Biomedical subjects

H Mazarguil

Publications and source records attributed to H Mazarguil.

At least 37 records · Page 2Linked to original sources

PubMed1997

Comparison of the structure-activity relationships of nociceptin and dynorphin A using chimeric peptides.

The aim of the present study was to delineate the functional domains of nociceptin (noc), a neuropeptide which is structurally related to dynorphin A (dyn). The binding and biological potencies towards the nociceptin (ORL1) and dynorphin A (kappa-opioid) receptors of twenty dyn/noc and noc/dyn hybrid peptides were compared with those of the parent heptadecapeptides. Replacement of as many as eleven residues in the C-terminus of dynorphin by the corresponding nociceptin sequence has no significant effect on binding and biological activity towards the kappa-opioid receptor. In marked contrast, replacement of as few as six residues (RKLANQ) in the C-terminus of nociceptin by the corresponding dynorphin sequence (LKWDNQ) dramatically impairs both affinity and activity towards the ORL1 receptor. This clearly indicates that the two neuropeptides have different functional architectures, despite the dual structural homology of both ligands and receptors. Moreover, the recombinant peptide approach led us to identify hybrids whose sequences differ only at positions 5 and 6 and displaying opposite or no receptor selectivity. One contains the dynorphin Leu5-Arg6 sequence and prefers the kappa-opioid receptor, whereas the other comprises the nociceptin Thr5-Gly6 sequence and prefers the ORL1 receptor. A third, containing the mixed dynorphin/nociceptin Leu5-Gly6 sequence, does not discriminate between the two types of receptor.

Amino Acid Sequence↗

PubMed1997

Extracellular cleavage of the vascular endothelial growth factor 189-amino acid form by urokinase is required for its mitogenic effect.

Alternative splicing of vascular endothelial growth factor (VEGF) mRNA results in three distinct molecular forms of 121 or 165 (V165) amino acids that are released in the conditioned medium of cultured cells and one longer isoform of 189 amino acids (V189) that remains cell-associated. V189 has been expressed in wild type CHO-K1 cells and in glycosaminoglycan-deficient pgsA-745 Chinese hamster ovary (CHO) mutant cells. It could be released from CHO-K1 cell membranes by heparin or a synthetic peptide designed on the sequence encoded by exon 6 but was freely released from CHO mutant cells. In both cases, the immunoreactive V189 was mainly released as a 40-kDa cleaved form, provided that the serine protease urokinase, but not plasmin, was active. Recombinant V189 was purified from insect cells infected with a recombinant baculovirus as a nonmitogenic 50-kDa precursor that binds to the receptor Flt-1 but not to Flk-1. It could be matured by urokinase as a 38-kDa fragment able to bind to Flk-1 and to trigger cell proliferation. V165 and V189, however, could be cleaved by plasmin as 34-kDa fragments that exhibit a decreased mitogenic activity. These findings indicate that the carboxyl-terminal domain of V189 masks its binding domain to Flk-1.

Alternative Splicing↗

PubMed1997

Recognition and activation of the opioid receptor-like ORL 1 receptor by nociceptin, nociceptin analogs and opioids.

Nociceptin, also known as orphanin FQ, was recently identified as the naturally occurring agonist of orphan opioid receptor-like ORL1 receptor (Meunier et al., 1995, Nature 377, 532; Reinscheid et al., 1995, Science 270, 792). Nociceptin is a heptadecapeptide which, although it resembles dynorphin A, the endogenous agonist of the kappa-opioid receptor, displays very low potency in competing with binding of [3H]diprenorphine to or inhibiting adenylate cyclase via mu-, delta- and kappa-opioid receptors. Tritium-labeled nociceptin ([3H]nociceptin) was used here to establish a pharmacological profile in vitro of the ORL 1 receptor. In membranes from recombinant Chinese hamster ovary (CHO) cells expressing the ORL 1 receptor, equilibrium binding of [3H]nociceptin is highly specific, saturable (Bmax in the range 1.3-1.8 pmol/mg protein) and of high affinity (Kd approximately equal to 0.1 nM). It is selectively decreased in the presence of Na+ ions and/or of the GTP analog 5'-guanylylimido-diphosphate, an allosteric regulation that is analogous to that of opiate binding to opioid receptors. A few opiates, namely lofentanil, a 4-anilinopiperidine derivative and etorphine, a 6,14-endo-ethenotetrahydrothebaine derivative, were found to be quite potent not only in competing with binding of [3H]nociceptin at the ORL 1 receptor but also in inhibiting forskolin-induced accumulation of cyclic AMP in intact recombinant CHO cells. In a preliminary attempt to delineate active parts of the neuropeptide, nociceptin analogs were also tested, including N- and C-terminal truncation products. Our results suggest that the highly basic, internal core of nociceptin might be essential in conferring on the peptide both affinity for and activity at the ORL 1 receptor. In this respect, the message and address division of dynorphin A, nociceptin's closest structural analog, do not seem to apply to nociceptin.

Adenylyl Cyclase Inhibitors↗

PubMed1997

Protein complexes containing gamma-tubulin are present in mammalian brain microtubule protein preparations.

The presence of gamma-tubulin in microtubule preparations, obtained by disassembly/ assembly cycles at 0degreesC/37degreesC from the brain of several mammals, is demonstrated by immunoblotting with specific antibodies directed against three distinct regions of the protein. In contrast gamma-tubulin was absent from pure tubulin obtained by chromatography on phosphocellulose, but was retained on the column with the other microtubule-associated proteins. A large part of the gamma-tubulin was present in cold stable material remaining after microtubule disassembly at OdegreesC and was partially solubilized using high salt, thus preventing its purification by the usual assembly/disassembly procedure used for alpha/beta-tubulin heterodimers. Brain gamma-tubulin was purified by affinity chromatography with gamma-tubulin antibodies raised against its carboxyl terminal region. Purified gamma-tubulin consisted of at least two polypeptides present in equal quantities and exhibiting a pI of 6.5 and 6.6, respectively. It was associated with the alpha/beta-tubulin heterodimer and with at least five other polypeptides of 75, 105, 130, 195, and 250 kDa. With the exception of the 250 kDa polypeptide, all of these proteins seem to be present in gamma-tubulin complexes isolated from Xenopus eggs. But, in contrast with Xenopus egg complexes, brain complexes exhibited a considerable heterogeneity of their apparent masses and composition in sucrose gradient centrifugation, in agreement with the absence of an homogeneous structure in electron microscopy. Despite this heterogeneity, gamma-tubulin complexes bind quantitatively to microtubule extremities. The possibility to further use mammalian brain gamma-tubulin and some of its associated proteins in biochemical and pharmacological experiments is of interest since brain microtubule protein preparations have been extensively used for studying both microtubule dynamics and the activity of microtubule poisons.

Animals↗

PubMed1997

Autoradiographic characterization of rat spinal neuropeptide FF receptors by using [125I][D.Tyr1, (NMe)Phe3]NPFF.

The binding properties of neuropeptide FF (NPFF) receptors were investigated in different laminae of the rat spinal cord by using quantitative autoradiography and [125I][D.Tyr1, (NMe)Phe3]NPFF as radioligand. In the superficial layers, the specific binding of [125I][D.Tyr1, (NMe)Phe3]NPFF was time-dependent, reversible, and saturable (KD = 0.1 nM). Preincubation of spinal sections increased the maximal number of [125I][D.Tyr1, (NMe)Phe3]NPFF binding sites. Bestatin, an inhibitor of aminopeptidases, increased significantly the apparent affinity of NPFF. Optimal binding of [125I][D.Tyr1, (NMe)Phe3]NPFF was observed in the presence of 120 mM NaCl in all laminae of the spinal cord. No significant differences were noted in the salt dependence in laminae I-II, IV-V, and X, and the pharmacological profile of [125I][D.Tyr1, (NMe)Phe3]NPFF binding was similar in each laminae. These results do not support the existence of NPFF receptors subtypes differentially localized in different area of the spinal cord. Our data reveal the effects of tissue treatments on binding characteristics of NPFF receptors and indicate that [125I][D.Tyr1, (NMe)Phe3]NPFF is a useful radioactive probe for the characterization of NPFF receptors in discrete brain areas.

Animals↗

PubMed1996

Strandedness discrimination in peptide-polynucleotide complexes.

Preferential binding to single- or double-stranded nucleic acids is important for the activity of many proteins that process RNA and DNA. We have investigated the mechanism of strandedness discrimination with peptides derived from the putative DNA-binding domain of the RecA protein, a bacterial recombinase that modulates its affinity for single-stranded DNA by means of ATP binding and hydrolysis. Contributions of electrostatic and non-electrostatic interactions to binding of these peptides with polynucleotides were evaluated by fluorescence spectroscopy as a function of salt concentration and peptide charge. Binding of these peptides to single- and double-stranded nucleic acids was dominated by non-electrostatic interactions. Small electrostatic contributions selectively enhanced peptide complexation with single-stranded nucleic acids. Similar results were observed in control experiments carried out with tripeptides containing charged and aromatic amino acid residues. It was possible to modify the strandedness preference of peptide-polynucleotide complexes by changing electrostatic contributions to the binding free energy. These observations suggest a mechanism whereby some proteins that interact with DNA or RNA might determine and regulate their relative affinity for single- and double-stranded nucleic acids.

Amino Acid Sequence↗

PubMed1996

Interaction studies between the p21Cip1/Waf1 cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen (PCNA) by surface plasmon resonance.

The cyclin-dependent kinase (CDK) inhibitor p21Cip1 consists of two domains that interact with CDKs and proliferating cell nuclear antigen (PCNA), respectively. We have investigated the interaction between p21Cip1 and PCNA using surface plasmon resonance (SPR) technology and compared the results with those obtained from other sources such as the yeast two-hybrid system. Whilst other methods are only semi-quantitative, the SPR technique allowed us to determine the kinetic parameters of the interaction. The apparent equilibrium constant KD calculated for these kinetic parameters was 3.2 x 10(-7) M. We further demonstrate the use of SPR to study the interaction between mutant proteins and to determine their actual KD. The interaction between p21Cip1/PCNA is shown to be dependent upon the trimeric conformation of PCNA since a point mutant that abolishes PCNA-PCNA interaction also abolishes PCNA's interaction with p21Cip1. Finally, we demonstrate that SPR can be used to characterise the interaction of p21Cip1 and PCNA in the presence of short competitive peptides.

Amino Acid Sequence↗

PubMed1996

Binding of viral antigens to major histocompatibility complex class I H-2Db molecules is controlled by dominant negative elements at peptide non-anchor residues. Implications for peptide selection and presentation.

Binding of viral antigens to major histocompatibility complex (MHC) class I molecules is a critical step in the activation process of CD8(+) cytotoxic T lymphocytes. In this study, we investigated the impact of structural factors at non-anchor residues in peptide-MHC interaction using the model of lymphocytic choriomeningitis virus (LCMV) infection of its natural host, the mouse. Altering viral genes by making reassortants, recombinants, and using synthetic peptides, CD8(+) cytotoxic T lymphocytes were shown to recognize only three H-2Db-restricted epitopes, GP amino acids 33-41/43, GP 276-286, and NP 396-404. However, LCMV NP and GP proteins contain 31 other peptides bearing the H-2Db motif. These 34 LCMV peptides and 11 other known H2-Db-restricted peptides were synthesized and examined for MHC binding properties. Despite the presence of the H-2Db binding motif, the majority of LCMV peptides showed weak or no affinity for H-2Db. We observed that dominant negative structural elements located at non-anchor positions played a crucial role in peptide-MHC interaction. By comparative sequence analysis of strong versus non-binders and using molecular modeling, we delineated these negative elements and evaluated their impact on peptide-MHC interaction. Our findings were validated by showing that a single mutation of a favorable non-anchor residue in the sequence of known viral epitopes for a negative element resulted in dramatic reduction of antigen presentation properties, while conversely, substitution of one negative for a positive element in the sequence of a non-binder conferred to the peptide an ability to now bind to MHC molecules.

Amino Acid Sequence↗

PubMed1996

Cardiovascular effects induced by the stimulation of neuropeptide FF receptors in the dorsal vagal complex: an autoradiographic and pharmacological study in the rat.

Previous studies have suggested that central administration of neuropeptide FF-related peptides may modulate cardiovascular parameters in the rat. In the present study, we investigated the role of dorsal vagal complex neuropeptide FF receptors in the central regulation of cardiovascular parameters. The fate of neuropeptide FF receptors in normal and nodose ganglionectomized rats was investigated using an autoradiographic approach with 125I-[DTyr1, (NMe)Phe3]NPFF as ligand for these receptors. We showed that neuropeptide FF binding sites are preferentially located postsynaptically with respect to the vagal afferent fibers in the nucleus tractus solitarius. Thus, ganglionectomy reduced by only 30% and 17% the density of peptide binding sites in the rostral and caudal regions of this nucleus, respectively. Bilateral microinjection of neuropeptide FF (1 nmol) into the commissural nucleus tractus solitarius produced an increase in blood pressure (+13.8 +/- 0.8 mmHg, n = 6), bradycardia (-29.0 +/- 3.2 bpm) and a significant inhibition (-47.6 +/- 3.1%) of the cardiac component of the baroreceptor reflex. Further studies with doses below 1 nmol indicate that NTS microinjections of the neuropeptide produced a dose-dependent decrease in heart rate. Similar cardiovascular effects were observed after bilateral NTS microinjections of one analog neuropeptide FF receptor agonist, [DTyr1, (NMe)Phe3]NPFF (1 nmol). Pretreatment with prazosin (100 micrograms/kg), an alpha 1-adrenoreceptor antagonist, inhibited the neuropeptide FF-evoked blood pressure effect. In addition, the neuropeptide FF-induced heart rate decrease was abolished by pretreatment with atropine (30 micrograms/kg), a muscarinic receptor antagonist. Taken together, these anatomical and pharmacological data suggest that neuropeptide FF receptors within the nucleus tractus solitarius, preferentially located on the postsynaptic component, are involved in the central reflex regulation of cardiovascular parameters.

Animals↗

PubMed1996

Characterization of an epitope of the human cytomegalovirus protein IE1 recognized by a CD4+ T cell clone.

CD4+ T cells specific for human cytomegalovirus (HCMV) IE1 protein are potential effectors of the control of HCMV infection through cytokine production. Better knowledge of major histocompatibility complex (MHC)-peptide-T cell receptor (TcR) interactions in the CD4+ T cell response should result in a better design of immunizing peptides and is a prerequisite for the development of vaccines or anti-cytomegalovirus therapy. In this study, the recombinant protein comprising residues 86-491 encoded by exon 4 of IE1 (GST-e4) was cleaved by enzymatic digestion and analyzed by high pressure liquid chromatography-mass spectroscopy (HPLC-MS). We identified the 14-residue epitope 162-DKREMWMACIKELH-175 recognized by an HLA-DR8-restricted clone, BeA3. Synthetic elongated, truncated and di-Ala-substituted peptides of the 18-mer IE1 158-IVPEDKREMWMACIKELH-175 sequence were used to analyze the amino acid motifs involved in binding to HLA-DR8 and recognition by the BeA3 clone. Substitutions which abolished (MW --> AA), or decreased (RE --> AA and MA --> AA) T cell clone proliferation, cytokine production and cytotoxicity were identified. Loss of T cell function induced by the MW --> AA substitution was associated with poor HLA-DR8 binding. Decreased T cell function (RE --> AA and MA --> AA) was associated with good HLA-DR8 binding, which suggested that these motifs were involved in TcR binding. Other substitutions induced potentiation of the T cell clone response: the IV --> AA substitution induced stronger proliferation, but equivalent cytokine production, when compared with the reference peptide IE1 (158-175). CI --> AA substitution induced strong potentiation of HLA-DR8 binding, proliferation and interferon-gamma and interleukin-4 production, possibly due to the removal of negative effects of Cys, Ile, or both side chains. Cytotoxicity was not improved by any substitution. Our results show modulation of the CD4+ T cell response according to the peptide residues involved in the HLA-DR8-peptide-TcR interaction.

Alanine↗

PubMed1996

The tubulin gene family of Paramecium: characterization and expression of the alpha PT1 and alpha PT2 genes which code for alpha-tubulins with unusual C-terminal amino acids, GLY and ALA.

The ciliated protozoan Paramecium harbours a particularly large diversity of microtubule networks, ranging from the elaborate and stable ciliary axonemes and basal bodies to very dynamic cytoplasmic, cortical or intranuclear arrays. Their organization and individual cycle of assembly/disassembly are well known and extensive immunocytochemical studies of the post-translational modifications in the various microtubule systems have been reported. However, in order to better understand the biogenesis of these multiple and diverse microtubule arrays, it seemed necessary to characterize the tubulin gene family. We show that P tetraurelia possesses four alpha- and three beta-genes and we report the cloning and sequencing of two intronless alpha-genes, alpha PT1 and alpha PT2, which code for very similar polypeptides, differing only by their unusual C-terminal amino acids, respectively GLY and ALA. Partial sequencing of the two other alpha-genes suggests an absence of any further isotype diversity. In an attempt to study the expression of alpha PT1 and alpha PT2, polyclonal antibodies were raised against the twelve C-terminal amino acids corresponding to the deduced polypeptide sequences. The reactivity of these anti-sequence antibodies was studied on blots of soluble tubulin and in situ and compared with that of other well characterized anti-alpha-tubulin antibodies. The molecular data show that in Paramecium, like in other ciliates, microtubule diversity does not arise from tubulin isotype diversity. The immunocytological data indicate that the native C-terminal sequences are predominantly detected in transient or nascent microtubule arrays and lead us to propose: 1) that the C-terminal TYR, absent in Paramecium and in most cilate species, has no intrinsic functional role; and 2) that post-translational modifications do not seem directly instrumental in the geometry and functions of microtubule arrays.

Alanine↗

PubMed1995

Relative implication of peptide residues in binding to major histocompatibility complex class I H-2Db: application to the design of high-affinity, allele-specific peptides.

The H-2Db peptide sequence SMIENLEYM was manipulated (N- and C-terminus truncation and alanine substitution) to determine the role of structural elements (peptide ends and residue side chains) in binding to H-2Db. We found that good binding affinity could be obtained by compensating the minimal binding condition for one element by the optimal condition of the other element. In particular, we showed, that although the minimal binding sequence could be as short as a heptamer (deletion of positions 1 and 2), it needed the presence of optimal amino acids at other positions (IENLEYM). Conversely, the structurally minimal peptide would accept multiple alanine residues, but required the optimal nonameric length (AAAENAEAA). Positions 1, 2, 3, 4, 5, 7 and 9, but not 6 and 8, were involved in the H-2Db-peptide interaction. Most residues interacted directly with the MHC molecule via their main chain (amino and carboxyl) atoms (positions 1 and 2), their side chains (positions 3 and 5), or both (position 9). Positions 4 and 7 were found to play an indirect role, probably by influencing the secondary structure. At the C-terminus, the presence of a residue at position 9, but not the hydrophobic nature of its side chain, was mandatory for binding. At the N-terminus, the role of the residue at position 1 was of either minor or critical importance depending on the presence or not of a strong auxiliary anchor at position 3. The indirect contribution of residue side chains at positions 4 and 7 reflected the importance of dynamic components in the binding process. Based on these results, we designed a series of high-affinity, H-2Db selective peptides derived from the sequence X1 AIX4NAEAL, where X1 = Y or K and X4 = E or K. After radioiodination or fluorescent (FITC) labelling, these peptides bound strongly and specifically to the surface of viable H-2Db-expressing cells. Rationally designed synthetic peptides, either alone or in a stable complex with MHC, might be of value for controlling CTL activity.

Alleles↗

PubMed1995

Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase.

Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.

Actin Cytoskeleton↗

PubMed1995

Optimal lymphocytic choriomeningitis virus sequences restricted by H-2Db major histocompatibility complex class I molecules and presented to cytotoxic T lymphocytes.

Infection with lymphocytic choriomeningitis virus induces the generation of CD8+ cytotoxic T lymphocytes (CTL). In the H-2b mouse, this cellular immune response is directed against three viral structural epitopes (GP1, GP2, and NP) presented by the major histocompatibility complex (MHC) class I H-2Db molecules. This study was undertaken to delineate which sequence of each of these three epitopes is optimal for MHC binding and CTL recognition. The first step was to synthesize the relevant peptides truncated at the N or C terminus and flanking the crucial H-2Db-anchoring Asn residue in position 5. These peptides were then tested (i) for their binding properties in two H-2Db-specific assays with viable cells (upregulation of H-2Db expression on the surface of RMA-S cells and competition against the Db-restricted peptide 125I-gp276-286 on T2-Db cells) and (ii) for their abilities to sensitize H-2b target cells for CTL lysis in vitro. For optimal antigenic presentation, all three epitopes required the MHC-anchoring Asn residue at position 5 of their sequences. The results clearly and unambiguously delineated optimal lengths for two of the epitopes and two options for the third. NP appeared as a conventional 9-amino-acid (aa)-long peptide, np396-404 (FQPQNGQFI). GP2 was defined as a longer peptide (11 aa), gp276-286 (SGVENPGGYCL). Characterization of the GP1 epitope was more complex: the 9-aa-long peptide gp33-41 (KAVYNFATC) and the carboxyl-extended 11-aa-long peptide gp33-43 (KAVYN FATCGI) were both established as possible optimal sequences depending on the cell line used to test binding and lysis.

Amino Acid Sequence↗

PubMed1994

Co-purification of a protein tyrosine phosphatase with activated somatostatin receptors from rat pancreatic acinar membranes.

We have previously shown that somatostatin promotes the stimulation of a membrane tyrosine phosphatase activity in pancreatic cells. To gain insight into the mechanism of somatostatin action, we purified somatostatin-receptor complexes from somatostatin 28-prelabelled rat pancreatic plasma membranes by immunoaffinity chromatography using immobilized antibodies raised against the N-terminal part of somatostatin 28, somatostatin 28 (1-14), which is not involved in receptor-binding-site recognition. After SDS gel electrophoresis a band with a molecular mass of 87 kDa was identified in the affinity-purified material as the somatostatin receptor. The 87 kDa protein was not observed when the membrane receptors were solubilized in a free unoccupied or somatostatin 14-occupied form, or when nonimmune serum replaced the anti-[somatostatin 28 (1-14)] anti-serum. Somatostatin 14 inhibited the appearance of the 87 kDa protein in the same range of concentrations that inhibit radioligand binding on pancreatic membranes. After somatostatin 28 treatment of membranes, purified somatostatin receptor preparations exhibited an elevated tyrosine phosphatase activity that dephosphorylated phosphorylated epidermal growth factor receptor and poly(Glu,Tyr). This activity was related to the presence of somatostatin receptors in purified material. It was increased by dithiothreitol and inhibited by orthovanadate. In purified material containing somatostatin receptors, anti-[Src homology 2 domains (SH2)]-containing tyrosine phosphatase SHPTP1 polyclonal antibodies identified a protein of 66 kDa which was not detected in the absence of somatostatin receptor. Furthermore, the anti-SHPTP1 antibodies immunoprecipitated specific somatostatin receptors from somatostatin-prelabelled pancreatic membranes and from untreated membranes. These results indicate that a 66 kDa tyrosine phosphatase related to SHPTP1 co-purifies with the pancreatic somatostatin receptors, and suggest that this protein is associated with somatostatin receptors at the membrane level.

Amino Acid Sequence↗

PubMed1994

Structure-activity study of neuropeptide FF: contribution of N-terminal regions to affinity and activity.

Twenty neuropeptide FF (NPFF) analogs having various lengths were synthesized by solid-phase peptide synthesis to gain more information on the role of N-terminal residues for the NPFF receptor affinity. The affinities were evaluated in the rat spinal cord membrane preparations, and the biological activities were measured on morphine analgesia in the mouse tail-flick test. Shortening of the NPFF sequence from the N-terminal produced only a moderate decrease in affinity until NPFF (4-8) was reached. In the same way, NPFF(3-8) significantly decreased morphine analgesia, while NPFF(4-8) had no significant effect at a dose of 22 nmol. The introduction in the N-terminal part of NPFF of a D-enantiomer at positions 2 and 1 or the presence of an N-methyl group on position 3 did not modify affinity and activity. Substitution of proline5 by the D-isomer decreased the affinity of NPFF analogs whatever their length, and [Tyr1,D-Pro5]NPFF(1-8) was 2.5-fold less potent than [Tyr1]NPFF(1-8) in reversing morphine-induced analgesia. In contrast, the presence of a glycine residue in position 5 did not influence the affinity toward NPFF receptors. Data provide evidence that the N-terminal segment of neuropeptide FF is responsible for high-affinity binding.

Amino Acid Sequence↗

PubMed1994

Annexin I as a potential inhibitor of insulin receptor protein tyrosine kinase.

Insulin receptor (IR) purified from human placenta by wheat germ agglutinin affinity chromatography was incubated in the presence of insulin, [gamma-32P]ATP and annexin I. In parallel to its own tyrosine phosphorylation, annexin I promoted a dose-dependent inhibition of IR autophosphorylation (IC50 0.5 microM). This effect was specific for insulin-stimulated tyrosine kinase activity and required the N-terminal end of the protein containing the phosphorylatable Tyr21 residue. A pentadecapeptide encompassing residues 16-30 of human annexin I displayed a similar activity, but at higher concentrations. These data underscore a specific interaction of IR with annexin I, which should be considered as a potential physiological regulator of the effects of insulin on its target tissues.

Adenosine Triphosphate↗

PubMed1994

Antibodies that can discriminate between dynein heavy chains and their HUV1 photoproducts.

Dyneins are multi-subunit enzymes that transduce chemical energy into the mechanical energy that makes cilia and flagella beat and moves organelles towards the minus end of microtubules. The ATPase activity is borne by heavy chains, and recent molecular analysis indicates that dynein heavy chain genes form an ancient multigene family: the similarity between the same isoform of two distantly related species is greater than that between different isoforms of the same species. We have exploited sequence identities between a Paramecium axonemal dynein heavy chain gene cloned in our laboratory and sequences of dynein heavy chains from other species to prepare antibodies against active-site peptides capable of recognizing dynein heavy chains regardless of species or isoform. One of the antibodies is perfectly specific for the larger product of V1 photolysis (HUV1) and thus incorporates a unique property of the hydrolytic ATP binding site of all known dynein heavy chains, the capacity for photocleavage in the presence of micromolar vanadate. Our characterization of these reagents suggests that they will be useful for biochemical and in situ studies of known dyneins as well as identification of potential new members of the family.

Amino Acid Sequence↗