Alan R. Day

Demonstration of a receptor on rabbit neutrophils for chemotactic peptides

Abstract [3H]formylNorleu-Leu-Phe, a potent leucocyte chemoattractant, binds specifically to a site on rabbit neutrophils with an affinity of 1.5 × 10−9 M. Other acylated peptide chemoattractants displace this binding at concentrations closely related to those levels required to elicit chemotaxis. The binding fulfills the major criteria for demonstration of specific receptor sites and indicates that a natural bacterial product and synthetic formyl-peptides produce chemotaxis by the same receptor mechanism.

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.

Some characteristics of the neutrophil receptor for chemotactic peptides

Formylated peptides have been shown to be potent chemoattractants for leukocytes [ 1,2]. With the aid of an intrinsically labelled chemoattractant, f-Nle-Leu-[‘HIPhe (ML[3H]P) [3], a high affinity binding site (Kd 1.5 nM) was demonstrated for formylated peptides on rabbit neutrophils [4]. The no. binding sites/cell was estimated to be -10’. In [S], 0.7 X 10’ sites/cell were estimated. This putative receptor, in addition to binding a number of peptide agonists and antagonists, interacted with a partially purified chemotactic factor produced by Es~h~~hiu cc& 141. Others have demonstrated in human cells a similar receptor for chemotactic peptides [6,7]. Here we present some characteristics of the chemoattractant binding site on the rabbit neutrophil. In an approach similar to that used to characterize other receptors such as the opiate [8] and fl-adrenergic [9] binding sites, we have investigated the correlation between binding and biological activity

Synthesis and binding characteristics of an intrinsically radiolabeled chemotactic acyl tripeptide Nα-Formyl—norleucyl—leucyl—phenylalanine

A large number of structurally unrelated compounds are known to be chemotactic to neutrophils [ 11. The heterogeneity of these molecules as well as the lack of structural information on many of them has prevented any definitive statements as to the nature of the cellular events controlling the chemotactic response. Recent studies in our laboratories have shown that a series of synthetic @-formy di-, triand tetrapeptides possess specific structural features which are translatable in terms of a specific receptor site on the cell surface of the neutrophil [2]. Identical structure-activity relationships were found for peptide mediated release of fl-glucuronidase, lysozyme and chemotactic response. The most potent of these synthetic chemoattractants was @formyl. Met.Leu.Phe.OH which had E&,-values of 7 X 10-r’

Secretagogues for lysosomal enzyme release as stimulants of arachidonyl phosphatidylinositol turnover in rabbit neutrophils

Summary [ 14 C]arachidonic acid incorporation into phosphatidylinositol from rabbit neutrophils pretreated with cytochalasin B was increased within 2 min by the synthetic peptide formyl-methionyl-leucyl-phenylalanine and the Ca 2+ ionophore A23187. A high concentration of the peptide elicited only a small increase in phosphatidylinositol turnover when [ 14 C]palmitic acid was employed as precursor. These data coincide with our earlier studies which identified a Ca 2+ -dependent increase in arachidonyl phosphatidylinositol turnover during adrenocorticotropin and A23187-induced activation of steroid production and release in adrenocortical cells. It is concluded that an increase in arachidonyl phosphatidylinositol turnover mediated by a Ca 2+ -dependent phospholipase A 2 may be a general mechanism for altering membrane function during secretory activity.

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.

A useful method for formylation of peptides

Abstract A method of formylation of peptides using formic acid and isovaleroyl chloride is outlined in this communication. Its application for synthesis of several model peptides is also presented.

Evidence for a single opioid receptor type on the field stimulated rat vas deferens

Abstract A series of opioids have been used to study the heterogeneity of the opioid receptor system in rat vas deferentia. β-Endorphin, etorphune, etonitazine, D-Ala 2 -Nle 5 (des-COOH) enkephalin and, sufentanil behave as full agonists in this tissue preparation. Ketobemidone, α(+)-N-allyl normetazocine, morphine and oxymorphone show little or no biological activity. In fact, the latter four drugs were able to inhibit the biological effects of β-endorphin, etorphine and sufentanil in a concentration dependent fashion. These data suggest that there is only one opioid receptor type in the rat vas deferens. These observations are discussed in terms of the binding modes for a series of drugs to an homogeneous receptor system.

Synthesis of several chemotactic peptide antagonists.

Abstract The following peptides have been synthesized using classical mixed anhydride methods: Boc-Leu-Phe-OH, Boc-Phe-Leu-Phe-OH, Boc-Leu-Phe-Leu-Phe-OH and Boc-Phe-Leu-Phe-Leu-Phe-OH. The tri, tetra and pentapeptides inhibit Formyl-Met-Leu-Phe-OH induced release of β-glucuronidase from rabbit peritoneal neutrophils. The antagonists exhibit ID 50 concentrations in the range 2.6−5.7×10 −7 M. The dipeptide was inactive at all concentrations tested.

NMR observation of the interaction of small oligopeptides with phospholipid vesicles

Abstract Using proton spin-lattice relaxation times, the interaction of small oligopeptides with sonicated vesicles of synthetic β-γ-dimyristoyl L-α-lecithin has been monitored at 29°C in D 2 O. The measured relaxation times for the lecithin choline methyl, alkyl chain, and terminal methyl protons were observed to shorten markedly with increasing concentration of peptide, the relaxation remaining exponential. Noticeable resonance broadening was observed at the highest peptide concentration studied. The data reported are for the effect of the pharmacologically active pentapeptide methionine-enkephalin. Similar results have been observed for the effect of tetraglycine. The relaxation of the observable resonances of the added peptide appear to be unaffected. The results are discussed in terms of peptide-vesicle interactions.