-enkephalin to opiate receptors in synaptosomal and microsomal fractions prepared from guinea pig ileum homogenates has been studied. It is found that the dissociation constants for etorphine from all fractions are the same. The binding capacity for etorphine for the purified synaptosomal fraction is greater than for other fractions by a factor of 5. For the enkephalin derivative binding to the microsomal fraction the dissociation constant is greater than for etorphine while the binding capacity is a factor of 3 lower. These results are in contrast to the case for binding to central nervous system subcellular fractions.