Brain natriuretic peptide receptors in the rat peripheral sympathetic ganglia

High affinity binding sites for brain natriuretic peptide were characterized in the rat superior cervical ganglia by quantitative autoradiography. In addition, the peptide increased the formation of cyclic GMP in the ganglia in vitro. Brain natriuretic peptide displaced atrial natriuretic peptide from its binding sites. Our results suggest that brain natriuretic peptide and atrial natriuretic peptide may share physiologically active receptors in sympathetic ganglia. Brain natriuretic peptide may modulate the synaptic transmission in sympathetic ganglia, in addition or in conjunction with atrial natriuretic peptide.     

Improving mass and liquid chromatography based identification of proteins using bayesian scoring

We present a method for peptide and protein identification based on LC-MS profiling. The method identified peptides at high-throughput without expending the sequencing time necessary for CID spectra based identification. The measurable peptide properties of mass and liquid chromatographic elution conditions are used to characterize and differentiate peptide features, and these peptide features are matched to a reference database from previously acquired and archived LC-MS/MS experiments to generate sequence assignments. The matches are scored according to the probability of an overlap between the peptide feature and the database peptides resulting in a ranked list of possible peptide sequences for each peptide submitted. This method resulted in 6 times more peptide sequence identifications from a single LC-MS analysis of yeast than from shotgun peptide sequencing using LC-MS/MS.     

Changes in lipid mobility associated with alamethicin incorporation into membranes

The binding state of the antibiotic peptide alamethicin with phospholipid bilayers was investigated in terms of the changes induced in lipid mobility. Fluorescence anisotropy was used for the study. It was found that an increase in peptide concentration induced different changes in lipid mobility above and below a critical peptide concentration. This concentration was also critical for an increase in the cooperative binding of the peptide, as detected by circular dichroism. Above the critical peptide concentration, the mobility of both lipid regions, around the polar head and hydrocarbon chain, became restricted with an increased peptide concentration. Below the critical level, however, an increased peptide concentration induced a "wobbling" of the lipid hydrocarbon chain. These results show that an increase in the cooperative binding of the peptide is accompanied by a change in the dominant configuration of the binding peptide. When the binding peptide increases, the dominant configuration appears to shift from surface association to deep incorporation within the membrane. This shift in configuration means that in the formation of ion-conductive pores, voltage-driven insertion of the peptide is a prominent step below a critical peptide concentration.     

Peptide heterodimers for molecular imaging

One main issue with peptide-based molecular imaging probes is their relatively low tumor affinity and short retention time. To improve peptide binding affinity, multivalency approach has been introduced. Traditionally, this approach involves the use of peptide homodimers or homomultimers in which peptide ligands of the same type are constructed with suitable linkers. Recently, a new approach using peptide heterodimers has emerged as a promising method for targeting multi-receptor over-expressed tumor cells. Significant affinity enhancements have been observed with peptide heterodimers compared with their parent peptide monomers. In a peptide heterodimer, two different peptide ligands capable of targeting two different receptors are covalently linked. The binding modes of peptide heterodimers can be monovalent or bivalent depending on whether simultaneous binding of two ligands can be achieved. Increased local ligand concentration and improved binding kinetics contribute to enhanced binding in both monovalent- and bivalent binding modes, while multivalency effect also plays an important role in bivalent binding mode. As many tumors overexpress multiple receptors, more peptide heterodimer-based molecular imaging probes are expected to be developed in future. This review article will discuss the peptide homodimers and heterodimers for molecular imaging with special emphasis on peptide heterodimers.     

Biophysical studies and anti-growth activities of a peptide, a certain analog and a fragment peptide derived from alpha-fetoprotein.

A chemically synthesized 34-amino acid peptide, an analog, and a fragment of the peptide have been purified and studied. Biophysical studies were carried out to determine some of the metal ion binding properties of the original peptide and an analog of this parent peptide, in which the two histidine residues were replaced by alanines. As shown by visible absorption spectroscopy, Co (II) forms a complex with the parent peptide, but not with the analog peptide, and one or two histidines in the parent peptide are ligands for Co (II) ion binding. The effects on disulfide bond formation in the peptide by Zn (II) and Co (II) ions were also examined for this analog. Anti-growth assays were performed using the original cysteine-containing peptide with Zn (II) ion complexed to the peptide through the two cysteine residues. These rat uterine growth assays showed that the complexing of Zn (II) ion to the peptide maintained the anti-growth activity of the peptide, while gel-filtration experiments showed the zinc ions maintained the peptide in its anti-growth form indefinitely in solution. A saliently important part of this research was the discovery that a fragment of the peptide consisting of a middle sequence of 14 amino acids was found to have significant anti-growth activity in the rat uterine assay. Its activity suggested that this fragment might be considered a viable candidate for testing in anti-cancer protocols.     

The construction of a bioactive peptide database in Metazoa

Bioactive peptides play critical roles in regulating most biological processes in animals, and have considerable biological, medical and industrial importance. A number of peptides have been discovered usually based on their biological activities in vitro or based on their sequence similarities in silico. Through searches in Swiss-Prot and Trembl protein databases using BLAST alignment tools and other in silico methods, all currently known bioactive peptides and their precursor proteins are extracted. In addition, 132 recently discovered putative peptide genes in Drosophila as well as their orthologs in other species are collected. In total, 20 027 bioactive peptides from 19 438 precursor proteins covering 2820 metazoan species are retained, and they, respectively, make up a peptide and a peptide precursor database. The peptides and peptide precursor proteins are further classified into 373 families, 178 of which are represented by Prosite Pfam or Smart motifs, or by typical peptide motifs that have been constructed recently. The remaining 195 families are novel peptide families. The motifs characterizing the 178 peptide families are saved into a peptide motif database. The peptide, peptide precursor and peptide motif databases (version 1.0) are the most complete peptide, precursor and peptide motif collection in Metazoa so far. They are available on the WWW at http://www.peptides.be/.     

Cationization of protein antigens. VI. Effects of cationization on the immunoregulatory properties of a bovine serum albumin peptide, a.a. 506-589.

Cationization of bovine serum albumin (BSA) causes a profound increase in its immunogenicity. To establish if immunoregulatory properties of an immunosuppressive peptide are affected by cationization, a BSA peptide, a.a. 506-583, was cationized and tested for its immunogenic properties. A greatly reduced amount of cationized peptide compared to native peptide was required to stimulate BSA-primed T cells to proliferate in vitro. Mice primed with the cationized peptide administered with an adjuvant responded with a significantly greater anti-BSA response than mice immunized with the native form of the peptide. In the absence of an adjuvant i.v. or i.p. administration of the native peptide was immunosuppressive, while the cationized form was immunoenhancing. Both forms of the peptide stimulated in vivo induction of L3T4+ (CD4), and Lyt-2+ (CD8) T cells. Removal of Lyt-2+ T cells from lymph node cultures following immunization with the native peptide caused a significant increase in the proliferation of the remaining T cells. This increase was not observed when the mice were immunized with the cationized peptide. No major BSA B cell determinants were present within the peptide sequence. Mice immunized with the peptide exhibited a negligible anti-BSA antibody response compared to those immunized with the whole BSA molecule. Furthermore, the peptide did not inhibit anti-BSA antibody binding to BSA. We demonstrated that cationization modifies immunoregulatory properties of an immunosuppressive BSA-derived peptide.     

A Novel Technique for the Effective Production of Short Peptide Analogs from Concatameric Short Peptide Multimers

We designed a basic unit of the modified chicken gonadotropin releasing hormone II (cGnRH-II) peptide containing a trypsin cleavable linker peptide at both ends of the original peptide. We made a synthetic DNA coding for the modified cGnRH-II peptide with asymmetric and complementary cohesive ends of linker nucleotides. A tandemly repeated DNA cassette for the expression of concatameric short peptide multimers was constructed by ligating the basic units. The expressed peptide multimers were purified and subject to amino-terminal sequence analysis, which displayed the amino acid sequences expected from the designed nucleotides of the expression cassette. The monomeric cGnRH-II peptide analogs were generated after trypsin digestion. The present results showed that the technique developed for the production of the concatameric peptide multimers with cleavable linker peptides can be generally applicable to the production of short peptide analogs.     

Limited validity of group additivity for the folding energetics of the peptide group

The principle of group additivity is a standard feature of analyses of the energetics of protein folding, but it is known that it may not always be valid for the polar peptide group. The neighboring residue effect shows that group additivity is not strictly valid for a heteropeptide. We show here that group additivity fails seriously for peptide groups close to either peptide end, even for a homopeptide that has blocked end groups with no formal charges involved. The failure of group additivity is caused by the electrostatic character of the solvation of peptide polar groups and is illustrated with values of the electrostatic solvation free energy (ESF) calculated by DelPhi. Solvation free energies and enthalpies are known experimentally for monoamides and are often used to model the solvation of peptide groups, but ESF results show that monoamide values are very different from those of peptide groups. A main cause of the difference is that peptide solvation depends on the dipole-dipole interactions made between adjacent peptide groups, which vary with peptide conformation. Ligands that interact with the peptide backbone by an electrostatic mechanism could show a similar peptide end effect, and hydrogen exchange results from the literature confirm that exchange rates are position-dependent close to peptide ends.     

The toxic alpha‐gliadin peptide 31–43 enters cells without a surface membrane receptor

Alpha‐gliadin peptide 31–43 is considered to be the main peptide responsible for the innate immune response in celiac disease patients. Recent evidence indicates that peptide 31–43 rapidly enters cells and interacts with the early endocytic vesicular compartment. However, the mechanism of its uptake is not completely understood. Our aim is to characterize, isolate and identify possible cell surface proteins involved in peptide 31–43 internalization by Caco‐2 cells. In this study, we used a chemical cross‐linker to block peptide 31–43 on cell surface proteins, and pulled‐down peptide‐proteins complexes using antibodies raised against peptide 31–43. Through this experimental approach, we did not observe any specific complex between cell proteins and peptide 31–43 in Coomassie‐stained denaturating gels or by Western blotting. We also found that type 2 transglutaminase was not necessary for peptide 31–43 internalization, even though it had a regulatory role in the process. Finally, we demonstrated that peptide 31–43 did not behave as a classical ligand, indeed the labeled peptide did not displace the unlabeled peptide in a competitive binding assay. On the basis of these findings and of previous evidence demonstrating that peptide 31–43 is able to interact with a membrane‐like environment in vitro, we conclude that membrane composition and organization, rather than a specific receptor protein, may have a major role in peptide 31–43 internalization by cells.     

Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation

A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones.     

Processing of exogenous antigens for presentation by class I MHC molecules involves post-Golgi peptide exchange influenced by peptide-MHC complex stability and acidic pH

Vacuolar alternate class I MHC (MHC-I) Ag processing allows presentation of exogenous Ag by MHC-I molecules with binding of antigenic peptides to post-Golgi MHC-I molecules. We investigated the role of previously bound peptides and their dissociation in generating peptide-receptive MHC-I molecules. TAP1-knockout macrophages were incubated overnight with an initial exogenous peptide, producing a large cohort of peptide-K(b) complexes that could influence subsequent peptide dissociation/exchange. Initial incubation with FAPGNYPAL, KVVRFDKL, or RGYVYQGL enhanced rather than reduced subsequent binding and presentation of a readout peptide (SIINFEKL or FAPGNYPAL) to T cells. Thus, K(b) molecules may be stabilized by an initial (stabilizing) peptide, enhancing their ability to bind readout peptide and implicating peptide dissociation/exchange. In contrast, incubation with SIINFEKL as stabilizing peptide reduced presentation of readout peptide. SIINFEKL-K(b) complexes were more stable than other peptide-K(b) complexes, which may limit their contribution to peptide exchange. Stabilizing peptides (FAPGNYPAL, KVVRFDKL, or RGYVYQGL) enhanced alternate MHC-I processing of HB101.Crl-OVA (Escherichia coli expressing an OVA fusion protein), indicating that alternate MHC-I Ag processing involves peptide dissociation/exchange. Stabilizing peptide enhanced processing of HB101.Crl-OVA more than presentation of exogenous OVA peptide (SIINFEKL), suggesting that peptide dissociation/exchange may be enhanced in the acidic phagosomal processing environment. Furthermore, exposure of cells to acidic pH increased subsequent binding and presentation of readout peptide. Thus, peptide dissociation/exchange contributes to alternate MHC-I Ag processing and may be influenced by both stability of peptide-MHC-I complexes and pH.     

Effect of insulin on ATP-citrate lyase phosphorylation: regulation of peptide A and peptide B phosphorylations

Insulin decreases multifunctional protein kinase (MFPK) activity in rat adipose tissue [Ramakrishna, S., & Benjamin, W. B. (1988) J. Biol. Chem. 263, 12677-12681]. Insulin also decreases the phosphorylation of peptide B but increases the phosphorylation of peptide A of ATP-citrate lyase (ATP-CL). The mechanism for this increase in peptide A phosphorylation was studied with purified ATP-CL from control and insulin- and isoproterenol-treated fat pads by using MFPK and the catalytic subunit of cAMP-dependent protein kinase (A-kinase). ATP-CL purified from insulin-treated fat pads is a better substrate for phosphorylation by MFPK compared to controls. This result is consistent with the hypothesis that insulin action decreases peptide B phosphorylation. To determine if the degree of phosphorylation at peptide B affects the phosphorylation rate of peptide A by A-kinase, ATP-CL was prepared with determined phosphate contents of peptides A and B. ATP-CL with a low phosphate content at peptide B is a better substrate for phosphorylation at peptide A by A-kinase than is ATP-CL with a high phosphate content at peptide B. These results suggest that the insulin-induced increase in ATP-CL phosphorylation at peptide A is due to a decrease in peptide B phosphorylation. ATP-CL prepared from isoproterenol-treated fat pads is also a better substrate for phosphorylation at peptide B by MFPK than controls. This increase in phosphorylation at peptide B by MFPK is due to positive second-site regulation by the isoproterenol-induced increase in peptide A phosphorylation.     

Affinity purification of proteins using ligands derived from peptide libraries

Peptide libraries can be used to identify ligands that bind specifically to a desired protein. These peptides may have significant advantages as specific ligands for affinity chromatography separations. This article describes the use of one of such peptide, Try-Asn-Phe-Glu-Val-Leu, as a ligand for the purification of S-protein using affinity chromatography. General strategies for peptide immobilization are discussed and the conditions for peptide immobilization to Emphazetrade mark gel are optimized. The effects of peptide orientation and peptide densities on protein binding are studied. Results indicate that the peptide affinity is not affected by the orientation of the peptide during immobilization, but association constants can be reduced by one order of magnitude when compared with the values in solution.With increased peptide density, the protein binding capacity of the gel increases, but both the percentage of peptide utilization and apparent binding constant between immobilized peptide and S-protein decrease. S-protein is separated from a mixture with BSA via affinity chromatography using specific elution with the peptide in solution.Finally, direct purification of S-protein from an enzymatic digestion mixture of ribonuclease A is demonstrated.(c) 1995 John Wiley & Sons, Inc.     

Amino acid residues on the surface of soybean 4-kDa peptide involved in the interaction with its binding protein.

Soybean 4-kDa peptide, a hormone-like peptide, is a ligand for the 43-kDa protein in legumes that functions as a protein kinase and controls cell proliferation and differentiation. As this peptide stimulates protein kinase activity, the interaction between the 4-kDa peptide (leginsulin) and the 43-kDa protein is considered important for signal transduction. However, the mechanism of interaction between the 4-kDa peptide and the 43-kDa protein is not clearly understood. We therefore investigated the binding mechanism between the 4-kDa peptide and the 43-kDa protein, by using gel-filtration chromatography and dot-blot immunoanalysis, and found that the 4-kDa peptide bound to the dimer form of the 43-kDa protein. Surface plasmon resonance analysis was then used to explore the interaction between the 4-kDa peptide and the 43-kDa protein. To identify the residues of the 4-kDa peptide involved in the interaction with the 43-kDa protein, alanine-scanning mutagenesis of the 4-kDa peptide was performed. The 4-kDa peptide-expression system in Escherichia coli, which has the ability to install disulfide bonds into the target protein in the cytoplasm, was employed to produce the 4-kDa peptide and its variants. Using mass spectrometry, the expressed peptides were confirmed as the oxidized forms of the native peptide. Surface plasmon resonance analysis showed that the C-terminal hydrophobic area of the 4-kDa peptide plays an important role in binding to the 43-kDa protein.     

Lipid-Induced Organization of a Primary Amphipathic Peptide: A Coupled AFM-Monolayer Study

To better understand the nature of the mechanism involved in the membrane uptake of a vector peptide, the interactions between dioleoylphosphatidylcholine and a primary amphipathic peptide containing a signal peptide associated with a nuclear localization sequence have been studied by isotherms analysis of mixed monolayers spread at the air-water interface. The peptide and the lipid interact through strong hydrophobic interactions with expansion of the mean molecular area that resulted from a lipid-induced modification of the organization of the peptide at the interface. In addition, a phase separation occurs for peptide molar fraction ranging from about 0.08 to 0.4 Atomic force microscopy observations made on transferred monolayers confirm the existence of phase separation and further reveal that mixed lipid-peptide particles are formed, the size and shape of which depend on the peptide molar fraction. At low peptide contents, round-shaped particles are observed and an increase of the peptide amount, simultaneously to the lipidic phase separation, induces morphological changes from bowls to filamentous particles. Fourier transform infrared spectra (FTIR) obtained on transferred monolayers indicate that the peptide adopts a β-like structure for high peptide molar fractions. Such an approach involving complementary methods allows us to conclude that the lipid and the peptide have a nonideal miscibility and form mixed particles which phase separate. Received: 31 July 1998/Revised: 4 November 1998     

Protease IV, a cytoplasmic membrane protein of Escherichia coli, has signal peptide peptidase activity

During export of the outer membrane lipoprotein across the cytoplasmic membrane, the signal peptide of the lipoprotein undergoes two successive proteolytic attacks, cleavage of the signal peptide by signal peptidase and digestion of the cleaved signal peptide by an enzyme called signal peptide peptidase(s) (Hussain, M., Ichihara, S., and Mizushima, S. (1982) J. Biol. Chem. 257, 5177-5182; Hussain, M., Ozawa, Y., Ichihara, S., and Mizushima, S. (1982) Eur. J. Biochem. 129, 233-239). Here we report that protease IV, a cytoplasmic membrane protease, exhibits the signal peptide peptidase activity. The signal peptide peptidase activity was cofractionated with protease IV throughout the entire process of purification of the latter enzyme. Only the signal peptide was digested by the peptidase among membrane proteins. Both the signal peptide peptidase activity and the protease IV activity were inhibited to similar degrees by antipain, leupeptin, chymostatin, and elastatinal that are known to inhibit the signal peptide peptidase activity in the cell envelope. From these results we conclude that protease IV is the signal peptide peptidase that is responsible for signal peptide digestion in the cytoplasmic membrane. The peptidase attacked the signal peptide only after its release from the precursor protein.     

Combinatorial solid phase peptide synthesis and bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.     

A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family. Molecular cloning, expression, and biological activity

A new peptide family (endothelin (ET] consisting of three members in mammals appears to be present in mice according to genomic Southern blot analysis. Two ET-related genes were identified by cloning and sequence analysis of a mouse genome. One encoded a peptide identical to porcine and human vasoconstrictor peptide ET, and the other encoded a novel peptide differing from ET in 3 amino acid residues, with 4 cysteines in the same positions as in ET. This novel peptide was synthesized and confirmed to have in vivo pressor activity similar to that of ET. Northern blot analysis, however, indicated the gene of this novel peptide to be expressed only in the intestine, and not in other tissues or cell lines, or endothelial cells. Furthermore, the peptide evoked a strong contractile response in the guinea pig ileum. This peptide may thus be reasonably classified as a gastrointestinal peptide, vasoactive intestinal contractor.     

ABRF ESRG 2006 Study: Edman Sequencing as a Method for Polypeptide Quantitation

The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for protein and peptide analysis. These studies provide a means for participating laboratories to compare their analyses against a benchmark of those from other laboratories that provide this valuable service. The main purpose of the 2006 study was to determine how accurate Edman sequencing is for quantitative analysis of polypeptides. Secondarily, participants were asked to identify a modified amino acid residue, N-epsilon-acetyl lysine [Lys(Ac)], present within one of the peptides. The ESRG 2006 peptide mixture consisted of three synthetic peptides. The Peptide Standards Research Group (PSRG) provided two peptides, with the following sequences: KAQYARSVLLEKDAEPDILELATGYR (peptide B), and RQAKVLLYSGR (peptide C). The third peptide, peptide C*, synthesized and characterized by ESRG, was identical to peptide C but with acetyl lysine in position 4. The mixture consisted of 20% peptide B and 40% each of peptide C and its acetylated form, peptide C*. Participating laboratories were provided with two tubes, each containing 100 picomoles of the peptide mixture (as determined by quantitative amino acid analysis) and were asked to provide amino acid assignments, peak areas, retention times at each cycle, as well as initial and repetitive yield estimates for each peptide in the mixture. Details about instruments and parameters used in the analysis were also collected. Participants in the study with access to a mass spectrometer (MALDI-TOF or ESI) were asked to provide information about the relative peak areas of the peptides in the mixture as a comparison with the peptide quantitation results from Edman sequencing. Positive amino acid assignments were 88% correct for peptide C and 93% correct for peptide B. The absolute initial sequencing yields were an average of 67% for peptide (C+C*) and 65.6 % for peptide B. The relative molar ratios determined by Edman sequencing were an average of 4.27 (expected ratio of 4) for peptides (C+C*)/B, and 1.49 for peptide C*/C (expected ratio of 1); the seemingly high 49% error in quantification of Lys(Ac) in peptide C* can be attributed to commercial unavailability of its PTH standard. These values compare very favorably with the values obtained by mass spectrometry.