Voltage-gated sodium channels are targets for toxins from the venom of the spider Heriaeus melloteei

Three novel peptides were isolated from the venom of the spider Heriaeus melloteei (Thomisidae) and characterized. The peptides named Hm-1, 2 and 3 blocked voltage-gated Na⁺ channels at concentrations in the order of 100 nM. Activity of the purified peptides was investigated in Na⁺ channel isoforms of mammals and insects. Hm-1 and 2 appeared to act as pore blockers, whereas Hm-3 modulated the channel activation process. The toxins described exhibit minor similarity with other known peptides and may therefore constitute new groups of Na⁺ channel ligands.     

The crystal state conformation of Aib-rich segments of peptaibol antibiotics

Ac-(Aib-Ala)₃-OH (a protected segment of the peptaibols gliodeliquescin and paracelsin), Z-Leu-Aib-Val-Aib-Gly-OtBu (a segment of [Leu]⁷-gliodeliquescin), Z-Val-Aib-Aib-Gln-OtBu (a common segment of alamethicin, paracelsin, and hypelcin), and Ac-Aib-Pro-(Aib-Ala)₂-OMe and Z-Aib-Pro-(Aib-Ala)₂-OMe, which represent differently N^α-protected 1–6 segments of alamethicin and hypelcin, have been synthesized by solution methods. The crystal-state conformations of these five Aib-containing peptides have been determined by X-ray diffraction analysis. We have confirmed that the 3₁₀-helical structure is preferentially adopted by Aib-rich short peptides. An experimentally unambiguous proof for the 3₁₀→α-helix conversion has been provided by the two differently N-blocked -Aib-Pro-(Aib-Ala)₂-OMe hexapeptides. The β-bend ribbon conformation, commonly observed in the (Aib-Pro)_n sequential oligopeptides, is not found in the -Aib-Pro-Aib-Ala-Aib-Ala- sequence. As expected on the basis of the l -configuration of the C^α-monoalkylated residues, a right-handed helix screw sense was found in all peptides investigated. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational studies of heterochiral peptides with diastereoisomeric residues: Crystal and molecular structures of linear dipeptides derived from leucine,isoleucine, and allo-isoleucine

The x-ray diffraction analyses of three N- and C-terminally blocked L , D dipeptides, namely t-Boc-D -Leu-L -Leu-OMe ( 1 ), t-Boc-L -Ile-D -alle-OMe ( 2 ), and t-Boc-D -aIle-L -Ile-OMe (3) containing enantiomeric or diastereomeric amino acid residues have been carried out. The structures were determined by direct methods and refined anisotropically to final R factors of 0.077. 0.058. and 0.072 for ( 1 ) ( 2 ) and ( 3 ), respectively. Peptides 1–3 all assume a similar U-shaped structure with ? and ψ torsion angles cosrresponding to one of the possible calculated minimum energy regions (regions E and G for L residues, and F*. D* and H* for D residues). The peptide backbones of 1-3 are almost super-imposable [provided that the appropriate inversion of the chiral centers of ( 2 ) is made]. Side-chain conformations of Leu residues in peptide ( 1 ) are g^? (tg^?) for the L -Leu residue and the mirrored g⁺ (tg⁺) for the D -Leu residue; however, in peptides ( 2 ) and ( 3 ) the conformations of the isoconfiguralional side chains of the Ile or allo-Ile residues are (g^?t) t and (tg⁺) tfor the L -Ile and the D -allo-Ile moieties, respectively. In all cases, these conformations correspond to the more populated conformers of β-branched residues statistically found in crystal structures of small peptides. The results seem to indicate that, at least in short peptides with enantiomeric or diastereoisomeric residues, the change in chirality in the main-chain atoms perturbs the backbone conformation to a lesser extent and the side chain conformation to a greater extent. © 1995 John Wiley & Sons, Inc.     

Ocellatins: New Antimicrobial Peptides from the Skin Secretion of the South American Frog <Emphasis Type="Italic">Leptodactylus ocellatus</Emphasis> (Anura: Leptodactylidae)

The emergence, in recent years, of microbial resistance to commonly used antibiotics has aroused a search for new naturally occurring bactericidal and fungicidal agents that may have clinical utility. In the present study, three new antimicrobial peptides were purified from the electrical-stimulated skin secretion of the South American frog Leptodactylus ocellatus by reversed-phase chromatographic procedures. Ocellatin 1 (¹GVVDILKGAGKDLLAHLVG ISEKV²⁵-CONH²), ocellatin 2 (¹GVVDILKGAGKDLLAHLVGKISEKV²⁵-CONH₂) and ocellatin 3 (¹GVLDILKNAAKNILAHAAEQI²¹-CONH₂) are structurally related peptides. These peptides present hemolytic activity against human erythrocytes and are also active against Escherichia coli. Ocellatins exhibit significant sequence similarity to other amphibian antimicrobial peptides, mainly to brevinin 2ED from Rana esculenta.     

β-Endorphin and ACTH related peptides in primary cultures of rat anterior pituitary cells: evidence for different intracellular pools

Acid extracts of rat anterior pituitary cells and cell-derived culture media were shown to contain three forms of β-endorphin immunoreactive peptides, corresponding in molecular size to the prohormone pro-opiomelanocortin (POMC), β-lipotropin and 3.5 kDa β-endorphin, and essentially two forms of adrenocorticotropin (ACTH) immunoreactivity, representing a 20 kDa intermediate fragment and 4.5 kDa ACTH. Under basal conditions the intracellular peptides contained a high proportion of the bioactive forms of β-endorphin and ACTH whereas the extracellular peptides contained a higher proportion of the inactive precursors. When the cells were incubated for 3 h in the presence of 10⁻⁸ M CRF, the levels of intracellular β-endorphin and ACTH immunoreactivity were reduced by 15–30% and there was a 4–5-fold increase in the level of the secreted peptides; furthermore, unlike the peptides released under basal conditions, the peptides secreted under the influence of CRF contained much higher proportions of 4.5 kDa ACTH and 3.5 kDa β-endorphin, reflecting the intracellular patterns of these peptides. Similar results were obtained when secretion was stimulated by 10⁻⁷ M epinephrine, which produced a 2-fold increase in peptide release. In the presence of 10⁻⁶ M dexamethasone the basal secretion of ACTH and β-endorphin related peptides, and the intracellular levels of these peptides, remained unaltered. The results point to the existence of different intracellular compartments from which peptides at different states of maturation can be released selectively.β-EndorphinACTHPituitary cell cultureProcessingCRFEpinephrine     

A31P NMR study of the interaction of amphibian antimicrobial peptides with the membranes of live bacteria

Amphibian skin is a rich source of peptides that are specificto pathogens and act by disrupting bacterial membranes. Threeantimicrobial peptides were isolated from the skin glands ofAustralian tree frogs, Litoria caerulea and Litoriagenimaculata. NMR spectroscopy was used to observe changesinduced by these peptides in the ³¹P resonances of bacterialmembranes in vivo. Caerin 1.1 and maculatin 1.1, both wide-spectrum antibiotics, disrupted the membranes ofBacillus cereus and Staphylococcus epidermidis (Gram-positive), leadingto an increase in the isotropic ³¹P NMR signal. Caerin 4.1, anarrow-spectrum antibiotic, however, did not affect the ³¹Pspectra of these organisms. The results demonstrate the use of³¹P NMR to study the effects of membrane-disrupting agents onthe membranes of live bacteria.     

A 31P NMR study of the interaction of amphibian antimicrobialpeptides with the membranes of live bacteria

Summary Amphibian skin is a rich source of peptides that are specific to pathogens and act by disrupting bacterial membranes. Three antimicrobial peptides were isolated from the skin glands of Australian tree frogs,Litoria caerulea andLitoria genimaculata. NMR spectroscopy was used to observe changes induced by these peptides in the³¹P resonances of bacterial membranes in vivo. Caerin 1.1 and maculatin 1.1, both wide-spectrum antibiotics disrupted the membranes ofBacillus cereus andStaphylococcus epidermidis (Gram-positive), leading to an increase in the isotropic³¹P NMR signal. Caerin 4.1, a narrow-spectrum antibiotic, however, did not affect the³¹P spectra of these organisms. The results demonstrate the use of³¹P NMR to study the effects of membrane-disrupting agents on the membranes of live bacteria.     

Two Novel Toxins from the Venom of the Scorpion Pandinus imperator Show that the N-terminal Amino Acid Sequence is Important for their Affinities towards Shaker B K+ Channels

Two novel peptides were purified from the venom of the scorpion Pandinus imperator, and were named Pi2 and Pi3. Their complete primary structures were determined and their blocking effects on Shaker B K⁺ channels were studied. Both peptides contain 35 amino acids residues, compacted by three disulfide bridges, and reversibly block the Shaker B K⁺ channels. They have only one amino acid changed in their sequence, at position 7 (a proline for a glutamic acid). Whereas peptide Pi2, containing the Pro7, binds the Shaker B K⁺ channels with a K _d of 8.2 nm, peptide Pi3 containing the Glu7 residue has a much lower affinity of 140 nm. Both peptides are capable of displacing the binding of ¹²⁵I-noxiustoxin to brain synaptosome membranes. Since these two novel peptides are about 50% identical to noxiustoxin, the present results support previous data published by our group showing that the amino-terminal region of noxiustoxin, and also the amino-terminal sequence of the newly purified homologues: Pi2, and Pi3, are important for the recognition of potassium channels. Received: 13 November 1995/Revised: 11 March 1996     

Evidence that Inhibition of Nicotine-Mediated Catecholamine Secretion from Adrenal Chromaffin Cells by Enkephalin, β-Endorphin, Dynorphin (1-13), and Opiates is not Mediated via Specific Opiate Receptors

Abstract: The opioid peptides Met- and Leu-enkephalin, dynorphin (1-13), and β-endorphin and the narcotic analgesics, morphine, levorphanol, and dextrorphan all produced a dose-dependent inhibition of nicotine (5 × 10^?6m )-mediated release of [³H]norepinephrine ([³H]NE) from bovine adrenal chromaffin cells in culture. None of these agents affected [³H]NE release induced by high K⁺ (56 mm ). Although the above results suggest that the opioid peptides and narcotic analgesics inhibit catecholamine release from adrenal chromaffin cells in culture, we suggest that these effects are not mediated by specific opiate binding sites, since (1) the inhibition was only produced with high concentrations of the agents—the threshold concentrations were 10^?7 to 10^?5m and higher; (2) the inhibition produced by the narcotic analgesics did not display stereospecificity, because the (d-isomer, dextrorphan, was slightly more active than the l-isomer, levorphanol; (3) the narcotic antagonists naloxone, naltrexone, and levallorphan did not reverse the inhibition produced by either the narcotic analgesics (e.g., morphine) or the opioid peptides (e.g., dynorphin). These three antagonists themselves inhibited the nicotine-mediated release of [³H]NE from the adrenal chromaffin cells in culture. Finally (4), the I₂-Tyr¹ substituted analogues of β-endorphin and dynorphin that are biologically less active than the parent compounds produced an inhibition of the nicotine-mediated [³H]NE release similar to that of their parent compounds. These results do not support the idea that high-affinity stereospecific opiate binding sites are involved in the inhibitory modulation of nicotinic evoked catecholamine release from bovine adrenal chromaffin cells in culture.     

Synthesis and conformational study of peptides possessing helically arranged ΔZPhe side chains

Z-Dehydrophenylalanine (Δ^zPhe) possessing four oligopeptides, Boc-(L -Ala-Δ^zPhe-Aib)_n-OCH₃ (n = 1–4: Boc, t-butoxycarbonyl; Aib, α-aminoisobutyric acid), were synthesized, and their solution conformations were investigated by ¹H-nmr, ir, uv, and CD spectroscopy and theoretical CD calculation. ¹H-nmr (the solvent accessibility of NH groups) and ir studies indicated that all the NH groups except for those belonging to the N-terminal L -Ala-Δ^zPhe moiety participate in intramolecular hydrogen bonding in chloroform. This suggests that the peptides n = 2–4 have a 4 → 1 hydrogen-bonding pattern characteristic of 3₁₀-helical structures. The uv spectra of all these peptides recorded in chloroform and in trimethyl phosphate showed an intense maximum around 276 nm assigned to the Δ^zPhe chromophores. The corresponding CD spectra of the peptides n = 2–4 showed exciton couplets with a negative peak at longer wavelengths, whereas that of the peptide n = 1 showed only weak signals. Theoretical CD spectra were calculated for the peptides n = 2–4 of several helical conformations, on the basis of exciton chirality method. This calculation indicated that the three peptides form a helical conformation deviating from the perfect 3₁₀-helix that contains three residues per turn, and that their side chains of Δ^z Phe residues are arranged regularly along the helix. The center-to-center distance between the nearest phenyl pair(s) was estimated to be ～ 5.5 Å. The chemical shifts of the Δ^zPhe side-chain protons (H^β and aromatic H) for the peptides n = 2–4 indicated anisotropic shielding effect of neighboring phenyl group(s); the effect also supports a regular arrangement of the Δ^z Phe side chains along the helical axis. © 1993 John Wiley & Sons, Inc.     

Efficient expression of isotopically labeled peptides for high resolution NMR studies: application to the Cdc42/Rac binding domains of virulent kinases in Candida albicans

The production of bioactive peptides and small protein fragments is commonly achieved via solid-phase chemical synthesis. However, such techniques become unviable and prohibitively expensive when the peptides are large (e.g., >30 amino acids) or when isotope labeling is required for NMR studies. Expression and purification of large quantities of unfolded peptides in E. coli have also proved to be difficult even when the desired peptides are carried by fusion proteins such as GST. We have developed a peptide expression system that utilizes a novel fusion protein (SFC120) which is highly expressed and directs the peptides to inclusion bodies, thereby minimizing in-cell proteolysis whilst maintaining high yields of peptide expression. The expressed peptides can be liberated from the carrier protein by CNBr cleavage at engineered methionine sites or through proteolysis by specific proteases for peptides containing methionine residues. In the present systems, we use CNBr, due to the absence of methionine residues in the target peptides, although other cleavage sites can be easily inserted. We report the production of six unfolded protein fragments of different composition and lengths (19 to 48 residues) derived from the virulent effector kinases, Cla4 and Ste20 of Candida albicans. All six peptides were produced with high yields of purified material (30–40 mg/l in LB, 15–20 mg/l in M9 medium), pointing to the general applicability of this expression system for peptide production. The enrichment of these peptides with ¹⁵N, ¹⁵N/¹³C and even ¹⁵N/¹³C/²H isotopes is presented allowing speedy assignment of poorly-resolved resonances of flexible peptides.     

Unexpected complexity of the dm1 mutation revealed in the structure of three H-2D/L-related antigens

The H-2L^dm1 and H-2D^dm1 MHC antigens of the B10.D2 (H-2 ^dm1 ) mutant mouse strain (formerly known as M504 or H-2 ^da ) have been compared to the H-2L^d and H-2D^d antigens of the B10.D2 (H-2 ^d ) mouse strain. L^dm1 and L^d are 45 000 M_r antigens and both are reactive with anti-H-2.28 (k/r anti-h2) serum and unreactive with anti-H-2.4 (k/b anti-a) serum which detects private determinants of the D^dm1 and D^d antigens. However, the tryptic peptide compositions of these two antigens are different and, based on the number of major tryptic peptides which coelute during ion-exchange chromatography, the estimated peptide homology between L^dm1 and L^d is 80 percent. A newly defined antigen (M_r = 39 000), designated gp39^dm1, was found in glycoprotein extracts of the ^dm1 strain but not of the d strain. This antigen coprecipitates with L^dm1 but does not coprecipitate with D^dm1 indicating that it lacks the H-2.4 determinant. In comparison with L^dm1, gp39^dm1 appears to contain far fewer Arg and Lys residues and is most likely not a simple proteolytic fragment of L^dm1. Finally, peptide maps of the D^dm1 antigen show that the majority of its Arg peptides are identical to D^d Arg peptides, whereas at least five of its Lys peptides and three of its Arg peptides correspond not to D^d peptides but to L^d and L^dm1 peptides. These data raise the possibility that the D^dm1 antigen is a hybrid molecule and they have also revealed an unexpected level of complexity in the dm1 mutant phenotype.     

Investigation of α/γ hybrid peptide self‐assembled structures with antimicrobial and antibiofilm properties

The present work describes the synthesis and characterization of α/γ hybrid peptides, Boc‐Phe‐γ⁴‐Phe‐Val‐OMe, P1 ; Boc‐Ala‐γ⁴‐Phe‐Val‐OMe, P2 ; and Boc‐Leu‐γ⁴‐Phe‐Val‐OMe, P3 together with the formation of self‐assembled structures formed by these hybrid peptides in dimethyl sulfoxide (DMSO)/water (1:1). The self‐assembled structures were characterized by infrared (IR) spectroscopy, circular dichroism (CD), and scanning electron microscopy (SEM). Further, α/γ hybrid peptide self‐assembled structures were evaluated for antibacterial properties. Among all, the self‐assembled peptide P1 exhibited the antimicrobial activity against Escherichia coli and Klebsiella pneumoniae, while self‐assembled peptide P3 inhibited the biofilms of Salmonella typhimurium and Pseudomonas aeruginosa. In this study, we have shown the significance of self‐assembled structures formed from completely hydrophobic α/γ hybrid peptides in exploring the antibacterial properties together with biofilm inhibition.     

Structured Functional Domains of Myelin Basic Protein: Cross Talk between Actin Polymerization and Ca-Dependent Calmodulin Interaction

The 18.5-kDa myelin basic protein (MBP), the most abundant isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the sheath. Solution NMR spectroscopy and a hydrophobic moment analysis of MBP's amino-acid sequence have previously revealed three regions with high propensity to form strongly amphipathic α-helices. These regions, located in the central, N- and C-terminal parts of the protein, have been shown to play a role in the interactions of MBP with cytoskeletal proteins, Src homology 3-domain-containing proteins, Ca²⁺-activated calmodulin (Ca²⁺-CaM), and myelin-mimetic membrane bilayers. Here, we have further characterized the structure-function relationship of these three domains. We constructed three recombinant peptides derived from the 18.5-kDa murine MBP: (A22–K56), (S72–S107), and (S133–S159) (which are denoted α1, α2, and α3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, isothermal titration calorimetry, transmission electron microscopy, fluorimetry, and solution NMR spectroscopy and chemical shift index analysis) to characterize the interactions of these peptides with actin and Ca²⁺-CaM. Our results show that all three peptides can adopt α-helical structure inherently even in aqueous solution. Both α1- and α3-peptides showed strong binding with Ca²⁺-CaM, and both adopted an α-helical conformation upon interaction, but the binding of the α3-peptide appeared to be more dynamic. Only the α1-peptide exhibited actin polymerization and bundling activity, and the addition of Ca²⁺-CaM resulted in depolymerization of actin that had been polymerized by α1. The results of this study proved that there is an N-terminal binding domain in MBP for Ca²⁺-CaM (in addition to the primary site located in the C-terminus), and that it is sufficient for CaM-induced actin depolymerization. These three domains of MBP represent molecular recognition fragments with multiple roles in both membrane- and protein-association.     

Characterization of the bioactive form of linear peptide antagonists at the δ-opioid receptor

The stereochemical requirements for δ-opioid receptor binding of a series of linear peptide antagonists with a novel conformationally restricted Phe analogue (Tic) as a second residue were examined by using a variety of computational chemistry methods. The δ-opioid receptor analogues with significant affinity, Tyr-Tic-NH₂ (TI-NH₂), Tyr-Tic-Phe-OH (TIP), Tyr-Tic-Phe-NH₂(TIP-NH₂), Tyr-Tic-Phe-Phe-OH (TIPP), Tyr-Tic-Phe-Phe-NH₂) (TIPP-NH₂), and the low affinity δ-opioid peptides Tyr-Pro-Phe-Pro-NH₂ (morphiceptin) and Tyr-Phe-Phe-Phe-NH₂ (TPPP-NH₂), were included in this study. The conformational profiles of these peptides were obtained by consecutive cycles of high and low temperature molecular dynamic simulations, coupled to molecular mechanical energy minimization carried out until no new conformational minima were obtained. Comparing the results for TPPP-NH₂ and TIPP-NH₂, the presence of the conformationally restricted Tic residue did not greatly reduce the number of unique low energy conformations, but did allow low energy conformers involving cis bonds between the first two residues. The conformational libraries of these peptides were examined for their ability to satisfy the three key ligand components for receptor recognition already identified by previous studies of high affinity cyclic (Tyr¹-D -Pen²-Gly³-Phe⁴-D -Pen⁵) enkephalin (DPDPE) type agonists: a protonated amine group, an aromatic ring, and a lipophilic moiety in a specific geometric arrangement. Two types of conformations common to the five high δ-opioid affinity L -Tic analogues were found that satisfied these requirements, one with a cis and the other with a trans peptide bond between the Tyr¹ and Tic² residues. Moreover, both the Tic² and Phe³ residues could mimic the hydrophobic interactions with the receptor of the Phe⁴ moiety in the cyclic DPDPE type agonists, consistent with the appreciable affinity of both di-and tripeptides. The low δ-opioid receptor affinity of morphiceptin can be understood as the result of conformational preferences that prevent the fulfillment of this pharmacophore for recognition. © 1996 John Wiley & Sons, Inc.     

Detection of nonopioid β‐endorphin receptor in the rat myocardium

Two selective agonists of nonopioid β‐endorphin receptor, synthetic peptides TPLVTLFK (octarphin) and SLTCLVKGFY (immunorphin), were labeled with tritium to specific activity of 29 and 25 Ci/mmol, respectively. Both labeled peptides were found to bind to high‐affinity naloxone‐insensitive binding sites on the membranes isolated from the rat myocardium (Kd = 2.0 ± 0.2 and 2.5 ± 0.3 nM, respectively). The [³H]octarphin specific binding to the myocardial membranes was inhibited by unlabeled β‐endorphin (Ki = 1.9 ± 0.2 nM) and immunorphin (Ki = 2.2 ± 0.3 nM). The [³H]immunorphin specific binding with the membranes was inhibited by unlabeled β‐endorphin (Ki = 2.3 ± 0.3 nM) and octarphin (Ki = 2.4 ± 0.3 nM). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin and [Leu⁵]enkephalin. Thus, β‐endorphin, immunorphin and octarphin bind to the common high‐affinity naloxone‐insensitive receptor of the rat myocardial membranes. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Evidence for Negative Charge in the Conduction Pathway of the Cardiac Ryanodine Receptor Channel Provided by the Interaction of K+ Channel N-type Inactivation Peptides

We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH₂-terminal domain of the Shaker K⁺ channel with purified, ryanodine-modified, cardiac Ca²⁺-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec⁻¹μm ⁻¹ for ShB and 2000 ± 200 sec⁻¹μm ⁻¹ for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied potential with the bulk of the voltage-dependence residing in K_off. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As is the case with inactivation and block of K⁺ channels, this is mediated by a large increase in K_on. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites which will contribute to the ion handling properties of this channel. Received: 15 December 1997/Revised: 13 March 1998     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.