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1.
J Recept Signal Transduct Res ; 18(2-3): 187-221, 1998.
Article in English | MEDLINE | ID: mdl-9651885

ABSTRACT

Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known. By manipulating these features we have developed new ligands to probe structural and mechanistic aspects of the peptide-receptor interaction. Three ligand groups have been developed: 1) ligands containing a Lys residue located in positions 2 through 7 that can be conjugated to FITC (N-formyl-Met1-Lys2-Phe3-Phe4, N-formyl-Met1-Leu2-Lys3-Phe4, N-formyl-Met1-Leu2-Phe3-Lys4, N-formyl-Met1-Leu2-Phe3-Phe4-Lys5, N-formyl-nLeu1-Leu2-Phe3-nLeu4-Tyr5-Lys6 and N-formyl-Met1-Leu2-Phe3-Phe4-Gly5-Gly6-Lys7; 2) fluorescent pentapeptide ligands (N-formyl-Met-X-Phe-Phe-Lys(FITC) where X = Leu, Ala, Val or Gly); and 3) small crosslinking ligands where the photoaffinity crosslinker 4-azidosalicylic acid (ASA) was conjugated to Lys in positions 3 and 4 and p-benzoyl-phenylalanine (Bpa) was located in position 2 in N-formyl-Met1-Bpa2-Phe3-Tyr4. The peptides were characterized according to activity and affinity in human neutrophils and cell lines transfected with FPR. All of the peptides were agonists, with parallel affinity and activity. In the first group, the peptide activity decreases as Lys is placed closer to the N-formyl group and the activity is improved by 1-3 orders of magnitude by conjugation with FITC. In the second group, the dissociation rate of the peptide from the receptor increases as position 2 is replaced by aliphatic amino acids with smaller alkyl groups. In the third group, crosslinking ligands remain biologically active, display nM affinity and covalently label the FPR.


Subject(s)
Cross-Linking Reagents/chemistry , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/chemistry , N-Formylmethionine Leucyl-Phenylalanine/metabolism , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Animals , Azides/chemistry , Binding, Competitive , Cell Line , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/radiation effects , Humans , In Vitro Techniques , Ligands , Light , Mice , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , N-Formylmethionine Leucyl-Phenylalanine/chemistry , N-Formylmethionine Leucyl-Phenylalanine/radiation effects , Neutrophils/metabolism , Receptors, Formyl Peptide , Receptors, Immunologic/biosynthesis , Receptors, Peptide/biosynthesis , Salicylates/chemistry , Structure-Activity Relationship
2.
J Pept Res ; 49(6): 476-83, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9266474

ABSTRACT

A 3D quantitative structure-activity relationship study (QSAR) of binding and activation of the human C5a receptor by peptide analogs of the C-terminal binding domain of C5a anaphylatoxin is reported. Using published C5a analog affinity and activity data, this paper seeks to elucidate the pharmacophore for the high affinity C-terminal binding domain of the C5a peptide with the molecular modeling technique of comparative molecular field analysis (CoMFA). In order to model peptides for which there was incomplete conformational data, an arbitrary linear conformation was imposed upon the highly flexible C5a analogs. The resulting models yield a crossvalidated q2 of 0.889 and 0.787, for receptor-ligand affinity and EC50 calcium release activity, respectively, suggesting these models have good predictive ability for other test peptides.


Subject(s)
Antigens, CD/chemistry , Complement C5a/metabolism , Peptides/chemistry , Peptides/metabolism , Receptors, Complement/chemistry , Amino Acid Sequence , Ligands , Models, Chemical , Molecular Sequence Data , Protein Conformation , Receptor, Anaphylatoxin C5a , Structure-Activity Relationship
3.
J Recept Signal Transduct Res ; 16(1-2): 59-75, 1996.
Article in English | MEDLINE | ID: mdl-8771531

ABSTRACT

The formyl peptide receptor on human neutrophils recognizes bacterial, N-formylated peptides and initiates a cascade of intracellular signals via a pertussis toxin sensitive Gi protein. We used fluorescence techniques to investigate the interactions of ligand (L), receptor (R), and G proteins (G), the ternary complex, in both live and fixed human neutrophils. By lightly fixing permeabilized neutrophils with a procedure that retained ligand binding, we were able to "capture' R and G in different configurations in the absence of ligand. Fixed receptors were trapped in a high affinity form (attributed to LRG) that could not be rapidly converted to low affinity by the addition of GTP[S]. Adding saturating nucleotide prior to fixation trapped receptors in a low affinity form (attributed to LR). The low affinity receptors retained the sensitivity of the native receptors to the presence of NA+. The distribution between high and low affinity receptors was modulated by GTP[S] in a dose dependent manner. The ability to redistribute low and high affinity receptor forms prior to fixation was unique to GTP[S], as compared to other non-activating nucleotides, suggesting that GTP[S] can regulate the distribution between R and RG. We suggest that precoupled receptors that give rise to high affinity ligand binding are likely to exist in native membranes in human neutrophils.


Subject(s)
Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/metabolism , Neutrophils/metabolism , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Guanosine Diphosphate/analogs & derivatives , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Guanylyl Imidodiphosphate/metabolism , Humans , Kinetics , Neutrophils/cytology , Receptors, Formyl Peptide , Receptors, Immunologic/chemistry , Receptors, Immunologic/drug effects , Receptors, Peptide/chemistry , Receptors, Peptide/drug effects , Sodium/metabolism , Spectrometry, Fluorescence , Substrate Specificity , Thionucleotides/metabolism
4.
J Exp Med ; 180(6): 2191-7, 1994 Dec 01.
Article in English | MEDLINE | ID: mdl-7964494

ABSTRACT

Analysis of synthetic tri- and tetrapeptides has previously indicated that N-formylation is required for high biological activity when they react with the phagocyte N-formylpeptide receptor, suggesting that the natural ligand for the receptor is from bacterial and/or mitochondrial sources. To explore this requirement further, we synthesized the pentapeptide methionyl-norleucyl-leucyl-phenylalanyl-phenylalanine (MNleLFF) and studied the effects of different NH2-terminal modifications on its activity. N-formyl-MNleLFF induced transient alterations of [Ca2+]i and superoxide production in human neutrophils with 10- and 100-fold greater potency, respectively, than the proto-type N-formylpeptide, N-formylmethionyl-leucyl-phenylalanine (fMLF). Surprisingly, N-acetyl-MNleLFF was a potent as N-formyl-MNleLFF. Moreover, the unacylated counterpart H-MNleLFF was also highly active, having an EC50 for calcium mobilization of 10 nM, and for respiratory burst activation of 100 nM. All three pentapeptides could completely desensitize calcium transients elicited by stimulation of neutrophils with fMLF, whereas the neutrophil chemoattractants C5a and interleukin 8 only weakly affected fMLF-induced transients, suggesting that they activate neutrophils via the same receptor as fMLF. Finally, all three pentapeptides activated the recombinant human N-formylpeptide receptor expressed in frog oocytes, but did not effectively activate related phagocyte receptors. These data broaden the potential sources of natural ligands for the N-formyl-peptide receptor from N-formylated bacterial and mitochondrial products to other nonformylated endogenous peptides.


Subject(s)
Calcium/blood , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/physiology , Oligopeptides/pharmacology , Receptors, Immunologic/agonists , Receptors, Peptide/agonists , Amino Acid Sequence , Animals , Cell Line , Complement C5a/pharmacology , Dose-Response Relationship, Drug , Female , Humans , In Vitro Techniques , Interleukin-8/pharmacology , Kinetics , Molecular Sequence Data , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , Neutrophils/drug effects , Oligopeptides/chemical synthesis , Oocytes/drug effects , Oocytes/physiology , Receptors, Formyl Peptide , Receptors, Immunologic/biosynthesis , Receptors, Peptide/biosynthesis , Respiratory Burst/drug effects , Structure-Activity Relationship , Time Factors , Tumor Cells, Cultured , Xenopus
5.
Cytometry ; 15(2): 148-53, 1994 Feb 01.
Article in English | MEDLINE | ID: mdl-8168401

ABSTRACT

Environmentally sensitive molecules have many potential cellular applications. We have investigated the utility of a pH sensitive ligand for the formyl peptide receptor, CHO-Met-Leu-Phe-Phe-Lys (SNAFL)-OH (SNAFL-seminaphtho-fluorescein), because in previous studies (Fay et al.: Biochemistry 30:5066-5075, 1991) protonation has been used to explain the quenching when the fluoresceinated formyl pentapeptide ligand binds to this receptor. Moreover, acidification in intracellular compartments is a general mechanism occurring in cells during processing of ligand-receptor complexes. Because the protonated form of SNAFL is excited at 488 nm with emission at 530 nm and the unprotonated form is excited at 568 nm with emission at 650 nm, the ratio of protonated and unprotonated forms can be examined by multiparameter flow cytometry. We found that the receptor-bound ligand is sensitive to both the extracellular and intracellular pH. There is a small increase in the pKa of the ligand upon binding to the receptor consistent with protonation in the binding pocket. Once internalized, spectral changes in the probe consistent with acidification and ligand dissociation from the receptor are observed.


Subject(s)
Flow Cytometry/methods , Fluoresceins/analysis , Hydrogen-Ion Concentration , Oligopeptides/analysis , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Amino Acid Sequence , Binding Sites , Extracellular Space/chemistry , Fluoresceins/metabolism , Humans , Intracellular Fluid/chemistry , Kinetics , Leukemia, Promyelocytic, Acute/metabolism , Leukemia, Promyelocytic, Acute/pathology , Ligands , Molecular Sequence Data , Neoplasm Proteins/metabolism , Oligopeptides/metabolism , Protein Binding , Receptors, Formyl Peptide , Spectrometry, Fluorescence , Tumor Cells, Cultured
6.
Pept Res ; 5(2): 102-5, 1992.
Article in English | MEDLINE | ID: mdl-1581636

ABSTRACT

Several molecular models of angiotensin II (AII) were constructed using information derived primarily from conformationally constrained AII analogues. In order to refine the original models, AII was compared using computer-aided molecular graphics with several of the recently reported non-peptide AII antagonists. As a first approximation, the imidazole, phenyl ring and acidic moiety (i.e., carboxyl, tetrazole) of the non-peptide AII antagonists were overlapped with the corresponding features of the peptide. AII and Dup 753 were overlapped using Multifit (SYBYL) and then both were optimized individually and refit; this procedure was repeated three times to generate a working model of AII. The less-potent non-peptide AII antagonists S 8308 and Exp 6155 could also be overlaid with the model of AII, albeit not as well as DuP 753. The working model of AII, which has a twisted U-shape, places the imidazole of His6 and the terminal carboxylate approximately 8.7 A apart.


Subject(s)
Angiotensin II/chemistry , Amino Acid Sequence , Angiotensin II/antagonists & inhibitors , Computer Simulation , Models, Molecular , Molecular Sequence Data , Protein Conformation
7.
Biochem J ; 276 ( Pt 3): 715-23, 1991 Jun 15.
Article in English | MEDLINE | ID: mdl-2064609

ABSTRACT

The characteristics of binding to the chemotactic receptors on rabbit peritoneal neutrophils were examined for seven formyl peptide analogues. These receptor-binding characteristics were compared with the abilities of the analogues to induce the biological responses of degranulation and chemotaxis. Five of the analogues showed distinct functional heterogeneity in their receptor-binding patterns, whereas the two most potent compounds displayed homogeneous binding patterns. The relative potencies of the formyl peptide analogues for stimulation of degranulation correlated well with their relative potencies for high-affinity, but not low-affinity, binding. The biphasic patterns for stimulation of chemotactic migration were similar for the less potent analogues, and their potencies paralleled those for both degranulation and receptor binding. In contrast, the most potent analogues induced a greater maximal extent of chemotactic migration than the other compounds, but displayed a lower than expected potency (i.e. they required higher than expected concentrations). These anomalies in the patterns of the chemotactic response cannot be reconciled with a simple receptor model comprising two independent classes of receptors. Instead, a model comprising interconvertible states of different affinities is proposed. The state of higher affinity appears to play a central role in initiation of both degranulation and chemotaxis. The more potent formyl peptide analogues are thought to stabilize an activated, higher-affinity, state of the receptor; this can explain their greater efficacy in stimulating chemotaxis. The proposed model may also be applicable to other receptors that are coupled by a guanine-nucleotide-binding regulatory protein to their associated effector.


Subject(s)
Cell Degranulation/drug effects , Chemotaxis, Leukocyte/drug effects , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/physiology , Receptors, Immunologic/physiology , Amino Acid Sequence , Animals , Molecular Sequence Data , Neutrophils/drug effects , Rabbits , Receptors, Formyl Peptide , Receptors, Immunologic/drug effects
8.
Agents Actions Suppl ; 35: 11-6, 1991.
Article in English | MEDLINE | ID: mdl-1781416

ABSTRACT

We have used spectrofluorometric and flow cytometric techniques to examine the interactions of formyl peptide ligands (L) with their cell surface receptors (R). Kinetic studies suggest that L binds to R at a diffusion limited rate and that R undergoes rapid transitions involving three states (LR, LRG, the ternary complex of L and R with the G protein, and a desensitized receptor "LRX" which forms within seconds) prior to internalization. A spectroscopic analysis of the interaction between L and R show that the binding pocket of R is large enough to contain no more than 6 amino acids and that a fluorescein-labelled pentapeptide is quenched upon binding to R. We hypothesize that histidine 90 (putatively located in the extracellular loop connecting the second and third transmembrane domains) protonates L and quenches the probe. New technology will extend the analysis of structure and dynamics to low affinity peptide receptors of living biological systems. Such technology will have implications in the design of peptidomimetic ligand and drug molecules.


Subject(s)
N-Formylmethionine Leucyl-Phenylalanine/metabolism , Receptors, Immunologic/chemistry , Animals , GTP-Binding Proteins/physiology , Humans , Protein Conformation , Receptors, Formyl Peptide , Receptors, Immunologic/metabolism
9.
Biochim Biophys Acta ; 1034(1): 67-72, 1990 Apr 23.
Article in English | MEDLINE | ID: mdl-2328263

ABSTRACT

The various diastereomers of the N alpha-formylated(CHO) and tert-butyloxycarbonylated (t-Boc) Phe-(Leu-Phe)n and (Leu-Phe)n methyl esters, where n = 1-2 and 1-3, respectively, have been newly synthesized and their physical properties described. The CHO-blocked peptides are all able to release beta-glucosaminidase from rabbit peritoneal neutrophils in a concentration-dependent manner. There is a strong effect of primary structure and of chirality on their biology activity; lengthening the peptide chain distinctly increases activity in each series and within a series the activity decreases in the order: all-L greater than D-L much greater than all-D. Of the t-Boc protected synthetic precursors, the all-L isomers have definite but weak agonist activity; the agonist activity of the other isomers is equivocal or not detectable. All the t-Boc peptides, however, are capable of acting as weak, specific antagonists. There is a dependence of antagonist activity on primary structure, but this is variable and contingent on the nature of the peptide. Similarly, an effect of chirality on antagonist activity, although present, also depends on the structure of the peptide. In the one instance directly tested, t-Boc-L-Phe-(D-Leu-L-Phe)2-OMe (OMe, methoxy) was found to be distinctly less active than the corresponding free acid.


Subject(s)
N-Formylmethionine Leucyl-Phenylalanine/antagonists & inhibitors , Neutrophils/drug effects , Peptides/pharmacology , Receptors, Immunologic/drug effects , Amino Acid Sequence , Animals , Molecular Sequence Data , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , Rabbits , Receptors, Formyl Peptide , Structure-Activity Relationship
10.
Biochemistry ; 29(2): 313-6, 1990 Jan 16.
Article in English | MEDLINE | ID: mdl-2105739

ABSTRACT

We have studied the topography of interaction of a family of fluorescent formyl peptides containing four (CHO-Met-Leu-Phe-Lys-fluorescein), five (CHO-Met-Leu-Phe-Phe-Lys- fluorescein), and six (CHO-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and CHO-Met-Leu-Phe-Phe-Phe-Lys- fluorescein) amino acids with their receptor using spectroscopic methods adapted to small sample volumes. Only the fluorescent peptides containing four and five amino acids were quenched upon binding to the receptor, indicating physical contact of the chromophore with the receptor. In contrast, only the hexapeptides were accessible to antibodies to fluorescein. Taken together, these results suggest that the carboxy terminus of the tetrapeptide or the pentapeptide is protected in the receptor binding pocket while the fluorescein on the carboxy terminus of either hexapeptide is exposed and recognized by the antibody to fluorescein. These results indicate that the binding pocket accommodates at least five but no more than six amino acids.


Subject(s)
Fluoresceins , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , Oligopeptides/metabolism , Receptors, Immunologic/metabolism , Amino Acid Sequence , Binding Sites , Fluorescein , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Humans , Immunologic Techniques , Molecular Sequence Data , Neutrophils/metabolism , Receptors, Formyl Peptide , Spectrometry, Fluorescence , Structure-Activity Relationship , Thiocyanates
11.
Pept Res ; 2(4): 275-81, 1989.
Article in English | MEDLINE | ID: mdl-2520765

ABSTRACT

The conformationally restricted CHO-L-Met-Xxx-L-Phe-OY (where Xxx = Aib, Ac3c, Ac5c, Ac6c, and Ac7c; Y = H, Me) tripeptides, analogs of the chemoattractant CHO-L-Met-L-Leu-L-Phe-OH, have been synthesized in solution by classical methods and fully characterized. Compounds were compared to determine the combined effect of backbone conformational preferences and side-chain bulkiness on the relation of three-dimensional structure to biological activity. Each peptide was tested for its ability to induce granule enzyme secretion from rabbit peritoneal polymorphonuclear leukocytes. In parallel, a conformational analysis on the CHO-blocked peptide and their tertbutyloxycarbonylated synthetic precursors was performed in the crystal state and in solution using X-ray diffraction, infrared absorption, and 1H nuclear magnetic resonance. The biological and conformational data are discussed in relation to the proposed model of the chemotactic peptide receptor of rabbit neutrophils.


Subject(s)
N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , N-Formylmethionine Leucyl-Phenylalanine/chemistry , Neutrophils/physiology , Oligopeptides/chemistry , Alkylation , Amino Acid Sequence , Animals , Glycine , Indicators and Reagents , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , N-Formylmethionine Leucyl-Phenylalanine/chemical synthesis , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Oligopeptides/chemical synthesis , Oligopeptides/pharmacology , Protein Conformation , Rabbits , Spectrophotometry, Infrared , Structure-Activity Relationship
12.
J Leukoc Biol ; 45(6): 529-37, 1989 Jun.
Article in English | MEDLINE | ID: mdl-2723537

ABSTRACT

Polymorphonuclear leukocytes (PMNs) from subjects diagnosed as having juvenile periodontitis (JP) have been categorized on the basis of their chemotactic (CTX) response to f-met-leu-phe (FMLP) when assayed concurrently with PMNs from periodontally healthy subjects (HP). When PMNs from JP groups demonstrating depressed CTX were assayed for lysosomal enzyme secretion (LES) in response to FMLP, there were no significant differences with respect to rate or amount. Significant differences were observed between HP and chemotactically depressed JP cells when assessed for FMLP receptor ligand binding at 23 degrees C, but not at 4 degrees C. Receptor differences observed at 23 degrees C in HP cells included an increase in amount of total binding, number of receptors, and available displaceable binding sites, compared with the chemotactically depressed JP PMNs, whereas the receptor affinities were similar. These data suggest that differences in FMLP receptor density in JP PMN that are chemotactically depressed may be related to processes that modulate receptor mobility and/or expression.


Subject(s)
Neutrophils/ultrastructure , Receptors, Immunologic/metabolism , Chemotaxis, Leukocyte , Hot Temperature , Humans , Lysosomes/enzymology , N-Formylmethionine Leucyl-Phenylalanine/metabolism , Neutrophils/pathology , Neutrophils/physiopathology , Periodontitis/metabolism , Periodontitis/physiopathology , Receptors, Formyl Peptide
13.
Biochem Biophys Res Commun ; 157(2): 569-74, 1988 Dec 15.
Article in English | MEDLINE | ID: mdl-3202870

ABSTRACT

Replacement of leucine in the chemotactic peptide For-Met-Leu-Phe by the sterically constrained amino acids alpha-aminoisobutyric acid and aminocyclohexanecarboxylic acid affords compounds of equal or greater activity than the parent. NMR studies indicate that the parent compound is present as a beta-sheet in solution, whereas the analogues prefer a beta-turn. Application of molecular modelling would indicate that the beta-turn conformer is energetically preferable and thus suggests that it is the orientation adopted by the peptides.


Subject(s)
N-Formylmethionine Leucyl-Phenylalanine , Computer Graphics , Computer Simulation , Hydrogen Bonding , Protein Conformation , Structure-Activity Relationship
14.
Peptides ; 9(6): 1195-205, 1988.
Article in English | MEDLINE | ID: mdl-3247246

ABSTRACT

The two diastereomeric sulphoxides and the sulphone derived from the formyl-methionyl tripeptide chemoattractant CHO-L-Met-L-Phe-OMe have been synthesized and fully characterized. The diastereomeric sulphoxide tripeptides have been separated at the stage of their N-tert-butyloxycarbonyl synthetic precursors. All of the oxidized sulphur derivatives induce secretion of granule enzymes with ED50s from 1-2 x 10(-9) M with no significant differences in activity among them. They are also active to the same relative extent in inducing chemotaxis. In parallel, a solution conformational analysis has been performed in solvents of widely different polarities and capabilities of H-bond formation using circular dichroism, infrared absorption and 1H nuclear magnetic resonance. No significant propensity for formation of intramolecularly (C = O...H-N) H-bonded folded forms has been detected in any of the four tripeptides. Intermolecular S = O...H-N interactions are postulated to tentatively explain the 1H nuclear magnetic resonance behavior of the Met and, particularly, Leu NH resonances of the two sulphoxide tripeptides in CDCl3 solution. The biological and conformational data agree with the recently proposed model of the chemotactic peptide receptor of rabbit neurotrophils, which involves the extended backbone of the integrity of the Met side-chain sulphide sulphur atom as a corollary point of ligand interaction.


Subject(s)
N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , N-Formylmethionine Leucyl-Phenylalanine/chemical synthesis , Circular Dichroism , Magnetic Resonance Spectroscopy , Oxidation-Reduction , Protein Conformation , Stereoisomerism , Structure-Activity Relationship , Sulfones , Sulfoxides
15.
J Leukoc Biol ; 43(5): 420-8, 1988 May.
Article in English | MEDLINE | ID: mdl-2836540

ABSTRACT

Binding of a potent chemotactic formyl tetrapeptide, formylmethionyl-leucyl-phenylalanyl-phenylalanine (fMet-Leu-Phe-Phe), to the formyl peptide receptors on the rabbit neutrophil was assessed by two approaches. A tritiated preparation of fMet-Leu-Phe-Phe was used for direct binding studies, whereas indirect studies comprised an assessment of the ability of the formyl tetrapeptide to competitively inhibit the binding of 35S-labeled formylmethionyl-leucyl-phenylalanine. These two approaches yielded analogous results. The formyl tetrapeptide fMet-Leu-Phe-Phe showed rapid and saturable binding to the same chemotactic receptors as the less potent formyl tripeptides with which it was compared. Its equilibrium-binding pattern, however, was different: fMet-Leu-Phe-Phe showed a homogeneous binding pattern, in contrast to the heterogeneity seen with the less potent compounds. The relative potencies for high-affinity binding of the two standard formyl tripeptides and fMet-Leu-Phe-Phe correlated well with their relative potencies for stimulating the biological response of degranulation; the relative potencies for low-affinity binding correlated less well.


Subject(s)
Chemotactic Factors/metabolism , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , Neutrophils/metabolism , Receptors, Immunologic/metabolism , Animals , Binding, Competitive , N-Formylmethionine Leucyl-Phenylalanine/metabolism , Oligopeptides/metabolism , Rabbits , Receptors, Formyl Peptide , Time Factors
17.
Biochim Biophys Acta ; 884(3): 545-9, 1986 Dec 10.
Article in English | MEDLINE | ID: mdl-3778938

ABSTRACT

The retro-all-D analog and retro isomer of the formyl-methionyl-carboxamide-tripeptide chemoattractant, CHO-L-Met-L-Leu-L-Phe-NH2, namely CHO-D-Phe-D-Leu-D-Met-NH2 and CHO-L-Phe-L-Leu-L-Met-NH2, respectively, have been synthesized in solution by classical methods and fully characterized. The tetrapeptide CHO-L-Phe-Gly-L-Leu-L-Met-NH2, representing the C-terminal portion of the tachykinin, Substance P, and resembling the sequence of the retro isomer, has also been synthesized and characterized. The three N alpha-formylated tripeptide amides, prepared in order to obtain a deeper insight into the model of binding at the formyl peptide chemotactic receptor on rabbit neutrophils, have been tested for their ability to induce granule enzyme secretion from rabbit peritoneal neutrophils. The retro isomer, CHO-L-Phe-L-Leu-L-Met-NH2 is approximately 100-fold less active, the retro-all-D analog, CHO-D-Phe-D-Leu-D-Met-NH2 approximately 10,000-fold less active and the Substance P analog CHO-L-Phe-Gly-L-Leu-L-Met-NH2 1000-fold less active than the parent formyl peptide chemoattractant, CHO-L-Met-L-Leu-L-Phe-NH2. We interpret these results to indicate that a precise alignment of amino acid side chains as well as backbone amide bonds is an important factor involved in the receptor recognition of the formyl tripeptide chemoattractant.


Subject(s)
Chemotaxis, Leukocyte , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , N-Formylmethionine Leucyl-Phenylalanine/chemical synthesis , Neutrophils/physiology , Receptors, Immunologic/metabolism , Animals , Kinetics , Muramidase/metabolism , N-Formylmethionine Leucyl-Phenylalanine/metabolism , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Rabbits , Receptors, Formyl Peptide , Stereoisomerism , Structure-Activity Relationship
18.
Int J Pept Protein Res ; 26(5): 482-92, 1985 Nov.
Article in English | MEDLINE | ID: mdl-4086165

ABSTRACT

Using solution methods, we have synthesized several series of L-methionine homo-oligopeptides from the dipeptide to the hexapeptide with either a free alpha-carboxyl function or different C-blocking groups (methoxy, benzyloxy, benzylamino) and with N-blocking groups of either the amide type (formyl, pivaloyl) or the urethane type (tert.-butyloxycarbonyl). Compounds were compared to determine the combined effect of main-chain length and presence and nature of N- and C-blocking groups on conformation-activity relationship. Each peptide was tested for its ability to induce rabbit peritoneal polymorphonuclear leukocytes in the presence of cytochalasin B to secrete granule enzymes. In parallel, a conformational analysis was carried out in the solid state and in solution, using infrared absorption and circular dichroism. The biological and conformational data are discussed in relation to a recently proposed model of the chemotactic peptide receptor of rabbit neutrophils.


Subject(s)
Chemotactic Factors/chemical synthesis , Chemotaxis, Leukocyte/drug effects , Oligopeptides/chemical synthesis , Animals , Chemotactic Factors/pharmacology , Circular Dichroism , In Vitro Techniques , Methionine , Neutrophils/drug effects , Neutrophils/enzymology , Oligopeptides/pharmacology , Protein Conformation , Rabbits , Spectrophotometry, Infrared , Structure-Activity Relationship
19.
Biochem J ; 222(2): 307-14, 1984 Sep 01.
Article in English | MEDLINE | ID: mdl-6089766

ABSTRACT

The relationship between receptor binding of the formylated peptide chemoattractant formylmethionylleucylphenylalanine (fMet-Leu-Phe), lysosomal enzyme secretion and metabolism of membrane phospholipids was evaluated in both human polymorphonuclear leucocytes (PMN) and the dimethyl sulphoxide (Me2SO)-stimulated human myelomonocytic HL-60 leukaemic cell line. In both cell types, exposure to fMet-Leu-Phe (100 nM) induced rapid lysosomal enzyme secretion (maximal release less than 30 s) and marked changes in the 32P-labelling of the inositol lipids phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as well as phosphatidic acid (PtdA). Specifically, levels of [32P]PtdIns and [32P]PtdIns(4,5)P2 decreased rapidly (peak decrease at 10-15s), with a subsequent increase at 30 s and later. PtdIns4P and PtdA showed only an increase. In Me2SO-differentiated HL-60 cells prelabelled with [3H]inositol for 20 h, fMet-Leu-Phe caused a net increase in the cellular content of [3H]inositol phosphates, including a rapid increase in [3H]inositol 1,4,5-trisphosphate, suggesting that PtdIns(4,5)P2 breakdown occurs by a phospholipase C mechanism. Both lysosomal enzyme secretion and changes in phospholipid metabolism occur over the same agonist concentration range with a similar time course. Binding of [3H]fMet-Leu-Phe, although occurring over the same concentration range, exhibited markedly slower kinetics. Although depletion of extracellular Ca2+ had no effect on ligand-induced polyphosphoinositide turnover, PtdIns turnover, PtdA labelling and lysosomal enzyme secretion were severely curtailed. These studies demonstrate a receptor-mediated enhancement of phospholipid turnover that correlates with a specific biological response to fMet-Leu-Phe. Further, the results are consistent with the idea that phospholipase C-mediated degradation of PtdIns(4,5)P2, which results in the formation of inositol trisphosphate, is an early step in the stimulus-secretion coupling pathway of the neutrophil. The lack of correlation between these two responses and the equilibrium-binding condition suggests that either these parameters are responsive to the rate of ligand-receptor interaction or only fractional occupation is required for a full biological response.


Subject(s)
Leukocytes/metabolism , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Phosphatidylinositols/blood , Calcium/pharmacology , Cell Line , Dimethyl Sulfoxide/pharmacology , Humans , Leukemia, Myeloid/metabolism , Leukocytes/drug effects , Lysosomes/enzymology , Neutrophils/drug effects , Neutrophils/metabolism , Phosphates/blood , Receptors, Cell Surface/metabolism , Receptors, Formyl Peptide
20.
Biochemistry ; 23(4): 698-704, 1984 Feb 14.
Article in English | MEDLINE | ID: mdl-6712920

ABSTRACT

Using solution peptide synthesis, we have made three series of N alpha-formylated homooligopeptides, from the dipeptide to the heptapeptide, derived from L-methionine, L-norleucine, and S-methyl-L-cysteine and related to the chemotactic peptide N alpha-formylmethionylleucylphenylalanine. Compounds were prepared to determine the combined effects of the main-chain length and the presence of a sulfur atom in side-chain gamma- and delta-positions. Each peptide was tested for its ability to induce rabbit peritoneal polymorphonuclear leukocytes in the presence of cytochalasin B to secrete granule enzymes. In parallel, a conformational analysis was carried out in the solid state and in solution, using infrared absorption and circular dichroism. We examined these peptides in solvents of widely different polarities, i.e., chloroform, 2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoropropan-2-ol, and mixed organic-aqueous media. The tendencies to form antiparallel-chain beta-associated and folded structures were determined. The biological and conformational data are described in terms of a model of the chemotactic peptide receptor of rabbit neutrophils recently proposed by Freer et al. (1982) [Freer, R.J., Day, A.R., Muthukumarswamy, N., Pinon, D., Wu, A., Showell, H.J., & Becker, E.L. (1982) Biochemistry 21, 257-263]. In the three N alpha-formylated C-methoxy homooligopeptide series tested, the highest level of activity attained is at the tetrapeptide or pentapeptide stage, confirming the suggestion that the formylpeptide receptor is large enough to accommodate a peptide with at least four amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Chemotactic Factors/pharmacology , Oligopeptides/pharmacology , Animals , In Vitro Techniques , Muramidase/metabolism , Neutrophils/drug effects , Neutrophils/enzymology , Protein Conformation , Rabbits , Structure-Activity Relationship
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