共查询到20条相似文献,搜索用时 31 毫秒
1.
The membrane lipid phase may be an important mediator of the peptide-receptor interaction. In order to understand the mechanism of this interaction, it is important to know the peptide structure, not only in the hydrophobic lipid bilayer environment, but also at the bilayer surface and in solution. To investigate this problem we have measured the secondary structure of the 11-residue neuropeptide substance P (SP) and its fragments in aqueous solutions, in membrane mimetic solvents, and associated with lipid bilayers using Raman and CD spectroscopy. Raman and CD spectra of SP bound to liposomes indicate a less than 20% helix content. We interpret these results to indicate that SP contains virtually no helix when bound to negatively charged liposomes. These spectra are similar to spectra of peptides in type I and III beta-turns. SP forms between 10 and 30% (1-3 residues) helical structure in sodium dodecyl sulfate micelles and less than 10% helix in methanol and trifluoroethanol. The binding of SP to negatively charged liposomes significantly changes the structure of the lipid acyl chains, decreasing order in some cases and increasing it in others. Raman spectra of SP in water indicates that SP near 30 mM forms an ensemble of structures in water that is distinct from completely unfolded peptide and from the aggregated beta-sheet form observed in saline solutions. We conclude from our CD results that methods used to quantitate secondary structure from CD spectra of short peptides cannot be used to distinguish between very short helical segments and beta-turns. 相似文献
2.
3.
Bouquié R Bonnin A Bernardeau K Khammari A Dréno B Jotereau F Labarrière N Lang F 《Cancer immunology, immunotherapy : CII》2009,58(4):553-566
HLA multimers are now widely used to stain and sort CD8 T lymphocytes specific for epitopes from viral or tumoral antigens
presented in an HLA class I context. However, the transfer of this technology to a clinical setting to obtain clinical grade
CD8 T lymphocytes that may be used in adoptive cell transfer (ACT) is hindered by two main obstacles: the first obstacle is
the use of streptavidin or derived products that are not available in clinical grade to multimerize HLA/peptide monomers and
the second is the reported high degree of apoptosis that eventually occurs when T cell receptors are crosslinked by HLA multimers.
In the present report, we describe new HLA multimers composed of immunomagnetic beads covalently coupled to a mAb specific
for the AviTag peptide and coated with HLA/peptide monomers bearing the non biotinylated AviTag at the COOH terminus of the
HLA heavy chain. Thus, all the components of this new reagent can be obtained in clinical grade. We compared these new multimers
with the previously described multimers made with streptavidin beads coated with biotinylated HLA/peptide monomers, in terms
of sorting efficiency, recovery of functional T cells, apoptosis and activation. We provide evidence that the new multimers
could very efficiently sort pure populations of T lymphocytes specific for three different melanoma antigens (Melan-A, gp100
and NA17-A) after a single peptide stimulation of melanoma patients’ PBMC. The recovered specific T cells were cytotoxic against
the relevant melanoma cell-lines and, in most cases, produced cytokines. In addition, in marked contrast with streptavidin-based
multimers, our new multimers induced very little apoptosis or activation after binding specific T lymphocytes. Altogether,
these new multimers fulfill all the necessary requirements to select clinical grade T lymphocytes and should facilitate the
development of ACT protocols in cancer patients. 相似文献
4.
Tehming Liang Margaret A. Cascieri 《Biochemical and biophysical research communications》1980,96(4):1793-1799
A biologically active 125I-substance P derivative (I125-BH-substance P), prepared by conjugation of substance P with [125I]Bolton-Hunter reagent, binds specifically to isolated rat parotid cells. The Kd is 4 nM for I-BH-substance P, 5 nM for substance P, 0.18 μM for substance P octa(4–11)peptide, and 1.6 μM for substance P [pyroglutamyl6]hexa(6–11)peptide. Substance P free acid and substance P penta(7–11)peptide are much weaker competitors and the C-terminal tri(9–11)peptide has no effect at 30 μM. The binding is also inhibited by 1 μM physalaemin, eledoisin and substance P methyl ester, but not by unrelated peptides. The selective inhibition of the binding by the biologically active analogs and fragments of substance P indicates that the 125I-labeled N(1)acylated substance P derivative may interact with a substance P receptor on parotid cells. 相似文献
5.
Robin Whelpton Adina T. Michael-Titus Susan M. Stephens Ka Wai Yau David Fengas 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1998,716(1-2)
Gradient elution reversed-phase high-performance liquid chromatographic and capillary electrophoretic separations were optimised to separate substance P (SP) and twelve of its fragments. The methods were applied to a study of the in vivo metabolism of substance P in the rat after intrastriatal injection of the peptide (10 nmol). SP and significant amounts of its N-terminal fragments, SP(1-7) and SP(1-4), were detected but no major C-terminal fragments could be identified. At the concentration studied, the metabolism of SP was shown to follow zero order elimination kinetics with a rate of decay of 0.2 nmol/min. As we have shown that SP(1–4) and SP(1–7) can be produced in vivo in the striatum in relatively large amounts, it is conceivable that these fragments contribute to the overall pharmacological pattern of activity of the parent peptide. 相似文献
6.
Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1) less than 0.1 nM and KD(2) approximately 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD approximately 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD approximately 1 nM) or its N-terminal fragment galanin-(1-13) (KD approximately 1 microM), which constitutes the N-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (KD = 0.4 microM), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
The presence of substance P (SP) in the immature rat ovary was determined by radioimmunoassay (RIA) of acidic extracts. The extracts produced an inhibition-displacement curve of 125I-SP binding parallel to that generated by authentic SP in the SP RIA. Initial chromatographic characterization of ovarian SP in Sephadex G-25 revealed the presence of a molecular form that coeluted with authentic SP and a more abundant component that eluted earlier, suggesting the presence of a heavier peptide, immunologically similar to SP. Nevertheless, further characterization of these two seemingly different components by reversed-phase high-performance liquid chromatography (HPLC) demonstrated that both of them had a retention time similar to that of authentic SP. The ovarian concentration of SP-like immunoreactivity (SP-LI) varied in relation to the onset of puberty, with values increasing significantly between the late juvenile phase and the day of first proestrus. Substance P seems to be devoid of steroidogenic capacity since SP itself and its stable analog [pGlu5,MePhe8,Sar9]-SP5-11 (SP-A) failed to stimulate steroid secretion from either granulosa cells in culture or ovarian fragments in short-term incubation. Substance P also failed to stimulate prostaglandin E2 release from whole ovaries and to modify the steroidal response of cultured granulosa cells to follicle-stimulating hormone and to the beta 2-adrenergic agonist Zinterol. Production of SP-LI from granulosa cells in culture could not be detected under either basal or gonadotropin-stimulated conditions. These observations and the distribution of the peptide within the ovary presented in the companion paper (Dees et al., this issue) strongly suggest that SP is not directly involved in regulating steroidogenesis. Instead, SP may be a component of the so-called sensory innervation of the ovary, and among other undisclosed functions it may contribute to the regulation of ovarian blood flow. 相似文献
8.
Wang LL Guo Z Han Y Wang PF Zhang RL Zhao YL Zhao FP Zhao XY 《Regulatory peptides》2011,167(2-3):185-191
Evidence suggests that substance P (SP) participates in the pathology of acute myocardial ischemia and infarction but the profiles of the peptide in regulation of cardiac functions are still elusive. The aim of this study was to investigate the role of substance P in regulation of cardiac functions and its association with adrenergic mechanism in acute myocardial ischemia and infarction with rodent models. The experiments were carried out in Sprague-Dawley rats. SP and norepinephrine were significantly up-regulated in myocardium at 15min, 30min and 60min of coronary artery occlusion. Pretreatment of the rats with a specific antagonist of neurokinin-1 receptor, D-SP, significant increased+dp/dt and decreased -dp/dt, compared with the controls, pretreated with 0.9% saline. Pretreatment of the isolated CAO hearts with substance P (10(-7)mol/L) significantly increased left ventricular end diastolic pressure. SP producing no effects on cardiac functions when given alone to isolated (non-CAO) heart caused significant attenuation of the changes in the contractility and diastolic functions induced by norepinephrine, when given with norepinephrine. SP attenuated the increase in the activity of PKA provoked by norepinephrine in cultured myocytes. In conclusion, the findings may indicate SP regulates cardiac functions via modulation of adrenergic activity, through suppression of over-activation of PKA. 相似文献
9.
《Life sciences》1994,54(19):PL327-PL332
Intravenous administration of the undecapeptide [Sar9]substance P (SP) sulfone (1.5 nmol/kg) and the hexapeptide [Glp6,Pro9]SP(6ndash;11) (septide; 0.4 nmol/kg) produced a comparable (about 30–40 % of maximal effect) increase of insufflation pressure (bronchospasm) in anesthetized guinea-pigs. The non peptide NK-1 receptor antagonist, (±)CP 96,345 and the peptide NK-1 receptor antagonist, GR 82,334 antagonized dose-dependently the response to both agonists. Both antagonists were more potent against peptide than against [Sar9]SP sulfone (9 and 4 fold difference in ED50 for (±)CP 96,345 and GR 82,334, respectively). These findings indicate that a ‘septide-sensitive’ mechanism mediates bronchoconstriction in vivo and it influences the estimate of the potency of NK-1 receptor antagonists. 相似文献
10.
NMR and CD studies have been used to analyze the model membrane-bound structure of the neuropeptide substance P (RPKPQQFFGLM-NH2, SP), which has previously been proposed as the NK1 receptor active form. Conformations were determined for the SP in the presence of aqueous solutions of zwitterionic dodecylphosphocholine (DPC) and anionic sodium dodecylsulfate (SDS) micelles. The two structures are similar, although fast exchange between free and bound forms was observed for SP with DPC micelles, and predominantly bound characteristics were found for SP in SDS. The addition of 150-200 mM NaCl had no observable effect on the bound conformation in either case. Thus, the structure of SP at a micelle surface is determined largely by hydrophobic forces, and the electrostatic interactions determine the amount of SP that is bound. 相似文献
11.
M G A?rapetiants K Gekht P Oeme R I Kruglikov I A Kolome?tseva 《Zhurnal vysshe? nervno? deiatelnosti imeni I P Pavlova》1983,33(3):493-499
As a result of chronic stress, anxiety appeared in the rats behaviour, motor activity increased, heart rate quickened, blood pressure raised, conditioned instrumental alimentary reflexes missed, the duration of deep phases of sleep lowered, time of falling asleep became longer, the number of awakening increased. The change in quantitative characteristics of sleep was accompanied by its worsening, especially of rapid sleep. Administration of substance P (SP) eledoisin hexopeptide (EH) (250 mcg/kg), 100-200 mcg/kg of delta sleep peptide and 10 mcg/kg of ethylcrotyl barbiturate improved the rats behaviour and sleep parameters. Calipnon accelerated falling asleep. Delta sleep peptide increased sleep duration. SP and EH restored not only the quantitative characteristics of deep phases of sleep but greatly improved their quality: lowered the blood pressure disrupted tachycardia, normalized the conditioned activity. 相似文献
12.
Synthesis, microbicidal activity, and solution structure of the dodecapeptide from bovine neutrophils 总被引:1,自引:0,他引:1
The dodecapepetide sequence R-L-C-R-I-V-V-I-R-V-C-R with a disulfide bridge between the cysteine residues found in bovine neutrophils was synthesized by solid-phase procedures. Its antimicrobial activity against oral microorganisms such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus mutans, and Streptococcus gordonii was examined, and its structural features were examined by CD and determined by two-dimensional (2D) nmr. The strains P. gingivalis (W50 and 381), A. actinomycetemcomitans (Y4 and 67), S. gordonii (DL1), and S. mutans (GS5) are found to be highly sensitive to this peptide at 2-2.5 microM concentrations, suggesting that the dodecapeptide is a potent antibiotic for oral pathogens. The weak negative n-sigma* band observed at approximately 265-270 nm in the CD spectra of this peptide provides evidence for the presence of a disulfide bridge. The negative n-pi* band at approximately 200 nm and the positive pi-pi* band at 185 nm suggest a folded structure for this peptide. The negative n-pi* shifts from 200 to 206 nm with an increase in intensity in dipalmitoylphosphotidylcholine vesicles, suggesting that the peptide might associate to form higher order aggregates in lipid medium. The assignment of backbone and side-chain proton resonances has been accomplished by the combined analysis of 2D total correlated and nuclear Overhauser effect spectroscopy. The temperature dependence of amide NH chemical shifts and (1)H-(2)H exchange effect on amide NH resonances indicate the involvement of amide NH groups of Cys3, Ile5, Ile8, Val10, and Arg12 in intramolecular hydrogen bonding. The coupling constant (J(NH-C(alpha)H)) values, the set of medium-, short-, and long-range nuclear Overhauser effects, and the results of restrained structure calculation using the distance geometry algorithm for nmr applications provide evidence for a folded, loop-like structure with a type I (III) beta-turn involving Ile5, Val6, Val7, and Ile8, and two antiparallel beta-strands involving the N-terminal Arg1, Leu2, Cys3, and Val4 and the C-terminal Arg9, Val10, Cys11, and Arg12 residues. The structure of the dodecapeptide mimics the amphiphilic structure of large 30-35 residue defensins and the peptide appears to exhibit similar antimicrobial potency. 相似文献
13.
The development and application of a novel N-terminal directed substance P antiserum 总被引:4,自引:0,他引:4
Most of the substance P (SP) antisera currently used for radioimmunoassay cross-react to a greater or lesser extent with C-terminal fragments of SP. With the availability of SP free acid it has been possible to produce an antiserum specific for the N-terminal part of the molecule. The amino groups on Arg1 and Lys3 were protected by trifluoracetylation and the peptide was then conjugated through its C-terminal carboxyl group to bovine serum albumin by 1-ethyl-3-(3-dimethyl-amino propyl)-carbodiimide (EDC). After conjugation, the amino protective groups were removed under alkaline conditions and the resulting SP-albumin conjugate was used for immunization of rabbits. The SP antiserum thus produced was found to possess only 0.1% and 0.01% cross-reactivity with SP(2–11) and SP(3–11) respectively, and none at all in the presence of 1000 fold molar excess of other smaller C-terminal fragments of SP. There was no significant difference in the brain content of SP like immunoreactivity (SPLI) measured with either the N- or C-terminal directed antiserum. Using this novel antiserum together with a C-terminal directed antiserum, it was shown that deamidation is unlikely to be a rate limiting step in SP inactivation by rat brain slices. 相似文献
14.
Neutron diffraction has been used to study the membrane-bound structure of substance P (SP), a member of the tachykinin family of neuropeptides. The depth of penetration of its C-terminus in zwitterionic and anionic phospholipid bilayers was probed by specific deuteration of leucine 10, the penultimate amino acid residue. The results show that the interaction of SP with bilayers, composed of either dioleoylphosphatidylcholine (DOPC), or a 50:50 mixture of DOPC and the anionic phospholipid dioleoylphosphatidylglycerol (DOPG), takes place at two locations. One requires insertion of the peptide into the hydrophobic region of the bilayer, the other is much more peripheral. The penetration of the peptide into the hydrophobic region of the bilayer is reflected in a marked difference in the water distribution profiles. SP is seen to insert into DOPC bilayers, but a larger proportion of the peptide is found at the surface when compared to the anionic bilayers. The positions of the two label populations show only minor differences between the two types of bilayer. 相似文献
15.
Calcitonin gene-related peptide: novel neuropeptide 总被引:5,自引:0,他引:5
Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide encoded in the calcitonin gene. Its expression is dependent on tissue-specific alternative RNA processing: mRNA for CGRP predominates in the brain, whilst calcitonin (CT) mRNA predominates in thyroid C cells. The existence of this hitherto unsuspected peptide was predicted by mRNA analysis and demonstrated using antibodies raised against a synthetic peptide corresponding to the predicted C-terminal sequence of CGRP. The distribution of CGRP in the central and peripheral nervous system and its co-localization in some neurons with substance P (SP) or acetylcholine suggests several possible roles in autonomic, sensory and motor functions. Its actions appear to depend on the existence of specific CGRP receptors in target tissues, distinct from the receptors for CT but bearing some resemblance to them. 相似文献
16.
Substance P (SP), a biologically active peptide, contains a carboxy-terminal methionine residue that can oxidize to methionine sulfoxide in dilute solutions exposed to air. The oxidized form of SP, SP sulfoxide, is readily separable from SP by reverse-phase high-pressure liquid chromatography (hplc). Oxidation of SP occurs during some conventional extraction and purification procedures. In 2 m acetic acid extracts of rat brain, SP oxidation was largely prevented by addition of 0.01 m 2-mercaptoethanol. SP sulfoxide can be reduced to SP in high yield by 4.2 m 2-mercaptoethanol at 80°C in 1.5 h. The activity of SP sulfoxide in some bio- and immunoassays for SP was examined. SP sulfoxide had 36% of the sialagogie and 40% of the hypotensive activity of SP. Relative to SP, the immunoreactivity of the sulfoxide with two different antisera against SP was 80 and 40%, respectively. Thus, spontaneous oxidation of substance P can affect estimates of its tissue concentration by such assays and must be taken into account when using reverse-phase hplc to identify or purify this peptide. 相似文献
17.
Many studies have suggested that CD8 Abs affect the binding of class I MHC tetramers/multimers to CD8(+) T cells, which has led to the interpretation that CD8 participates directly in multimer binding. In contrast, a recent publication has argued that CD8 Abs instead cause reorganization of TCR distribution and hence have an indirect effect on multimer binding to the TCR alone. We address these issues by testing the role of CD8 and the impact of CD8 Abs on the binding of normal and mutant multimers to Ag-specific mouse T cells. Our data suggest that, in this system, CD8 Abs act directly on CD8 and only mediate their effects on multimer binding when CD8 is capable of binding to the multimer. These data reinforce the paradigm that CD8 plays an active and direct role in binding of class I MHC multimers. 相似文献
18.
C. Curtain Frances Separovic Katherine Nielsen David Craik Yong Zhong Alan Kirkpatrick 《European biophysics journal : EBJ》1999,28(5):427-436
We have studied the interactions with neutral phospholipid bilayers of FPI, the 23-residue fusogenic N-terminal peptide of
the HIV-1LAI transmembrane glycoprotein gp41, by CD, EPR, NMR, and solid state NMR (SSNMR) with the objective of understanding how it
lyses and fuses cells. Using small unilamellar vesicles made from egg yolk phoshatidylcholine which were not fused or permeabilised
by the peptide we obtained results suggesting that it was capable of inserting as an α-helix into neutral phospholipid bilayers but was only completely monomeric at peptide/lipid (P/L) ratios of 1/2000 or lower.
Above this value, mixed populations of monomeric and multimeric forms were found with the proportion of multimer increasing
proportionally to P/L, as calculated from studies on the interaction between the peptide and spin-labelled phospholipid. The
CD data indicated that, at P/L between 1/200 and 1/100, approximately 68% of the peptide appeared to be in α-helical form. When P/L=1/25 the α-helical content had decreased to 41%. Measurement at a P/L of 1/100 of the spin lattice relaxation effect on the 13C nuclei of the phospholipid acyl chains of an N-terminal spin label attached to the peptide showed that most of the peptide
N-termini were located in the interior hydrocarbon region of the membrane. SSNMR on multilayers of ditetradecylphosphatidyl
choline at P/Ls of 1/10, 1/20 and 1/30 showed that the peptide formed multimers that affected the motion of the lipid chains
and disrupted the lipid alignment. We suggest that these aggregates may be relevant to the membrane-fusing and lytic activities
of FPI and that they are worthy of further study.
Received: 8 June 1998 / Revised version: 18 November 1998 / Accepted: 28 December 1998 相似文献
19.
M A Purbhoo J M Boulter D A Price A L Vuidepot C S Hourigan P R Dunbar K Olson S J Dawson R E Phillips B K Jakobsen J I Bell A K Sewell 《The Journal of biological chemistry》2001,276(35):32786-32792
Recognition of antigen by cytotoxic T lymphocytes (CTL) is determined by interaction of both the T cell receptor and its CD8 coreceptor with peptide-major histocompatibility complex (pMHC) class I molecules. We examine the relative roles of these receptors in the activation of human CTL using mutations in MHC class I designed to diminish or abrogate the CD8/pMHC interaction. We use surface plasmon resonance to determine that point mutation of the alpha3 loop of HLA A2 abrogates the CD8/pMHC interaction without affecting the affinity of the T cell receptor/pMHC interaction. Antigen-presenting cells expressing HLA A2 which does not bind to CD8 fail to activate CTL at any peptide concentration. Comparison of CTL activation by targets expressing HLA A2 with normal, abrogated, or diminished CD8/pMHC interaction show that the CD8/pMHC interaction enhances sensitivity to antigen. We determine that the biochemical basis for coreceptor dependence is the activation of the 23-kDa phosphoform of the CD3zeta chain. In addition, we produce mutant MHC class I multimers that specifically stain but do not activate CTL. These reagents may prove useful in circumventing undesirable activation-related perturbation of intracellular processes when pMHC multimers are used to phenotype antigen-specific CD8+ lymphocytes. 相似文献
20.
Numerous photoaffinity studies of the NK-1 receptor have been carried out with peptide agonist analogues of substance P (SP). However, no information is available with regard to the domain interaction of peptide antagonists within this receptor. We describe herein the photoaffinity labelling of the SP receptor with a peptide antagonist analogue, Bapa(0)[(pBzl)Phe(8),DPro(9),MePhe(10),Trp(CHO)(11)]SP. Photolabelling, enzymatic or chemical cleavage of the covalent complex, purification via streptavidin-coated beads and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis led us to show that the methyl of Met174 side chain, within the receptor's second extracellular loop, is covalently linked to the antagonist photoreactive at position 8. 相似文献