Jay A. Glasel

Deuteron magnetic resonance studies on the microdynamical behavior of partially deuterated oxytocin with neurophysin

Abstract With the use of specifically deuteron-labeled oxytocin and related molecules, deamino oxytocin and prolyl-leucyl-glycinamide, some of the intramolecular micro-mica of the free molecules and their interactions with their biological carriers, the neurophysins, have been studied. The work involves contemporary synthetic and isolation chemical techniques and computer processed nuclear magnetic resonance spectroscopy. The results show that there is considerable intramolecular motion in the “tail” of oxytocin with respect to the remainder of the molecule and that there is a rapid dynamical equilibrium between neurophysin-bound and free hormone in solution. The implications of this work on the biological action of the hormone-protein complex are discussed as well as the interpretation of high resolution nuclear magnetic resonance data in the light of these results.

Systematic elimination of RF pulse defect errors in Fourier transform spin-lattice relaxation measurements

Abstract The elimination of rf pulse defect errors in Fourier transform spin-lattice relaxation measurements is discussed. The standard 180°−τ−90°−t pulse sequence is used. However, the phase of the 90° pulse is shifted by 180° on every repetition of the sequence, while the phase of the 180° pulse is unaltered. Implementation of this phase shifted pulse sequence is discussed for a JEOL PFT-100 single phase detector spectrometer.

360 MHz proton NMR spectra of active and inactive derivatives of methionine-enkephalin. Assignments, derived parameters, conformational implications

Abstract The proton NMR spectra in aqueous and dimethylsulfoxide solutions have been obtained at 360 MHz for: methionine-enkephalin, leucine-enkephalin, (Phe 1 )-methionine-enkephalin, norleucine-enkephalin, and des-amino-methionine-enkephalin. Resonance assignments, derived chemical shifts, spin coupling constants, and amide proton temperature dependencies (in dimethylsulfoxide) are presented.

A comparison of solution, solid state and theoretical conformations of morphine.

Summary Using very high frequency 1 H nuclear magnetic resonance, nitrogen inversion in the N-substituted piperidine ring of a fused polycyclic molecule is demonstrated for the first time. The molecule discussed is the potent opiate agonist morphine where the piperidine ring is known to be of vital importance in receptor mediated pharmacological action. Previously, it has been accepted that the conformation of the piperidine ring was solely N-R, equatorial, on the basis of solid state and theoretical conformation studies. The present work demonstrates a relatively slow rate of interchange between the axial and equatorial forms and therefore requires a reappraisal of the previous literature as applied to the receptor active conformation of morphine.

Rabbit anti-idiotypic antibodies raised against monoclonal anti-morphine IgG block μ & δ opiate receptor sites

Abstract Rabbit antibodies have been raised against murine monoclonal anti-morphine Fab fragments. Following affinity purification, these antibodies competitiveky 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.

The sensitivity of opiate receptors and ligands to short wavelength ultraviolet light

Abstract The sensitivity of the opiate receptor, opiates and opioid ligands to ultraviolet light has been examined with the view of using this property to aid in characterizing the receptor. It is demonstrated that the opiate receptor is very sensitive to short wavelength ultraviolet light which causes rapid destruction of opiate binding activity. It is also shown that tritiated and cold opiate ligands are sensitive to light of this wavelength and when the receptor and opiate ligands are irradiated together, irreversible binding occurs. Along with the sensitivity of the receptor system to proteases and phospholipase the photosensitivity will aid in receptor isolation as well as in development of specific photoaffinity labels.

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.

The quantitative conformations of some dinucleoside phosphates in solution

Abstract Using the lanthanide ion probe technique the NMR spectra of five naturally occurring dinucleoside phosphates have been investigated in aqueous solution. The conformations derived by computer analysis of this data are shown and discussed. The conformations are all anti-anti .

NMR rotating frame relaxation studies of intramolecular motion in peptides. Tyrosine ring motion in methionine-enkephalin

Summary Proton, rotating frame, nuclear magnetic spin-lattice relaxation measurements have been performed in aqueous solution on the unlabelled tyrosyl ring nuclei in [1-(α,ββ,δδ- 2 H 5 )-L-Tyr,5-L-Met]-enkephalin. In the temperature range measured, 7°C to 30°C, the chemical shift difference between the two epsilon protons is modulated by motions of the main chain and the ring with an average rate of the order of ∼ 400 sec. −1 This work confirms an earlier suggestion by us that the ring of the tyrosyl residue in Methionine-enkephalin is quite rigidly fixed with respect to the peptide main chain compared with overall molecular tumbling.

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).