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PubMed2001

Reversed-phase electrochromatography of amino acids and peptides using porous polymer monoliths.

Efficient and rapid separation of minute levels of amino acids and bioactive peptides is of significant importance in the emerging field of proteomics as well as in the clinical and pharmaceutical arena. We have developed novel UV-initiated acrylate-based porous polymer monoliths as stationary phases for capillary- and chip-electrochromatography of cationic, anionic, and neutral amino acids and peptides, followed by absorbance or laser-induced fluorescence detection. The rigid monoliths are cast-to-shape and are tunable for charge and hydrophobicity. For separations at low pH, monoliths containing quaternary amine moieties were used to achieve high electroosmotic flow, and for high pH separations monoliths with acidic sulfonic acid groups were employed. Efficient and reproducible separations of phenylthiohydantoin-labeled amino acids, native peptides, and amino acids and peptides labeled with naphthalene-2,3-dicarboxaldehyde (NDA) were achieved using both negatively- and positively-charged polymer monoliths in capillaries. Separation efficiencies in the range of 65,000-371,000 plates/m were obtained with capillary electrochromatography. Buffer composition and the degree of column hydrophobicity were studied systematically to optimize separations. The monoliths were also cast in the microchannels of glass chips and electrochromatographic separation followed by laser-induced fluorescence detection of three NDA-labeled bioactive peptides was obtained.

Amino Acids↗

PubMed2004

Bioactivity of a peptide derived from acetylcholinesterase: involvement of an ivermectin-sensitive site on the alpha 7 nicotinic receptor.

A peptide fragment of 14 amino acids, derived from the C-terminus of acetylcholinesterase (AChE), might underlie the now well-established noncholinergic effects of the enzyme. This peptide is bioactive in a variety of systems including acute (brain slices) and chronic (organotypic culture) preparations of hippocampus, a pivotal area in Alzheimer's disease (AD); invariably, the action of the peptide is mediated specifically via an as yet unknown receptor. In this study, the allosteric alpha 7 agent, ivermectin (IVM), had a modest inhibitory effect, whilst that of the peptide was significantly more marked. However, ivermectin rendered ineffective the toxicity of high doses of the peptide, that is, when the two were co-applied, only the smaller effects of ivermectin were seen. Ivermectin, therefore, is presumably acting at a site that is identical to, or at least strongly interactive with, the normal binding site for AChE-peptide. This observation could have important implications for eventual therapeutic targeting of the action of AChE-peptide, in neurodegeneration.

Acetylcholinesterase↗

PubMed2003

Rapid detection and identification of angiotensin-converting enzyme inhibitors by on-line liquid chromatography-biochemical detection, coupled to electrospray mass spectrometry.

An analytical method based on on-line liquid chromatography-biochemical detection (LC-BCD) coupled to electrospray mass spectrometry was developed for the detection and identification of angiotensin-converting enzyme (ACE) inhibitors in complex mixtures, such as hydrolyzed whey proteins. ACE inhibitory activity was detected by coupling a homogeneous, substrate conversion based bioassay on-line to high-performance liquid chromatography (HPLC). Chemical information was obtained by directing part of the HPLC effluent towards a mass spectrometer. After correlating the biochemical and chemical data, the accurate molecular masses of the bioactive peptides were used as search queries in protein databases. Combined with the recorded mass spectrometry (MS)-MS fingerprints, bioactive peptides were selected from the database search results. The results of LC-BCD-MS analyses were verified by establishing a bioactivity balance. Reference samples, containing several peptides at concentration levels similar to those observed in the hydrolyzed milk samples, were analyzed by LC-BCD-MS. High recoveries of biological activity were obtained, indicating that the correct ACE inhibitors were identified and that no co-elution of significantly bioactive molecules had occurred. Approximately, 30 ACE inhibitors were detected and identified. IC50 values of ACE inhibitors, reported in literature, ranged between 43 and 580 microM.

Amino Acid Sequence↗

PubMed2000

Peptides comprising the bulk of rat brain extracts: isolation, amino acid sequences and biological activity.

Chromatographic separation of rat brain extracts followed by automatic Edman sequencing of the major individual components resulted in identification of 61 endogenous peptides derived from known functional proteins (hemoglobin, myelin basic protein, cytochrome-c oxidase, etc.) or unknown precursors. The results are compared with the data obtained earlier for bovine brain. Although the sequences of bovine and rat hemoglobin contain about 20% of amino acid substitutions, the families of structurally related peptides are very similar in both extracts. Several other proteins also give rise to identical or closely related peptide fragments in the two mammalian species. The outlined similarity extends almost exclusively to the most abundant peptides present in the extracts. The minor components show less overlap. Four hemoglobin-derived peptides isolated from rat brain were shown to be biologically active in tumor cells. Eleven are identical to bioactive peptides from other species. Ten structurally overlap with bioactive peptides from other sources. The data obtained show similar biosynthetic pathways of pool components in different species, the resultant peptides being aimed at fulfilling related functions.

Amino Acid Sequence↗

PubMed1996

X-ray crystallography of peptides: the contributions of the Italian laboratories.

The review article summarizes the most relevant solid state structural and conformational results obtained in the laboratories involved in Italy in the studies of synthetic and natural peptides by x-ray diffraction analyses. Some of the topics will include research studies carried out in other European countries, whereas in other cases studies carried out in Italy will be included in other review articles included in this volume. The review deals with peptides containing symmetrically achiral and unsymmetrically chiral C alpha,alpha-dialkylated glycine residues, peptides containing beta-alanine residues, alpha,beta-dehydroamino acid residues, and aminosuccinyl residues, peptides containing the thioamide surrogate, heterochiral peptides and several bioactive peptides systems with the proposed relationships between function and structure.

Amino Acid Sequence↗

PubMed1994

Isolation and identification of cecropin antibacterial peptides from the extracellular matrix of the insect integument.

A method for the screening and isolation of antibacterial peptides from the extracellular insect cuticle using ultrafiltration and reverse-phase high-performance liquid chromatography is presented. Using this method we were able to conveniently isolate cuticular antibacterial peptides belonging to the cecropin family in 1 day. The procedure should facilitate the rapid screening of antibacterial peptides or other bioactive peptides from extracellular matrices.

Amino Acid Sequence↗

PubMed2002

Proinsulin C-peptide and its analogues induce intracellular Ca2+ increases in human renal tubular cells.

Based on the findings that proinsulin C-peptide binds specifically to cell membranes, we investigated the effects of C-peptide and related molecules on the intracellular Ca2+ concentration ([Ca2+]i) in human renal tubular cells using the indicator fura-2/AM. The results show that human C-peptide and its C-terminal pentapeptide (positions 27-31, EGSLQ), but not the des (27-31) C-peptide or randomly scrambled C-peptide, elicit a transient increase in [Ca2+]i. Rat C-peptide and rat C-terminal pentapeptide also induce a [Ca2+]i response in human tubular cells, while a human pentapeptide analogue with Ala at position 1 gives no [Ca2+]i response, and those with Ala at positions 2-5 induce responses with different amplitudes. These results define a species cross-reactivity for C-peptide and demonstrate the importance of Glu at position 1 of the pentapeptide. Preincubation of cells with pertussis toxin abolishes the effect on [Ca2+]i by both C-peptide and the pentapeptide. These results are compatible with previous data on C-peptide binding to cells and activation of Na-,K+ATPase. Combined, all data show that C-peptide is a bioactive peptide and suggest that it elicits changes in [Ca2+]i via G-protein-coupled pathways, giving downstream enzyme effects.

Animals↗

PubMed1999

Design and application of multimodular peptide synthetases.

Nonribosomal peptide synthetases produce bioactive peptides of great structural diversity. Their modular organization makes them amenable to the construction of hybrid enzymes that synthesize novel products. New strategies for combinatorial approaches are being developed from the recent advances in nonribosomal peptide synthesis on the genetic, biochemical and structural level.

Biotechnology↗

PubMed1992

Abundant expression of parathyroid hormone-related protein in human amnion and its association with labor.

In animal models, parathyroid hormone-related protein (PTHrP) increases placental calcium transport and inhibits contraction of uterine smooth muscle. The present studies were undertaken to characterize the expression of PTHrP in human uteroplacental tissues. PTHrP mRNA was identified by Northern analysis as a single species (approximately 1.8 kilobases) in human amnion, chorion, placenta, decidua, and myometrium. The most abundant signal was seen in amnion, where it was 10-400 times that in the other uteroplacental tissues. PTHrP mRNA abundance was decreased in amnion (but not in the other tissues) following the onset of labor (P less than 0.001). PTHrP mRNA in amnion appeared to be translated to a bioactive peptide, as PTHrP bioactivity and immunoreactive PTHrP in amnion correlated closely with PTHrP mRNA content (r = 0.86 and 0.95, respectively; P less than 0.05 and P less than 0.01). Amniotic fluid contained PTHrP, 21 +/- 6 pmol/liter (n = 10) at 16 weeks and 41 +/- 9 pmol/liter (n = 7) at 38 weeks (P = 0.05). These concentrations equaled or exceeded those found in plasma of patients with hypercalcemia secondary to PTHrP. After rupture of the fetal membranes, PTHrP mRNA in amnion was decreased by 78% (P less than 0.0001). This decrease appeared to be specific for PTHrP mRNA, as glyceraldehyde-3-phosphate dehydrogenase mRNA was unchanged following rupture of membranes. Like PTHrP mRNA, PTHrP bioactivity and immunoreactive PTHrP in amnion decreased significantly following rupture of membranes (P less than 0.03 and P less than 0.01, respectively). Since PTHrP is a potent antagonist of uterine muscle contraction, the decrease of PTHrP following rupture of the fetal membranes may play a key role in the onset of labor.

Amnion↗

PubMed1999

Kalirin, a multifunctional PAM COOH-terminal domain interactor protein, affects cytoskeletal organization and ACTH secretion from AtT-20 cells.

The production and regulated secretion of bioactive peptides require a series of lumenal enzymes to convert inactive precursors into bioactive peptides plus several cytosolic proteins to govern granule formation, maturation, translocation, and exocytosis. Peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme essential for biosynthesis of many peptides, is an integral membrane protein with trafficking information in both its lumenal and cytosolic domains. Kalirin, a PAM cytosolic domain interactor protein with spectrin-like repeats and GDP/GTP exchange factor activity for Rac1, is expressed with PAM in neurons but is not expressed in the anterior pituitary or AtT-20 corticotrope cells. Expression of Kalirin alters the cytoskeletal organization of Chinese hamster ovary and AtT-20 cells expressing membrane PAM. Expression of membrane PAM also alters cytoskeletal organization, demonstrating the presence of endogenous proteins that can mediate this effect. Significant amounts of both PAM and Kalirin fractionate with cytoskeletal elements. Since cytoskeletal organization is critical for exocytosis, constitutive-like and regulated secretions were evaluated. Whereas the constitutive-like secretion of adrenocorticotropic hormone (ACTH) is increased by expression of membrane PAM, regulated secretion is eliminated. Expression of Kalirin in AtT-20 cells expressing membrane PAM restores stimulated secretion of ACTH. Thus, Kalirin or its homologue may be essential for regulated secretion, and the PAM-Kalirin interaction may coordinate intragranular with cytosolic events.

Actins↗

PubMed2005

Antimicrobial peptide therapeutics for cystic fibrosis.

Greater than 90% of lung infections in cystic fibrosis (CF) patients are caused by Pseudomonas aeruginosa, and the majority of these patients subsequently die from lung damage. Current therapies are either targeted at reducing obstruction, reducing inflammation, or reducing infection. To identify potential therapeutic agents for the CF lung, 150 antimicrobial peptides consisting of three distinct structural classes were screened against mucoid and multidrug-resistant clinical isolates of P. aeruginosa, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and Staphylococcus aureus. Five peptides that retained potent antimicrobial activities in physiological salt and divalent cation environment were further characterized in vivo using a rat chronic lung infection model. All animals were inoculated intratracheally with 10(4) P. aeruginosa mucoid PAO1 cells in agar beads. Three days following inoculation treatment was initiated. Animals were treated daily for 3 days with 100 microl of peptide solution (1 mg/ml) in 10 mM sodium citrate, which was deposited via either intratracheal instillation or aerosolization. Control animals received daily exposure to vehicle alone. At the end of the treatment the lungs of the animals were removed for quantitative culture. Four peptides, HBCM2, HBCM3, HBCPalpha-2, and HB71, demonstrated significant reduction in Pseudomonas bioburden in the lung of rats. Further in vivo studies provided direct evidence that anti-inflammatory activity was associated with three of these peptides. Therefore, small bioactive peptides have the potential to attack two of the components responsible for the progression of lung damage in the CF disease: infection and inflammation.

Amino Acid Sequence↗

PubMed2001

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity.

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized beta-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the beta-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.

Bacterial Proteins↗

PubMed1992

Control of tumor cell biology through regulation of peptide hormone processing.

Control of the biology of individual cells, organs, and organisms is achieved through an interplay of a host of specific interactions, many involving peptide hormones as modulators or effectors. In tumor cells these processes may result in uncontrolled growth as a consequence of autocrine and/or paracrine growth effects. Most peptide hormones are bioactive only after processing of the precursor prohormone by posttranslational processing enzymes. The enzymes involved with the production of bioactive peptides, in particular those associated with peptide alpha-amidation, provide potential targets for disruption of posttranslational processing mechanisms as a means of regulation of the progression of tumor cells. This information provides a rational basis for consideration of inhibition of autocrine neuropeptide synthesis as a strategy for chemointervention.

Amino Acid Sequence↗

PubMed1988

The cDNA sequence coding for prepro-PGS (prepro-magainins) and aspects of the processing of this prepro-polypeptide.

Amphibian skin is well known as a source of peptides homologous to bioactive peptides found in mammalian gut and brain. A systematic investigation of the skin secretions from Xenopus laevis revealed several peptides not derivable from known precursors. The sequence elucidation, utilizing fast atom bombardment/mass spectrometry, of two peptides, PGS and PGS Gly-10;Lys-22, is reported. These have been independently characterized and named magainins and found to have antimicrobial activity. A mixed sequence oligonucleotide probe complementary to the mRNA sequence coding for PGS was synthesized and used to screen a Xenopus skin cDNA library. A full length cDNA species encoding prepro-PGS was isolated and characterized, and its sequence is reported. The deduced precursor sequence was found to contain one copy of PGS Gly-10;Lys-22 and five copies of PGS. The proteolytic processing of this prepro-polypeptide is discussed.

Amino Acid Sequence↗

PubMed2003

Production of C-terminal amidated recombinant salmon calcitonin in Streptomyces lividans.

Salmon calcitonin (sCT) is one of the many bioactive peptides that require C-terminal amidation for full biologic activity. To produce fully bioactive sCT in large scale, we constructed Streptomyces lividans [pMSA], an engineering Streptomyces strain. In the expression vector, glycine-extended sCT, the substrate for amidation, and rat alpha-amidating enzyme cDNA were cloned under the control of the strong constitutive promoter from the Streptomyces fradiae aph gene in pIJ680. Both were expressed in a secretory manner by the recombinant strain using the expression and secretion signals of melC1. Extracellularly expressed recombinant sCT was purified to near homogeneity and characterized by enzyme immunoassay, followed by direct amino-terminal sequencing. High-performance liquid chromatography, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and bioassay in vivo demonstrated purified product to be equivalent to synthetic standard. Thus, the engineered Streptomyces strain can produce bioactive, C-terminal amidated recombinant sCT in the culture supernatant directly. The ease of the recombinant process, as well as its potential for scale-up, makes it adaptable to production demands for sCT, and it may be applied to other bioactive peptides that need C-terminal amidation.

Animals↗

PubMed1995

Isolation and properties of an angiotensin II-cleaving peptidase from mesquite pollen.

Biochemical studies of pollen proteins have been focused, primarily, in investigating their roles as allergens. These molecules, some of which have enzymatic activity, act as antigens and initiate the production of IgE antibodies, leading to allergic and/or asthmatic responses. Included in this mixture of proteins are proteinases which, although they may or may not be allergenic, could still be involved in airway dysfunction. We have isolated an arginine-specific endopeptidase to homogeneity from mesquite (Prosopis velutina) pollen, a known wind-borne allergen, which has a molecular mass near 84 kDa by NaDodSO4-gel electrophoresis, a pH optimum in the neutral to alkaline range, and a requirement for Ca2+ for stabilization. The enzyme is inhibited by diisopropyl fluorophosphate (DFP) and N-p-tosyl-L-lysine chloromethylketone but not by N-p-tosyl-L-phenylalanine chloromethylketone, EDTA, or iodoacetamide. It was also not inhibited by human plasma proteinase inhibitors nor several other naturally occurring plant and animal inhibitors. Cleavage by the endopeptidase was primarily on the carboxy-terminal side of arginine residues in peptides, whereas proteins such as kallikrein and prothrombin were only activated and/or degraded extremely slowly. Several bioactive peptides that may be involved in maintaining normal lung function were readily fragmented, including angiotensin II, a vasoconstrictor, and atrial natriuretic peptide, a modulator of vascular permeability, both of which were rapidly cleaved at low enzyme:substrate molar ratios. Thus, the pollen endopeptidase could be involved in exacerbating the development of asthma by inactivating bioactive peptides that have ameliorating effects in maintaining lung airway homeostasis.

Amidohydrolases↗

PubMed1999

Cathepsin A/protective protein: an unusual lysosomal multifunctional protein.

Cathepsin A/protective protein [3.4.16.5], carboxypeptidase A, is a lysosomal serine protease with structural homology to yeast (Saccharomyces cerevisiae) carboxypeptidase Y. Cathepsin A is a member of the alpha/beta hydrolase fold family and has been suggested to share a common ancestral relationship with other alpha/beta hydrolase fold enzymes, such as cholinesterases. Several lines of evidence indicate that cathepsin A is a multicatalytic enzyme with deamidase and esterase in addition to carboxypeptidase activities. Cathepsin A was recently identified in human platelets as deamidase. In vitro, it hydrolyzes a variety of bioactive peptide hormones including tachykinins, suggesting that extralysosomal cathepsin A plays a role in regulation of bioactive peptide functions. Recent reports emphasize the lysosomal protective function of cathepsin A rather than its protease function. The protective function of cathepsin A is distinct from its catalytic function. Human lysosomal beta-galactosidase and neuraminidase exist as a high molecular weight enzyme complex, in which there is a 54-kDa glycoprotein termed 'lysosomal protective protein'. Based on cell culture studies, protective protein was found to protect both beta-galactosidase and neuraminidase from intralysosomal proteolysis by forming a multienzyme complex and was shown to be deficient in patients with galactosialidosis, a combined deficiency of beta-galactosidase and neuraminidase. Molecular cloning and gene expression studies have disclosed that protective protein is cathepsin A. The cathepsin A precursor has the potential to restore both beta-galactosidase and neuraminidase activities in fibroblasts from patients with galactosialidosis. Cathepsin A knockout mice showed a phenotype similar to human galactosialidosis and the deficient phenotype found in the mutant mice was corrected by transplanting erythroid precursor cells overexpressing cathepsin A. Collectively, these findings demonstrate the significance of cathepsin A as a key molecule in the onset of galactosialidosis and also highlight the therapeutic potential of the cathepsin A precursor for patients with galactosialidosis.

Age of Onset↗

PubMed2006

Thermodynamics of binding interactions between bovine beta-lactoglobulin A and the antihypertensive peptide beta-Lg f142-148.

The binding capacity of bovine beta-lactoglobulin variant A (beta-Lg A) for six peptides derived from beta-Lg was evaluated using an ultrafiltration method under the following conditions: pH 6.8, 40 degrees C, and a beta-Lg A/peptide molar ratio of 1:5. Only peptides beta-Lg f102-105, f142-148, and f69-83 bound in significant amounts to beta-Lg A corresponding to 1.5, 1.1, and 0.7 mol of peptide per mole of beta-Lg A, respectively. The interaction between beta-Lg A and the antihypertensive peptide beta-Lg f142-148 was investigated further by isothermal titration calorimetry. The binding isotherms at pH 6.8 and 25 degrees C confirmed that beta-Lg f142-148 bound to beta-Lg A and that the interaction followed a sequential three-site binding model with constants of association of 2 x 10(3), 1 x 10(3), and 0.4 x 10(3) M(-1) for the first, second, and third binding sites, respectively. The enthalpy of binding was exothermic for the first and second binding sites and endothermic for the third binding site. Binding of the peptide to all three sites was spontaneous as shown by the negative free energy values. These results show for the first time that beta-Lg A can bind bioactive peptides. This potential could be exploited to transport bioactive peptides and protect them in the gastrointestinal tract following their oral administration as nutraceuticals.

Animals↗