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At least 397 records · Page 22Linked to original sources
PubMed1994

TAP (transporter associated with antigen processing)-independent presentation of endogenously synthesized peptides is enhanced by endoplasmic reticulum insertion sequences located at the amino- but not carboxyl-terminus of the peptide.

Under most circumstances, cell surface MHC class I molecules display peptides derived from a cytosolic pool of proteins. The efficient presentation of such peptides requires the functioning of two MHC gene products [TAP1 and TAP2 (transporter-associated with Ag processing 1 and 2)] that form a complex that facilitates transmembrane movement of peptides from the cytosol to the endoplasmic reticulum, the site of peptide association with class I molecules. It has been previously shown that peptides can be presented in a TAP-independent manner in association with HLA A2.1 or H-2 Kd if they are expressed COOH-terminal to an endoplasmic reticulum insertion/signal sequence derived from the adenovirus E3/19K glycoprotein (Anderson et al., 1991. J. Exp. Med. 174: 489; Eisenlohr et al., 1992. Cell 71: 963). We show that: 1) the E3/19K signal sequence greatly enhances the presentation of each of four additional peptides tested in association with H-2 Kb or Kk, 2) the E3/19K signal sequence can be substituted by a signal sequence derived from beta-IFN, and 3) the E3/19K signal sequence does not function when located at the COOH terminus of antigenic peptides. These findings indicate that first, many peptides require TAP for efficient presentation to T cells, second, expression of peptides COOH-terminal to signal sequences is a generally applicable method of bypassing the TAP-dependence of peptide presentation and third, the leader sequence does not act to bypass TAP simply by increasing the hydrophobic nature of peptides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1985

Glucagon-like peptide-1 but not glucagon-like peptide-2 stimulates insulin release from isolated rat pancreatic islets.

Glucagon-like peptide-1 and glucagon-like peptide-2 are encoded by the m-RNA of pancreatic preproglucagon. They show high conservation in different species and substantial sequence homology to glucagon. Because no definite biological activity of these peptides has been reported, we investigated the effect of synthetic C-terminally amidated glucagon-like peptide-1 [1-36] and synthetic human glucagon-like peptide-2 [1-34] with a free C-terminus on insulin release from isolated precultured rat pancreatic islets in the presence of glucose. Glucagon-like peptide-1 stimulates insulin release at 10 and 16.7 mmol/l glucose in a dose-dependent manner. Significant stimulation starts at 2.5 nmol/l in the presence of 10 mmol/l glucose and near maximal release is observed at 250 nmol/l, with approximately 100% increase over basal at both glucose concentrations. The peptide reaches 63% of the maximal stimulatory effect of glucagon. No stimulation occurs in the presence of 2.8 mmol/l glucose. Glucagon-like peptide-2 has no effect on insulin secretion at any glucose concentration tested. It is concluded that glucagon-like peptide-1, in contrast to glucagon-like peptide-2, exhibits a glucose-dependent insulinotropic action on isolated rat pancreatic islets similar to that of glucagon and gastric inhibitory polypeptide.

Amino Acid Sequence↗
PubMed1996

Vasoactive intestinal peptide and gastrin-releasing peptide play distinct roles in the suprachiasmatic nucleus.

Vasoactive intestinal peptide and gastrin-releasing peptide levels were measured in the punched-out suprachiasmatic nucleus tissue from rats kept under a prolonged dim light (in vivo). Vasoactive intestinal peptide content increased from 4 to 8 h, returned to the baseline level at 12 to 16 h, and then increased again until 36 h after the light was switched off (dim light). Gastrin-releasing peptide level, in contrast, showed no significant changes, but a slight decrease from 1 to 4 h was detected under the dim light. In suprachiasmatic nucleus explant-slice culture, in vitro, Arg-vasopressin release increased transiently or showed a decrease at 30 min after exposure to vasoactive intestinal peptide or gastrin-releasing peptide, respectively. Treatment with anti-vasoactive intestinal peptide and anti-gastrin-releasing peptide antibodies reversed these effects. These findings suggest reciprocal roles of vasoactive intestinal peptide and gastrin-releasing peptide in Arg-vasopressin release.

Animals↗
PubMed2004

New ouabain-conjugated peptide found from phage displayed peptide library.

BACKGROUND: Recently, numerous investigations have shown that endogenous ouabain plays an important role in primary and secondary hypertension. The purpose of this study was to find ouabain-conjugated peptides (OCP) to block or to antagonize actions between endogenous ouabain (EO) and sodium pump, to serve as theoretical and experimental bases of EO in hypertension. METHODS: The study involved screening the phage displayed 12-peptide library by biopanning for OCP. The DNA sequence of each selected peptide was determined and the amino acid sequences were deduced. The highest consistent of the polypeptide was synthesized by peptide synthesizer. Synthetic OCP was identified, its binding activity determined by radioligand binding assay, and bioactivity of ouabain conjugated peptide was measured by erythrocyte 86Rb uptake. RESULTS: Three kinds of peptides were identified. Peptide A (12 peptide, Leu-Leu-Ala-Asp-Thr-Thr-His-His-Arg-Pro-Trp-Thr) was the highest consistence of peptide sequences, occupied in 66.7% (8/12). Peptide A was synthesized. The results verified that there was binding activity between synthetically OCP and 3H-ouabain, and that OCP was capable of suppressing the inhibition action between ouabain and sodium pump on the surface of erythrocyte. The results showed that efficacy was in a dose-dependent manner. CONCLUSION: It is important that the results not only obtain distinctive OCP, but also supply valuable experimental data in detection of ouabain and therapy in hypertension.

Dose-Response Relationship, Drug↗
PubMed1997

Molding a peptide into an RNA site by in vivo peptide evolution.

Short peptides corresponding to the arginine-rich domains of several RNA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities. In the case of the HIV-1 Rev-Rev response element (RRE) complex, the peptide forms a single alpha-helix that binds deeply in a widened, distorted RNA major groove and makes a substantial set of base-specific and backbone contacts. Using a reporter system based on antitermination by the bacteriophage lambda N protein, it has been possible to identify novel arginine-rich peptides from combinatorial libraries that recognize the RRE with affinities and specificities similar to Rev but that appear to bind in nonhelical conformations. Here we have used codon-based mutagenesis to evolve one of these peptides, RSG-1, into an even tighter binder. After two rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity and 15-fold higher specificity than the wild-type Rev peptide, and in vitro competition experiments show that RSG-1.2 completely displaces the intact Rev protein from the RRE at low peptide concentrations. By fusing RRE-binding peptides to the activation domain of HIV-1 Tat, we show that the peptides can deliver Tat to the RRE site and activate transcription in mammalian cells, and more importantly, that the fusion proteins can inhibit the activity of Rev in chloramphenicol acetyltransferase reporter assays. The evolved peptides contain proline and glutamic acid mutations near the middle of their sequences and, despite the presence of a proline, show partial alpha-helix formation in the absence of RNA. These directed evolution experiments illustrate how readily complex peptide structures can be evolved within the context of an RNA framework, perhaps reflecting how early protein structures evolved in an "RNA world."

Amino Acid Sequence↗
PubMed2005

A membrane-permeable peptide containing the last 21 residues of the G alpha(s) carboxyl terminus inhibits G(s)-coupled receptor signaling in intact cells: correlations between peptide structure and biological activity.

Cell-penetrating peptides are able to transport covalently attached cargoes such as peptide or polypeptide fragments of endogenous proteins across cell membranes. Taking advantage of the cell-penetrating properties of the 16-residue fragment penetratin, we synthesized a chimeric peptide that possesses an N-terminal sequence with membrane-penetrating activity and a C-terminal sequence corresponding to the last 21 residues of G alpha(s). This G alpha(s) peptide was an effective inhibitor of 5'-N-ethylcarboxamidoadenosine (NECA) and isoproterenol-stimulated production of cAMP in rat PC12 and human microvascular endothelial (HMEC-1) cells, whereas the carrier peptide had no effect. The maximal efficacy of NECA was substantially reduced when PC12 cells were treated with the chimeric peptide, suggesting that it competes with G alpha(s) for interaction with receptors. The peptide inhibited neither G(q)- nor G(i)-coupled receptor signaling. The use of a carboxy-fluorescein derivative of the peptide proved its ability to cross the plasma membrane of live cells. NMR analysis of the chimeric peptide structure in a membrane-mimicking environment showed that the G alpha(s) fragment assumed an amphipathic alpha-helical conformation tailored to make contact with key residues on the intracellular side of the receptor. The N-terminal penetratin portion of the molecule also showed an alpha-helical structure, but hydrophobic and hydrophilic residues formed clustered surfaces at the N terminus and center of the fragment, suggesting their involvement in the mechanism of penetratin internalization by endocytosis. Our biological data supported by NMR analysis indicate that the membrane-permeable G alpha(s) peptide is a valuable, nontoxic research tool to modulate G(s)-coupled receptor signal transduction in cell culture models.

Adenosine-5'-(N-ethylcarboxamide)↗
PubMed1998

Peptide YY, glucagon-like peptide-1, and neurotensin responses to luminal factors in the isolated vascularly perfused rat ileum.

Exposure of the ileum to nutrients markedly inhibits several upper gastrointestinal functions. Hormonal peptides of the ileal wall, i.e. peptide YY (PYY), glucagon-like peptide-1 (GLP-1), and neurotensin (NT), are thought to play a role in this negative feedback mechanism. The present study was conducted to comparatively assess the secretion of PYY, GLP-1, and NT upon luminal infusion of a variety of individual luminal factors in the isolated vascularly perfused rat ileum preparation. PYY, GLP-1, and NT were measured in the portal effluent with specific RIAs. Glucose (250 mM) induced a pronounced release of the three peptides, whereas a physiological concentration of 5 mM did not induce peptide secretion. Peptone (5%, wt/vol) evoked a sustained release of PYY, GLP-1, and NT. Only NT secretion was increased upon luminal administration of 100 mM sodium oleate. Short chain fatty acids (20 mM) evoked an early and transient release of the three peptides. In contrast, taurocholate (20 mM) induced a sustained release of PYY, GLP-1, and NT, but the threshold concentration for peptide release was lower for NT than for PYY or GLP-1. Cellulose or pectin (0.5%, wt/vol) did not modify peptide secretion. In conclusion, glucose and peptone are potent stimulants of PYY, GLP-1, and NT release. Only NT is released upon oleic acid stimulation. Finally, taurocholate is a potent stimulant of the release of the three peptides. Overall, PYY, GLP-1, and NT may participate cooperatively in the ileal brake. As relatively high concentrations of the various stimulants were required to elicit peptide release, it seems likely that this mechanism operates in cases of maldigestion or malabsorption.

Animal Nutritional Physiological Phenomena↗
PubMed2000

Recombinant human single-chain MHC-peptide complexes made from E. coli By in vitro refolding: functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens.

Soluble recombinant MHC-peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies. In this study, soluble recombinant single-chain human MHC (scMHC)-peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA-A2 in the presence of tumor-associated or viral peptides. The scMHC molecule was composed of beta2-microglobulin connected to the first three domains of the HLA-A2 heavy chain through a 15-amino acid flexible linker. Highly purified scMHC-peptide complexes were obtained in high yield using several peptides derived from the melanoma antigens gp100 and MART-1 or a viral peptide derived from HTLV-1. The scMHC complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA-A2-restricted peptides. We also generated scMHC-peptide tetramers, which were biologically functional; they induced a peptide-specific CTL clone to be activated and secrete IFN-gamma, and were able to stain specifically CTL lines. Such recombinant soluble scMHC-peptide complexes and tetramers should prove of great value for characterization of immune responses involving CTL, for visualization of antigen-specific immune responses, for in vitro primary CTL induction, and for peptide binding assays and structural studies.

Antigens, Neoplasm↗
PubMed2002

Peptide analysis, stability studies, and structural modeling explain contradictory peptide motifs and unique properties of the NOD mouse MHC class II molecule H2-A(g7).

The MHC class II molecule H2-A(g7) is the chief genetic determinant in insulin-dependent diabetes mellitus of the non-obese diabetic (NOD) mice. Poor peptide binding ability, as well as presentation of a unique subset of peptides by this molecule was suggested to promote autoimmunity in this strain. However, several laboratories have presented results in favor of an H2-A(g7) molecule that can avidly bind many different peptides. The crystal structures of H2-A(g7) in complex with two different peptides did not completely resolve this issue. To analyze the peptide binding capacity and the motif requirements of H2-A(g7), we eluted natural ligands from purified H2-A(g7) molecules isolated from the H2-A(g7)-transfected M12-C3 cells. A low peptide yield dominated by a few peptide ligands was found. Pool sequencing and alignment of individual ligands on the basis of molecular modeling revealed a peptide-binding motif with basic/aliphatic/small hydrophilic amino acids at relative position 1 (p1), aliphatic amino acids at p4, Ala at p6, and acidic amino acids and Ser/Gly at p9, as well as acidic residues at p10/11. Though weak, the binding of individual ligands, as well as the importance of an acidic C-terminal residue was confirmed by peptide binding studies to isolated H2-A(g7) molecules. Furthermore, the H2-A(g7) molecule incompletely dissociated into its constituent chains in SDS-electrophoresis under nonreducing conditions. This provides additional evidence of its weak affinity for peptides, which probably arises from the combination of beta56His/beta57Ser/beta78Ala and other unique H2-A(g7) residues in contact with the antigenic peptide. These results allow a better understanding of the role of this molecule in the development of autoimmunity and the identification of epitopes relevant to diabetes.

Amino Acid Motifs↗
PubMed1991

R15 alpha 1 and R 15 alpha 2 peptides from Aplysia: comparison of bioactivity, distribution, and function of two peptides generated by alternative splicing.

The mRNA precursor encoded by the R15 gene is alternatively spliced in different neurons to form two related variants, R15-1 and R15-2 mRNA. One of the peptides encoded by the R15-2 mRNA, the R15 alpha 1 peptide, is expressed in the endogenously bursting neuron R15 and mediates some of its central and peripheral synaptic actions. In this study we found that the R15 alpha 2 peptide, which is encoded by the R15-1 mRNA, is synthesized in other neurons in the abdominal ganglion and is also bioactive. The R15 alpha 1 and R15 alpha 2 peptides were found to exert many similar actions on the cardiovascular, digestive, respiratory, and reproductive systems. However, the differences between many of the pharmacological effects of the R15 alpha 1 and R15 alpha 2 peptides indicate that alternative splicing in this system results in two functionally different peptides. Widespread immunoreactivity was found for an antibody directed against the R15 alpha 2 peptide, both in the central nervous system and the periphery. But because of the shared sequence with the R15 alpha 1 peptide, the antibody cross-reacts with the R15 alpha 1 peptide. To distinguish immunocytochemically between the two peptides, we also raised a second antibody that recognizes only the R15 alpha 1 peptide. This antibody labeled the cell body of only one neuron in the central nervous system, R15, although widespread immunoreactivity was found in axons and varicosities in the periphery.

Animals↗
PubMed1988

Relationships between the FMRFamide-related peptides and other peptide families.

The relationships between peptide families are recognized in terms of structural similarity and immunological and biological activity. Most of the currently known FMRFamide-related peptides (FaRPs) of molluscs were tested in a radioimmunoassay (RIA) and in the two standard bioassays for FMRFamide: the radula protractor muscle of the whelk Busycon contrarium, and the isolated heart of the clam Mercenaria mercenaria. Some peptides were also tested on the heart of the snail Helix aspersa. The responses of the different assays to these peptides were generally similar, but substantial diversity precluded an absolute resolution of relationships, even among molluscan FaRPs. Nevertheless, this set of responses does constitute a standard against which to estimate the relative affinities of putative FaRPs from other animal groups. Many of the non-molluscan FaRPs (e.g., the pancreatic polypeptide-related peptides, gastrin/CCK, and the opioid peptides) are relatively inactive on the molluscan assays, but others (e.g., LPLRFamide, a peptide isolated from chicken brain; the opioid receptor-modulating peptides A18Fa and F8Fa; and gamma 1-MSH) were relatively potent. Several arthropod FaRPs have substantial FMRFamide-like sequence similarity and immunoreactivity, and they may be homologous members of the molluscan peptide family. However, those structural and functional aspects of peptide families that transcend phyletic lines probably reflect basic principles of binding between peptides and membrane proteins rather than homology.

Amino Acid Sequence↗
PubMed1995

A bradykinin-potentiating peptide (peptide K12) isolated from the venom of Egyptian scorpion Buthus occitanus.

A nontoxic peptide with bradykinin-potentiating activity was isolated from the dialyzed venom of the scorpion Buthus occitanus by reverse-phase high performance liquid chromatography (RP-HPLC). The pharmacological activity of the peptide was bioassayed by its ability to potentiate added bradykinin (BK) on the isolated guinea pig ileum as well as the isolated rat uterus for contraction. Moreover, the peptide potentiates in vivo the depressor effect of BK on arterial blood pressure in the normotensive anesthetized rat. Chemical characterization of the peptide was also performed. The amino acid composition of the peptide showed 21 amino acid residues per molecule including three proline residues. The amino acid sequence of the purified peptide was confirmed by mass spectrometry. Either N- or C-terminal ends were free. The sequence does not show a homology with bradykinin-potentiating peptides isolated from either scorpion or snake venoms. Furthermore, we did not find a significant sequence homology between the sequence of the isolated peptide and any of proteins or peptides in GenPro or NBRF data banks. The peptide also inhibited angiotensin-converting enzyme (ACE), and could not serve as substrate for the enzyme. It could be concluded that the mechanism of bradykinin-potentiating peptide (BPP) activity may be due to ACE inhibition.

Amino Acid Sequence↗
PubMed2000

Vessel dilator, long acting natriuretic peptide, and kaliuretic peptide increase circulating prostaglandin E2.

Prostaglandin E2 (PGE2) increases in the circulation of persons with congestive heart failure (CHF), but the cause of this increase is unknown. Prostaglandins are not stored, therefore, they cannot be released in response to congestive heart failure itself but rather need to have their synthesis stimulated by a hormone or some other substance. Prostaglandin E2's biologic properties are nearly identical to four peptide hormones originating from amino acids 1-30 [long acting natriuretic peptide], 31-67 [vessel dilator], 79-98 [kaliuretic peptide] and 99-126 [atrial natriuretic peptide, ANP] of the 126 amino acid ANP prohormone. ANP previously has been found to have no effect on circulating PGE2 concentrations in persons with CHF. The present investigation was designed to determine if one or more of the other three atrial natriuretic peptides might increase PGE2 when infused at their respective 100 ng/kg body weight/minute concentrations for 60 minutes in persons with congestive heart failure. Vessel dilator increased PGE2 8-fold (P<0.001) in the first 20 minutes of its infusion with PGE2 remaining 2-3 fold increased (P<0.05) for 60 minutes after stopping its infusion. Long acting natriuretic peptide did not increase PGE2 until 40 minutes of its infusion but it caused the maximal increase (27-fold; P<0.001) of PGE2 of the three peptide hormones tested. Kaliuretic peptide's stimulated increase of PGE2 also began in a delayed fashion but its effects lasted the longest, with PGE2 being increased (P<0.05) for two hours after the cessation of kaliuretic peptide's infusion. This investigation demonstrates that 1) three endogenous peptide hormones increase PGE2 in the circulation and 2) suggests that the known increase in PGE2 in CHF may be in part secondary to these peptides.

Adult↗
PubMed2001

The distribution of a kinin-like peptide and its co-localization with a CRF-like peptide in the blood-feeding bug, Rhodnius prolixus.

Rhodnius prolixus, a blood-feeding hemipteran insect, ingests large meals which are followed by rapid diuresis to eliminate excess water and salt. In Rhodnius, serotonin and an unidentified peptide(s) [33,34] have been shown to act as neurohormonal diuretic factors. In other insects, two families of diuretic peptides, the corticotropin releasing factor (CRF)-like, and kinin peptides [9], have been identified and sequenced. Recently, we demonstrated the presence of a CRF-like diuretic peptide in the CNS and digestive system of Rhodnius [47] using immunohistochemistry and bioassay. In this study, combining immunohistochemistry and radioimmunoassay (RIA) techniques, we show the presence of leucokinin-like peptide(s) in the CNS and digestive system of Rhodnius 5th instar. Additionally, double-label immunohistochemistry demonstrates that the leucokinin-like and CRF-like peptides are co-localized in the posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM) and in neurohaemal areas on abdominal nerves one and two, suggesting the possibility of co-release of the peptides into the hemolymph.Partially purified extracts of the CNS and neurohaemal tissue were tested in vitro on Malpighian tubule secretion and cAMP assays. The factors eluting with increasing acetonitrile percentages from Sep-Pak cartridges were assayed in the presence or absence of ketanserin, a serotonin antagonist which blocks the effects of serotonin on Malpighian tubules. The results indicate activity of serotonin and a CRF-like diuretic peptide on Rhodnius Malpighian tubules, but fail to demonstrate activity of the leucokinin-like peptide(s). The rapid diuresis following feeding is a highly coordinated event, requiring the movement of water and salt across the epithelial cells of the crop into the hemolymph, and from the hemolymph across the cells of the Malpighian tubules. The urine then travels along the Malpighian tubules into the hindgut in order to be expelled. The presence of a leucokinin-like peptide(s) in the CNS and digestive system, which co-localizes with a CRF-like peptide(s), suggests that kinins may play a role in the rapid diuresis, although possibly not directly on the Malpighian tubules.

Animals↗
PubMed1997

Novel peptide-binding proteins and peptide transport in normal and TAP-deficient microsomes.

Most major histocompatibility complex (MHC) class I-binding peptides are translocated by TAP heterodimers, but some enter the ER lumen by alternative pathways. To further define mechanisms of peptide handling, we developed a system for the analysis of peptide-binding components in the ER membrane and lumen using iodinated cross-linkable peptide derivatives. Here we demonstrate that at least three proteins bind peptides in the ER lumen. Peptide cross-linking to these lumenal proteins can be used as an alternative method to monitor peptide transport. TAP and one other protein bind peptides on the cytoplasmic face of the ER. The presence of multiple peptide-binding proteins necessitates caution in interpreting traditional peptide-binding and transport assays. Finally, we demonstrate sequence-specific peptide transport in TAP-deficient cells transfected with only rat TAP1.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

Helix formation in alpha,gamma- and beta,gamma-hybrid peptides: theoretical insights into mimicry of alpha- and beta-peptides.

Alpha,gamma- and beta,gamma-hybrid peptides, which are composed of two different homologous amino acid constituents in alternate order, are suggested as novel classes of peptide foldamers. On the basis of a systematic conformational search employing the methods of ab initio MO theory, the possibilities for the formation of periodic secondary structures in these systems are described. The conformational analysis provides a great number of helix conformers widely differing in energy, which can be arranged into three groups: (i) helices with all hydrogen bonds formed in forward direction along the sequence, (ii) helices with all hydrogen bonds in backward direction, and (iii) helices with alternate hydrogen-bond directions (mixed or beta-helices). Most stable are representatives of beta-helices, but their stability decreases considerably in more polar environments in comparison to helix conformers from the other two classes. There is a great similarity between the overall topology of the most stable hybrid peptide helices and typical helices of peptides which are exclusively composed of a single type of homologous amino acids. Thus, the helices of the beta,gamma-hybrid peptides mimic perfectly those of the native alpha-peptides as, for instance, the well-known alpha-helix, whereas the most stable helix conformers of alpha,gamma-hybrid peptides correspond well to the overall structure of beta-peptide helices. The two suggested novel hybrid peptide classes expand considerably the pool of peptide foldamers and may be promising tools in peptide design and in material sciences.

Amino Acids↗
PubMed1987

Flow cytometric resonance energy transfer measurements support the association of a 95-kDa peptide termed T27 with the 55-kDa Tac peptide.

Two monoclonal antibodies (OKT27 and OKT27b) have been produced that react with distinct epitopes of a 95-kDa peptide. The T27 antigen is widely distributed, being expressed on B lymphocytes, monocytes, and adult T-leukemic cells but not on polymorphonuclear leukocytes or platelets. There was a low level of T27 expression on resting T cells that increased on T-cell activation. In preliminary studies, the OKT27b antibody coprecipitated a 55-kDa peptide, as well as the 95-kDa peptide, from the radiolabeled cells of the HuT 102B2 cell line. Preclearance with anti-Tac, a monoclonal antibody to the 55-kDa peptide of the multichain interleukin 2 receptor, removed the 55-kDa but not the 95-kDa peptide from subsequent OKT27b immunoprecipitates of HuT 102B2 extracts, suggesting the possibility that the T27 peptide was associated with the Tac peptide. However, the precipitation of the p55 Tac peptide by OKT27b was quite inconsistent. Thus, additional information was sought using a flow cytometric energy transfer technique to provide a physical estimation of the proximity between the Tac and the T27 peptides. The flow cytometric version of the fluorescence resonance energy transfer technique permits the determination of inter- and intramolecular distances at 2- to 10-nm levels on a cell-by-cell basis. Using this approach, there was a mean energy transfer of 7.3% with HuT 102B2 cells when fluorescein isothiocyanate anti-Tac served as the donor and tetramethylrhodamine isothiocyanate OKT27 served as the acceptor. In contrast, there was no energy transfer in comparable studies observed when fluorescein anti-Tac and rhodamine anti-transferrin receptor antibodies were used. These observations support the conclusion that there is a close nonrandom proximity in HuT 102B2 cells between the 95-kDa peptide identified by the OKT27 monoclonal antibody and the p55 Tac peptide of the multichain interleukin 2 receptor.

Antibodies, Monoclonal↗
PubMed1996

Identification of the peptides that stimulate the phosphoinositide hydrolysis in lymphocyte cell lines from peptide libraries.

Peptides which stimulate the formation of inositol phosphates (InoPs) in lymphocyte cell lines were identified by screening synthetic peptide libraries composed of random sequences of hexapeptides. The peptides containing the consensus sequence XKYX(P/V)M were found to be most active in the phospholipase C (PLC)-mediated formation of InoPs in a human B myeloma cell line, U266. The peptides also stimulated the phosphoinositide hydrolysis and the release of [Ca2+]i in HL60 and U937 cell lines. On the other hand, these peptides showed no effect in the following cell lines: NIH3T3, PC12, Daudi, Sp2, Jurkat, H9, Molt-4, SupT-1, K562, and RBL-2H3. The result suggests the possibility that the peptides may have cell type specificity. Experiments with one of the active peptides, WKYMVM-NH2 showed that its action mimics the effect of AlF4- which is a G-protein activator in the InoPs generation, and pertussis toxin partially blocked the InoPs accumulation and [Ca2+]i release induced by the peptide in the U266 cells. Binding assays with the peptide labeled with 125I showed that U266 cells have a saturable number of binding sites for the peptide. Taken together, these results suggest that the peptides could activate PLC-mediated signal transduction via a pertussis toxin-sensitive G-protein coupled receptor in certain cell types.

Amino Acid Sequence↗