The LKEKK synthetic peptide as a ligand of rat intestinal epithelial cell membranes

A tritium-labeled synthetic LKEKK pentapeptide corresponding to the sequences 16–20 of human thymosin-α₁ and 131–135 of human interferon-α₂ was obtained with a specific activity of 28 Ci/mmol. [³H]LKEKK was found to bind with high affinity (K _d 3.7 ± 0.3 nM) to the membranes isolated from epithelial cells of rat small intestinal mucosa. The trypsin treatment of the membranes did not affect the binding, thus supporting the nonprotein nature of the peptide receptor. The binding of the labeled peptide was inhibited by unlabeled thymosin-α₁, interferon-α₂, and cholera toxin B subunit (K _i 4.2 ± 0.4, 3.5 ± 0.3, and 4.7 ± 0.3 nM respectively). The pentapeptide did not affect the adenylate cyclase activity within the concentration range of 1–1000 nM.     

Binding of a synthetic targeting peptide to a mitochondrial channel protein

Membrane crystals of the mitochondrial outer membrane channel VDAC (porin) fromNeurospora crassa were incubated with a 20-amino-acid synthetic peptide corresponding to the N-terminal targeting region of subunit IV of cytochrome oxidase. The peptide caused disordering and contraction of the crystal lattice of the membrane arrays. Also, new stain-excluding features were observed on the peptide-treated arrays which most likely correspond to sites at which the peptide accumulates. The stain exclusion zones associated with binding of the targeting peptide (and with binding of apocytochromec in an earlier study) have been localized on a two-dimensional density map of frozen-hydrated, crystalline VDAC previously obtained by cryo-electron microscopy. The results indicate that both the peptide and cytochromec bind to protein arms which extend laterally between the channel lumens. The finding that imported polypeptides bind to a specific region of the VDAC protein implicates this channel in the process by which precursor proteins are recognized at and translocated across the mitochondrial outer membrane.     

Binding of Zn-chlorin to a synthetic four-helix bundle peptide through histidine ligation

We have used a two histidine-containing synthetic peptide (Sharp et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 10465-10470) as a scaffold to bind Zn(II) chlorin e6 (ZnCe6) through histidine ligation. Protocols for the preparation and purification of the peptide using an Escherichia coli expression system are presented. Size-exclusion chromatography and circular dichroism measurements indicate that the peptide self-assembles into a four-helix bundle protein. Two variants of the peptide lacking either one or both of the histidine residues were used to demonstrate the stoichiometry of ZnCe6 binding. Comparison of the titration profiles determined by UV-vis spectroscopy for the purified one- and two-histidine peptides suggests that the two-histidine peptide can bind two ZnCe6. The binding stoichiometry of ZnCe6 was verified by gel chromatography and native gel electrophoresis using the peptide variant lacking histidine residues as the control. Like many other chlorophyll analogue molecules, ZnCe6 can be photooxidized. The light-induced electron transfer between the ZnCe6-peptide complex and the added phenyl-p-benzoquinone was measured using time-resolved EPR spectroscopy and shown to be faster and have a higher yield than the electron transfer between unbound ZnCe6 and quinone. The implications of constructing a ZnCe6-peptide complex in terms of artificial photosynthesis are discussed.     

Binding of synthetic beta-human atrial natriuretic peptide to cultured rat vascular smooth muscle cells

We have studied the effects of synthetic beta-human atrial natriuretic peptide (beta-hANP), an antiparallel dimer of alpha-hANP, on receptor binding and cGMP generation in cultured rat vascular smooth muscle cells and compared the effects with those of alpha-hANP. Characteristics of temperature-dependent binding and degradation of 125I-beta-hANP were similar to those of 125I-alpha-hANP. Scatchard analysis indicated a single class of binding sites for beta-hANP with a maximal binding capacity one-half that of alpha-hANP. Parallel and antiparallel dimers were equipotent in inhibiting the binding and stimulating intracellular cGMP formation, of which the maximal effect was about one-half that of alpha-hANP. Reverse-phase high performance liquid chromatography revealed that most of beta-hANP added to cells was converted to a small molecular mass component corresponding to alpha-hANP after incubation. These data suggest that the less potent effect of beta-hANP in receptor binding and cGMP generation may be partly accounted for by the possible conversion of beta-hANP to alpha-hANP at the site of target cells.     

Binding mode of SARS-CoV-2 fusion peptide to human cellular membrane

Infection of human cells by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) relies on its binding to a specific receptor and subsequent fusion of the viral and host cell membranes. The fusion peptide (FP), a short peptide segment in the spike protein, plays a central role in the initial penetration of the virus into the host cell membrane, followed by the fusion of the two membranes. Here, we use an array of molecular dynamics simulations that take advantage of the highly mobile membrane mimetic model to investigate the interaction of the SARS-CoV2 FP with a lipid bilayer representing mammalian cellular membranes at an atomic level and to characterize the membrane-bound form of the peptide. Six independent systems were generated by changing the initial positioning and orientation of the FP with respect to the membrane, and each system was simulated in five independent replicas, each for 300 ns. In 73% of the simulations, the FP reaches a stable, membrane-bound configuration, in which the peptide deeply penetrated into the membrane. Clustering of the results reveals three major membrane-binding modes (binding modes 1–3), in which binding mode 1 populates over half of the data points. Taking into account the sequence conservation among the viral FPs and the results of mutagenesis studies establishing the role of specific residues in the helical portion of the FP in membrane association, the significant depth of penetration of the whole peptide, and the dense population of the respective cluster, we propose that the most deeply inserted membrane-bound form (binding mode 1) represents more closely the biologically relevant form. Analysis of FP-lipid interactions shows the involvement of specific residues, previously described as the “fusion-active core residues,” in membrane binding. Taken together, the results shed light on a key step involved in SARS-CoV2 infection, with potential implications in designing novel inhibitors.     

Binding of synthetic peptide TPLVTLFK to nonopioid beta‐endorphin receptor on rat brain membranes

The synthetic peptide TPLVTLFK corresponding to the sequence 12–19 of β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (K_d = 2.6 ± 0.2 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, as well. The [³H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (K_i = 2.4 ± 0.2 nM ) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (K_i = 2.9 ± 0.2 nM ). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [³H]immunorphin with membranes (K_i = 2.8 ± 0.2 nM ). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Activation of human T-lymphocytes

Summary Stereological data of phytohaemagglutinin (PHA)-activated human T-lymphocytes were recorded at intervals (12 to 72 h) together with biochemical (isotope-uptake, lymphotoxin-release) and morphological measurements. About 98 % of the cells were activated 12 h after PHA-stimulation. The activation phase lasted less than 48 h, i.e., cells entering the activation phase within 12 h were at their activation maximum by 48 h. The activated cell increased in size. The nuclear/cytoplasmic-ratio decreased. Most of the cytoplasmic organelles developed in phase with the increase of cytoplasmic volume. After 48 h, mitotic figures were frequently seen. Due to the increasing number of secondary, activated daughter cells, parameters of most cytoplasmic components declined between 48 and 72 h. Structural changes in the nucleus preceded the ³H-leucine uptake, which had not reached its maximum after 72 h of incubation. The ³H-leucine uptake started as early as 12 h after culture initiation, and its increase was proportional to the increasing polyribosome density. No maximum uptake was reached up to 72 h, but the development of structural components related to this uptake was at its maximum at the end of the activation phase (48 h). The formation of bound ribosomes occurred subsequent to the enlargement of the surface of the rough endoplasmic reticulum. Initial polysome formation occurred at the expense of existing free ribosomes.     

Interaction of a synthetic peptide of the interferon alpha-2 C-terminal part with human blood leukocytes. Binding to peripheral blood mononuclear cells.

A biologically active synthetic peptide, 2438, representing the 124-138 amino acid sequence of the human interferon alpha-2 (IFN alpha-2) molecule, which is known to possess IFN-like antiproliferative activity, specifically binds to human blood leukocytes. Scatchard plots reveal two different Kd values, for the 'low' and 'high' affinity binding. The interaction of the 125I-labelled peptide 2438 with the cells is not impaired by human IFN alpha-2 or cholera toxin.     

Interaction of synthetic peptide octarphin with human blood lymphocytes

The synthetic peptide octarphin (TPLVTLFK, fragment 12–19 of β-endorphin), a selective agonist of nonopioid β-endorphin receptor, was prepared with specific activity 28 Ci/mmol. The binding of [³H]octarphin to T and B lymphocytes isolated from the blood of donors was studied. It was found that [³H]octarphin binds both to T and B cells with high affinity: K _d = 3.0 ± 0.2 and 3.2 ± 0.3 nM, respectively. The specific binding of [³H]octarphin to T and B lymphocytes was competitively inhibited by unlabeled β-endorphin (K _i = 1.9 ± 0.2 and 2.2 ± 0.3 nM, respectively) and was not inhibited by unlabeled naloxone, [Met⁵]enkephalin, [Leu⁵]enkephalin, α-endorphin, and γ-endorphin. Thus, T and B lymphocytes of human blood possess a nonopioid β-endorphin receptor whose binding is provided by the fragment 12–19 (the octarphin sequence).     

Binding of netropsin to DNA and synthetic polynucleotides

The interaction of netropsin with DNA and synthetic polydeoxyribonucleotides was studied by absorption spectrophotometry and circular dichroism. The results are consistent with a model in which a netropsin molecule occupies five base pairs in binding and carries three reaction sites each capable of interacting with one AT base pair. We associate these reaction sites with the antibiotic peptide groups which probably interact with AT base pairs by a hydrogen bonding mechanism.     

Binding of sulfobromophthalein to rat and human ligandins: characterization of a binding-site peptide

Photoaffinity techniques were employed to affect the covalent binding of [35S]sulfobromophthalein to proteins of rat and human liver cytosol. In rat liver cytosol at low concentrations, sulfobromophthalein bound to the 22 kDa subunit of ligandin. In human liver cytosol, binding to a 23.5 kDa subunit was observed. At higher concentrations, sulfobromophthalein also bound to 12, 23.5, 37, and 42 kDa peptides. When the peptides resulting from CNBr cleavage of [35S]sulfobromophthalein-ligandin complex were resolved by high-performance liquid chromatography, radioactivity was associated with two peptides. The peptide containing 80% of the radioactivity was isolated and characterized. Its molecular weight is 3.4 kDa, it contains the single tryptophan residue of ligandin and has a glutamate (glutamine) as the N-terminal amino acid.     

Binding of a histidine-rich peptide to Porphyromonas gingivalis

Porphyromonas gingivalis 381 cells were incubated with 125I-histidine-rich polypeptide (histatin) 5 in the presence or absence of unlabeled histatin 5, to evaluate the histatin-binding capacity of the cells. The binding of histatin 5 was rapid, reversible, saturable and specific. The number of histatin 5-binding sites per cell was 3,600, and the dissociation constant (Kd) was in the order of 10(-6) M. These findings suggest that histatin interacts with certain bacterial cells through specific binding sites on their surface, and will allow the development of a histatin radioreceptor assay.     

A synthetic peptide of human apoprotein E with antibacterial activity

In recent years, several endogenous mammalian antibacterial peptides have been described. An amphipathic cationicalpha-helix is a common feature in many cases; therefore, other peptides with this characteristic might also possess antibiotic activity. In fact, a 30-mer peptide of apoprotein E 133-162 (LRVRLASHLRKLRKRLLRDADDLQKRLAVY) was found to have antibiotic activity comparable to those of a classic antibiotic (Gentamicin) and a neutrophil-derived antibiotic peptide (CAP18). Calculation of cationicity, hydrophobicity, and hydrophobic moment and the helical wheel diagram of apoprotein E 133-162 revealed close similarities to CAP18.     

Non-cross-reactive monoclonal antibodies to human chorionic gonadotropin generated after immunization with a synthetic peptide

Noncross-reactive monoclonal antibodies specific for human chorionic gonadotropin (hCG) were obtained after pre-selection for submolecular specificity with a synthetic peptide immunogen. Mice were immunized with a synthetic peptide representing a segment unique to the beta-subunit of hCG (amino acid residues 109-145), conjugated to diphtheria toxoid. We then derived nine different hybridomas that secreted monoclonal antibodies reactive with both native hCG and isolated C-terminal peptide, after somatic cell hybridization of immune spleen cells with a nonsecretory myeloma cell line. None of the nine monoclonal antibodies, termed beta-hCG-CTPa1----a9, reacted with hLH, hFSH, or hTSH, although these pituitary hormones display extensive amino acid sequence homology with hCG. The noncross-reactive anti-beta-hCG monoclonal antibodies show apparent association constants on the order of 10(9) to 10(10) M-1. A sandwich-type enzyme-linked immunosorbent assay was set up with cut-off values of around 5 mIU/ml. These antibodies might have important implications for: a) improving the diagnosis and clinical management of pregnancy; b) monitoring the course of development of carcinomas which secrete the hormone, through in vitro assays or in vivo radioimmunodetection; c) evaluating the antibodies' therapeutic potential against such carcinomas; d) studying the biologic functions of the C-terminal segment of beta-hCG; and e) addressing the anti-fertility effect of antibodies raised against that segment.     

Effect of synthetic beta-carotene on the formation of cytolytic T-lymphocytes

The effect of intraperitoneal injection of beta-carotene in different doses on the formation of cytolytic T lymphocytes (CTL) in a one-way mixed lymphocyte culture (MLC) of allogeneic mice was studied. The maximal cytotoxic activity of lymphocytes was attained in the MLC with splenocytes of mice which received 10 mg/kg beta-carotene 6 days before experimentation. The correlation was studied between the beta-carotene ability to stimulate CTL formation and antineoplastic activity. It was discovered that injection of beta-carotene in doses and times provoking maximal CTL induction had no effect on the animals' lifespan and the size of transplanted sarcoma 180.     

Binding of chemotactic peptide to the outer surface and to whole human spermatozoa with different affinity states

Binding of N-formyl-methionyl-L-leucyl-[³H]phenylalanine (fML[³H]Ph) to human ejaculated spermatozoa and to its isolated plasma membrane was studied. Our data confirm the presence of specific receptors for f-MLPh in the human spermatozoa and suggest that whole spermatozoa receptors exist in two affinity states, one high-affinity, low-capacity specific receptor (K_d = 12.3 ± 0.5 nM, n = 22,285 ± 65,008 binding sites per sperm cell) and a second one (K_d = 700 ± 47 nM) that is not saturable, indicating a low-affinity, high-capacity nonspecific site. In contrast, sperm membrane showed only one class of binding site (K_d = 6.4 ± 0.12 nM), which was statistically different from that of the high-affinity binding site of intact spermatozoa. To explain this difference we discuss the possibility that first, the two binding affinities represent two interconvertible states of a single receptor population, which, depending on the metabolic activity of spermatozoa, may change its physicochemical properties; or second, they reflect two different processes, binding and/or transport into the spermatozoa.