Identification by photoaffinity labeling of the extracellular N-terminal domain of PAC1 receptor as the major binding site for PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts many crucial biological functions through the interaction with its specific PAC1 receptor (PAC1-R), a class B G protein-coupled receptor (GPCR). To identify the binding sites of PACAP in the PAC1-R, three peptide derivatives containing a photoreactive p-benzoyl-phenylalanine (Bpa) residue were developed. These photosensitive PACAP analogs were fully biologically active and competent to displace radiolabeled Ac-PACAP27 from the PAC1-R. Subsequently, the ¹²⁵I-labeled photoprobes were used to anchor the PAC1-R expressed in Chinese hamster ovary cells. Photolabeling led to the formation of two protein complexes of 76 and 67 kDa, representing different glycosylated forms of the receptor. Proteinase and chemical cleavages of the peptide-receptor complexes revealed that ¹²⁵I[Bpa⁰, Nle¹⁷]PACAP27, ¹²⁵I[Bpa⁶, Nle¹⁷]PACAP27 and ¹²⁵I[Nle¹⁷, Bpa²²]PACAP27 covalently labeled the Ser⁹⁸ - Met¹¹¹ segment, the Ser¹²⁴ - Glu¹²⁵ dipeptide and the Ser¹⁴¹ - Met¹⁷² fragment, respectively. Taking into account the topology of the PAC1-R, these segments are mainly located within the extracellular N-terminal domain, indicating that this PAC1-R domain is the major binding site of PACAP27. The present study constitutes the first characterization of the binding domains of PACAP to its specific receptor and suggests heterogeneity within the binding mode of peptide ligands to class B GPCRs.     

Identification of a hexapeptide binding region in the nociceptin (ORL1) receptor by photo-affinity labelling with Ac-Arg-Bpa-Tyr-Arg-Trp-Arg-NH2

The interaction of Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH₂ (HP1), a high-affinity partial agonist of the opioid receptor like (ORL1) receptor, has been investigated using the photo-labile analogue [p-benzoyl-l-Phe (Bpa)²]-HP1. In recombinant CHO cells expressing the human ORL1 receptor, [Bpa²]-HP1 binds the receptor with high affinity (K; ∼3 nM) and is as potent as HP1 in stimulating GTPγS binding (50-60% of nociceptin maximal effect). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radio-iodinated [Bpa²]-HP1 results in the irreversible labelling of a glycoprotein of M_r∼66 kDa, as determined by SDS-PAGE. Cyanogen bromide (CNBr) and enzymatic footprints of the photo-labelled receptor and an engineered receptor mutant (L113M), containing an additional CNBR cleavage site, allowed the photoreactive region to be identified as ORL1[107-113] at the C-terminal of TM helix II. In addition the presence of a disulphide bridge between Cysl23 and Cys200 has been confirmed biochemically.     

[D‐(p‐Benzoylphenylalanine)13, tyrosine19]‐melanin‐concentrating hormone,a potent analogue for MCH receptor crosslinking

A photoreactive analogue of human melanin‐concentrating hormone was designed, [d‐ Bpa¹³,Tyr¹⁹]‐MCH, containing the d‐ enantiomer of photolabile p‐benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous‐flow solid‐ phase methodology using Fmoc‐strategy and PEG‐PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [d ‐Bpa¹³,Tyr¹⁹]‐MCH at its Tyr¹⁹ residue was carried out enzymatically using solid‐ phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed‐ phase mini‐column and HPLC. Saturation binding analysis of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH with G4F‐7 mouse melanoma cells gave a K_D of 2.2±0.2×10⁻¹⁰ mol/l and a B_max of 1047±50 receptors/cell. Competition binding analysis showed that MCH and rANF(1–28) displace [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH from the MCH binding sites on G4F‐7 cells whereas α‐MSH has no effect. Receptor crosslinking by UV‐irradiation of G4F‐7 cells in the presence of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH followed by SDS‐polyacrylamide gel electrophoresis and autoradiography yielded a band of 45–50 kDa. Identical crosslinked bands were also detected in B16‐F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS‐7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein‐coupled receptor, most likely with a varying degree of glycosylation. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of the dimerisation interface of human interleukin-8 by IL-8-variants containing the photoactivatable amino acid benzoyl-phenylalanine

The three-dimensional structure of human interleukin-8 (hIL-8) was determined by the use of NMR and X-ray methodology. At high concentrations interleukin-8 and many other chemokines form a non-covalent homodimer. Several studies have been performed to investigate the relevance of the dimer on receptor activation and led to contradictory results. In order to obtain a better understanding of the dimerisation process, covalently linked homo- and heterodimers were produced by photo-induced dimerisation of hIL-8 analogues that contain the photo-activatable amino acid p-benzoyl-phenylalanine (Bpa) at different positions. Whereas the N-terminal fragment (1–54) was expressed as recombinant thioester, the C-terminal fragments (55–77) that contain Bpa either at position 65 or 74 were obtained by solid-phase peptide synthesis. The segments were combined by expressed protein ligation and led to full length IL-8 variants containing the non-proteinogenic amino acid Bpa at single positions. IP₃ activity tests showed high biological activity for the CXCR1–GFP receptor for both variants comparable to that of the native ligand. The refolded and purified ligation-products were used for dimer formation by UV-irradiation. The analysis of the reaction mixture was performed by gel-electrophoresis and mass spectrometry and showed that dimer formation of IL-8 occurred in a position dependent manner. [Bpa⁷⁴]hIL-8 has a high tendency to form covalent dimers whereas no dimer formation was observed for the variant with Bpa at position 65. Accordingly one residue of the dimerisation interface could be identified. Dedicated to Prof. K Arnold on the occasion of his 65th birthday.     

Conformations of bombolitins I and III in aqueous solutions: circular dichroism, 1H NMR, and computer simulation studies

The heptadecapeptides bombolitin I and bombolitin III are two of a series of peptides postulated to be biologically active within a membrane environment. In the preceding paper [Bairaktari, E., Mierke, D.F., Mammi, S., & Peggion, E. (1990) Biochemistry (preceding paper in this issue)] the conformational preferences of these peptides in the presence of SDS surfactant micelles, a mimetic for biological membranes, were examined. During these studies the conformations of these peptides were investigated in aqueous solutions by circular dichroism and nuclear magnetic resonance. A large difference was observed for the two peptides. Bombolitin I lacks any observable secondary structure in aqueous solution, independent of temperature, pH, and concentration. In striking contrast, bombolitin III adopts a well-defined alpha-helix at concentrations greater than 1.3 mM. This is indeed surprising given the great similarity of the two peptides. The alpha-helix of bombolitin III is pH dependent, with a great decrease in the observed secondary structure at pH values below 3.5. This observation could only be due to the protonation of the Asp residue at the fifth position. These findings suggest that the secondary structure arises from molecular aggregation of bombolitin III through the formation of a salt bridge involving the Asp side chain. The alpha-helix observed at "high" concentration (greater than 2.5 mM) has been characterized by CD and by the NOE's measured throughout a majority of the peptide. The experimentally determined structure has been energy refined with restrained molecular dynamics. The conformational results from this study are then compared with the conformations found in the presence of surfactant micelles.     

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H_I and H_IV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca²⁺ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca²⁺ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca²⁺ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca²⁺. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.     

Conformation and interactions of bombolitin I analogues with SDS micelles and phospholipid vesicles: CD,fluorescence, two-dimensional NMR and computer simulations

Bombolitins are five structurally related heptadecapeptides acting at the membrane level able to lyse erythrocytes and liposomes and to enhance the activity of phospholipase A ₂(PLA₂). In the presence of SDS or phospholipid vesicles bombolitins are able to form amphiphilic α-helical structures and this property seems to be the major determinant of bioactivity. In order to test the model of interaction between bombolitin I and membranes, an analogue was synthesized in which all the lysines were replaced by arginines: ([Arg^2,9,12, Ile¹⁰] bornbolitin I). The design ofthis sequence allowed the synthesis of a second analogue through a specijic postsynthetic dansylation at the ?-amino group qf a lysine residue replacing the original leucine residue at position 7. The, first analogue was, fiilly characterized by CD and two-dimensional nmr in the presence of SDS or phospholipid vesicles. The peptide, folds into an amphiphilic α-helical confbrrnation with the helical segment spanning the central part of the sequencefrom Ile³ to His¹⁶. This behavior is identical to that observed for the native sequence. The replacement of Iysine residues by arginine hus no detectable effect on the conformational prderence of the peptide chain. By CD and fluorescence spectroscopy measurements, the fluorophore-containing analogue [Arg^2,9,12, Lys⁷(?-dansyl)] bombolitin I also folded into the α-helical conformation in the presence of SDS micelles or phospholipid vesicles. In particular, the dansyl fluorophore, which is located approximately in the middle of the apolar surface ojthe amphiphilic helix, is clearly buried in a hydrophobic environment when the peptide is bound to phospholipid vesicles. These findings support the hypothesis that the peptide helices are oriented parallel to the vesicle surface. © 1995 John Wiley & Sons, Inc.     

Conformational studies by circular dichroism, 1H NMR, and computer simulations of bombolitins I and III in aqueous solution containing surfactant micelles

The heptadecapeptides bombolitin I and bombolitin III are two members of a series of biologically active peptides postulated to be membrane active. In order to understand the effects of the membrane on the secondary structure of the peptides, we have carried out the conformational characterization of bombolitins I and III in the presence of SDS micelles using circular dichroism, nuclear magnetic resonance, and computer simulations. The characteristic bands in the circular dichroism spectra indicate an alpha-helix content of approximately 60% in bombolitin III and 70% in bombolitin I. The observation of NOE's quite distinctive for such secondary structure strongly supports the CD results. The conformational preferences of the two bombolitins derived from CD and NMR were then energetically refined with molecular dynamics simulations. The results from the spectroscopic examination were utilized as input for the simulations, the CD results for generation of the initial structure, and the NOE's as constraints during the simulations. The results from the different techniques employed are in complete agreement.     

Molecular cloning and antibacterial activity of bombolitin isolated from the venom of a bumblebee, Bombus terrestris

Three major components of bumblebee venom are bombolitin, phospholipase A₂, and a serine protease, with bombolitin being the most abundant. Here, we describe the molecular cloning of bombolitin isolated from the venom of a bumblebee, Bombus terrestris, and demonstrate its antibacterial activity. The B. terrestris bombolitin gene consists of 2 exons encoding 56 amino acid residues. Comparative analysis shows that mature B. terrestris bombolitin consists of 18 amino acid residues, which are identical to those of B. ignitus bombolitin. B. terrestris bombolitin displayed antibacterial activity against both the Gram-negative bacterium Klebsiella pneumoniae and the Gram-positive bacterium Staphylococcus aureus, indicating that B. terrestris bombolitin may be a potential antimicrobial agent.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

Genetic mapping of the mouse X chromosome in the region homologous to human Xq27–Xq28

The four loci Gabra3, DXPas8, CamL1, and Bpa, located near the murine X-linked visual pigment gene (Rsup), have been ordered using 248 backcross progeny from an interspecific mating of (B6CBA-A^w-J/A-Bpa) and Mus spretus. One hundred twenty backcross progeny have been analyzed at seven anchor loci spanning the X chromosome and form a regional mapping panel. An additional 128 progeny have been screened for recombination events between Cf-9 and Dmd. Eighteen recombinants between these loci have been detected in the 248 animals; all of the recombinants were screened at the other anchor loci to identify any double crossovers. Pedigree analysis using these recombinants strongly favors a gene order of (Cf-9)-Gabra3-(DXPas8, Bpa)-CamL1-(Rsvp, P3, Cf-8)-Dmd for the loci studied. Synteny with human Xq27–Xq28 is retained, although the relative order of some loci may differ between the two species.     

An early event in the transport mechanism of LacY protein: interaction between helices V and I

Helix V in LacY, which abuts and crosses helix I in the N-terminal helix bundle of LacY, contains Arg¹⁴⁴ and Trp¹⁵¹, two residues that play direct roles in sugar recognition and binding, as well as Cys¹⁵⁴, which is important for conformational flexibility. In this study, paired Cys replacement mutants in helices V and I were strategically constructed with tandem factor Xa protease cleavage sites in the loop between the two helices to test cross-linking. None of the mutants form disulfides spontaneously; however, three mutants (Pro²⁸ → Cys/Cys¹⁵⁴, Pro²⁸ → Cys/Val¹⁵⁸ → Cys, and Phe²⁹ → Cys/Val¹⁵⁸ → Cys) exhibit cross-linking after treatment with copper/1,10-phenanthroline (Cu/Ph) or 1,1-methanediyl bismethanethiosulfonate ((MTS)₂-1), 3–4 Å), and cross-linking is quantitative in the presence of ligand. Remarkably, with one mutant, complete cross-linking with (MTS)₂-1 has no effect on lactose transport, whereas quantitative disulfide cross-linking catalyzed by Cu/Ph markedly inhibits transport activity. The findings are consistant with a number of previous conclusions suggesting that sugar binding to LacY causes a localized scissors-like movement between helices V and I near the point where the two helices cross in the middle of the membrane. This ligand-induced movement may act to initiate the global conformational change resulting from sugar binding.     

Conformationally constrained D,L-alternating oligopeptides

The possibility of selectively reducing the number of β-helical structures theoretically possible for a D ,L -alternating peptide by using a N-methyl group as conformational constraint is considered. Some ¹H-nmr data regarding Boc(L -Nle-D -Nle)₃-L -Nle-D -MeNle -L -Nle-D -Nle-L -Nle-OMe (I), its formyl analogue (II), and the pentadecapeptide Boc(D -Leu-L -Leu)₅-D -MeLeu -(L -Leu-D -Leu)₂-OMe (III) are presented. It is shown that these alternating stereocooligopeptides with a N-methyl group in the (n ? 3) (I and II) or (n ? 4) position (III) differ drastically in their behavior from the corresponding nonmethylated compounds. In chloroform, I and II form predominantly ↑↓ β^7.2-helices and III forms almost exclusively ↑↓ β^5.6 or ↑↓ β^7.2-helices. The helices are in every case those having the maximum possible number of interchain H bonds.     

Characterization of Aβ aggregation mechanism probed by congo red

β-Amyloid peptide ^{1 1}These authors contributed equally to this work. Communicated by Ramaswamy H. Sarma (Aβ) aggregates are toxic to neuron and the main cause of Alzheimer’s disease (AD). The role of congo red (CR) on Aβ aggregation is controversial in aqueous solution. Both prevention and promotion of Aβ aggregation have been proposed, suggesting that CR may interact with Aβ of different structural conformations resulting in different effects on Aβ aggregation behavior. CR with these characteristics can be applied to probe the molecular mechanism of Aβ aggregation. Therefore, in the present study, we used CR as a probe to study the Aβ aggregation behavior in sodium dodecyl sulfate (SDS) condition. Our results show that Aβ₄₀ adopts two short helices at Q15-S26 and K28-L34 in the SDS environment. CR can interact with the helical form of Aβ₄₀, and the main interaction site is located at the first helical and hydrophobic core region, residues 17–25, which is assigned as a discordant helix region. Furthermore, CR may prevent Aβ₄₀ undergoing α-helix to β-strand conversion, and therefore aggregation through stabilizing the helical conformation of discordant helix in SDS environment, suggesting that the discordant helix plays a key role on the conformational stabilization of Aβ. Our present study implies that any factors or molecules that can stabilize the discordant helical conformation may also prevent the Aβ aggregation in membrane associated state. This leads to a new therapeutic strategy for the development of lead compounds to AD.     

Calcium Ions Promote Superoxide Dismutase 1 (SOD1) Aggregation into Non-fibrillar Amyloid: A LINK TO TOXIC EFFECTS OF CALCIUM OVERLOAD IN AMYOTROPHIC LATERAL SCLEROSIS (ALS)?*

Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca²⁺ dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca²⁺. We show that at physiological pH, Ca²⁺ induces conformational changes that increase SOD1 β-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca²⁺ boosts the onset of SOD1 aggregation. In agreement, Ca²⁺ decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca²⁺ induced aggregates consisting preferentially of antiparallel β-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca²⁺-induced aggregates, thus indicating that Ca²⁺ diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca²⁺ levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca²⁺ may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions.     

Ligand substitution reactions and cytotoxic properties of [Au(L)Cl2] and [AuCl2(DMSO)2] complexes (L = ethylenediamine and S-methyl-l-cysteine)

We have studied the kinetics of the complex formation of gold(III) complexes, [AuCl₂(en)]⁺ (dichlorido(ethylenediamine)aurate(III)-ion) and [AuCl₂(SMC)] (dichlorido (S-methyl-l-cysteine)aurate(III)) with four biologically N-donor nucleophiles. It was shown that studied ligands have a high affinity for gold(III) complex, which may have important biological implications, since the interactions of Au(III) with DNA is thought to be responsible for the anti-tumour activity. The [AuCl₂(SMC)] complex is more reactive than [AuCl₂(en)]⁺. L-His reacts faster than the other N-donor nucleophiles in the reaction with [AuCl₂(en)]⁺, but in the reaction with [AuCl₂(SMC)] 5′-GMP is the best nucleophile. Gold(III) complexes are much more reactive than Pt(II) complexes with the same nucleophiles. The activation parameters for all studied reactions suggest an associative substitution mechanism. The cytotoxicity of gold(III) complexes, [AuCl₂(en)]⁺, [AuCl₂(SMC)] and [AuCl₂(DMSO)₂]⁺ was evaluated in vitro against chronic lymphocytic leukemia cells, obtained from blood of patients with chronic lymphocytic leukemia (CLL). The [AuCl₂(en)]⁺ complex show comparable cytotoxicity profiles compared to cisplatin.     

Crystal structure of plant light‐harvesting complex shows the active,energy‐transmitting state

Plants dissipate excess excitation energy as heat by non‐photochemical quenching (NPQ). NPQ has been thought to resemble in vitro aggregation quenching of the major antenna complex, light harvesting complex of photosystem II (LHC‐II). Both processes are widely believed to involve a conformational change that creates a quenching centre of two neighbouring pigments within the complex. Using recombinant LHC‐II lacking the pigments implicated in quenching, we show that they have no particular role. Single crystals of LHC‐II emit strong, orientation‐dependent fluorescence with an emission maximum at 680 nm. The average lifetime of the main 680 nm crystal emission at 100 K is 1.31 ns, but only 0.39 ns for LHC‐II aggregates under identical conditions. The strong emission and comparatively long fluorescence lifetimes of single LHC‐II crystals indicate that the complex is unquenched, and that therefore the crystal structure shows the active, energy‐transmitting state of LHC‐II. We conclude that quenching of excitation energy in the light‐harvesting antenna is due to the molecular interaction with external pigments in vitro or other pigment–protein complexes such as PsbS in vivo, and does not require a conformational change within the complex.     

Enumeration of interacting species and sedimentation properties of fd-DNA at low ionic strength

The ionic strength dependent aggregation of f_d-DNA was studied by ultracentrifugal field experiments. Compressibility studies indicate a conformational change in the f_d-DNA aggregates with a change in the ionic environment. Principal component or factor analysis, a technique to determine the number of linearly independent components, indicated that three aggregating species are present in the f_d-DNA system at low ionic strength.     

A high-affinity,radioiodinatable neuropeptide FF analogue incorporating a photolabile p-(4-hydroxybenzoyl)phenylalanine

A new radioiodinated photoaffinity compound, [¹²⁵I]YE(Bpa)WSLAAPQRFNH₂, derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF₂ receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction (K_D = 0.24 nM) with a single class of binding sites. It was also able to affinity label NPFF₂ receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [¹²⁵I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF₂ receptor with apparent molecular weights of 140 and 95 kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF₂ receptor is observed. [¹²⁵I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF₂ receptors in vitro.