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3.
Biopolymers ; 92(1): 65-75, 2009.
Article in English | MEDLINE | ID: mdl-18975382

ABSTRACT

Glycine-rich proteins (GRPs) serve a variety of biological functions. Acanthoscurrin is an antimicrobial GRP isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana. Aiming to contribute to the knowledge of the secondary structure and stepwise solid-phase synthesis of GRPs' glycine-rich domains, we attempted to prepare G(101)GGLGGGRGGGYG(113)GGGGYGGGYG(123) GGY(126)GGGKYK(132)-NH(2), acanthoscurrin C-terminal amidated fragment. Although a theoretical prediction did not indicate high aggregation potential for this peptide, repetitive incomplete aminoacylations were observed after incorporating Tyr(126) to the growing peptide-MBHA resin (Boc chemistry) at 60 degrees C. The problem was not solved by varying the coupling reagents or solvents, adding chaotropic salts to the reaction media or changing the resin/chemistry (Rink amide resin/Fmoc chemistry). Some improvement was made when CLEAR amide resin (Fmoc chemistry) was used, as it allowed for obtaining fragment G(113)-K(132). NIR-FT-Raman spectra collected for samples of the growing peptide-MBHA, -Rink amide resin and -CLEAR amide resin revealed the presence of beta-sheet structures. Only the combination of CLEAR-amide resin, 60 degrees C, Fmoc-(Fmoc-Hmb)Gly-OH and LiCl (the last two used alternately) was able to inhibit the phenomenon, as proven by NIR-FT-Raman analysis of the growing peptide-resin, allowing the total synthesis of desired fragment Gly(101)-K(132). In summary, this work describes a new difficult sequence, contributes to understanding stepwise solid-phase synthesis of this type of peptide and shows that, at least while protected and linked to a resin, this GRP's glycine-rich motif presents an early tendency to assume beta-sheet structures.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Insect Proteins/chemical synthesis , Peptide Fragments/chemical synthesis , Temperature , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Benzhydryl Compounds , Chromatography, High Pressure Liquid , Insect Proteins/chemistry , Molecular Sequence Data , Peptide Fragments/chemistry , Spectrum Analysis, Raman , Spiders
4.
Anal Chim Acta ; 628(2): 123-32, 2008 Nov 03.
Article in English | MEDLINE | ID: mdl-18929000

ABSTRACT

The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with o-phthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C(18) stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L(-1) phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L(-1) phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, 50:50 and 65:35. At a flow rate of 10 microL s(-1) a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 microL s(-1) such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 micromol L(-1) for Tyr to 0.51 micromol L(-1) for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%.


Subject(s)
Amino Acids/isolation & purification , Chlorophyta/chemistry , Chromatography, Liquid/methods , Flow Injection Analysis/methods , Solvents/chemistry , Mercaptoethanol/chemistry , Sensitivity and Specificity , Spectrometry, Fluorescence/methods
5.
J Pept Sci ; 13(6): 386-92, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17492717

ABSTRACT

The preparation of small-sized protected peptide acids related to cholecystokinin and gomesin was attempted using peptide-Kaiser oxime resins (KOR) as starting materials. For comparison, peptide-2-Cl-trityl resin (CLTR) was also employed. While peptide detachment from KOR was achieved through the oxime ester bond hydrolysis mediated by DBU, hydroxide ion or Ca(+2) ion, peptide release from CLTR was accomplished through acid-catalysed hydrolysis of the peptide-resin ester linkage. Amino acid analysis of the peptide-resins before and after peptide release allowed the calculation of the reaction yields. RP-HPLC and LC/ESI-MS of the resulting crude peptides allowed estimation of their quality. The data collected indicated that: (i) among the procedures used for peptide displacement from KOR, the one employing DBU was the most efficient since it furnished all model protected peptide acids with the highest quality in a very short time; (ii) although slow, Ca(+2)-assisted peptide detachment from KOR was selective and was suitable for generating high-quality protected peptide acids containing up to five residues; (iii) even though the protocols employed for peptide release from CLTR have shown to be appropriate, the one employing 1% TFA/DCM was the most productive; (iv) in terms of product quality, DBU-catalysed peptide detachment from KOR was similar to TFA-catalysed peptide release from CLTR although the latter was more productive. These findings are relevant to peptide chemists in general, but especially to those interested in preparing acyl donors for convergent peptide syntheses by the t-Boc chemistry.


Subject(s)
Acids/chemistry , Peptides/chemistry , Catalysis , Chromatography, High Pressure Liquid , Spectrometry, Mass, Electrospray Ionization
6.
Biopolymers ; 88(3): 413-26, 2007.
Article in English | MEDLINE | ID: mdl-17245752

ABSTRACT

Peptides derived from endogenous hemoglobin play important biological roles in a variety of living systems. In previous works we showed that the fragment 33-61 of bovine alpha-hemoglobin (Hb33-61) and its C-terminus amidated analogue (Hb33-61a) exhibit antimicrobial activity and we determined the 3D structure of Hb33-61a bound to sodium dodecyl sulfate micelles. Here we report that Hb33-61a is lethal to Candida albicans at 6.25 microM probably through disruption of its plasma membrane. In addition, we show that, even when used at 50 microM, Hb33- 61a produces low hemolysis (16% +/- 3.0%). Recognizing that one of the key steps to study new compounds with potential pharmaceutical application is to identify the structural elements essential to express biological activity, we also investigated the anticandidal activity of Hb33- 61a fragments. The results indicated that Hb40-61a exhibits the same minimal inhibitory concentration as Hb33-61a, whereas Hb33-52a and Hb48-61a are significantly less active. Noteworthy, for all the peptides tested, we observed that C-terminus amidation produces a potentiation of their anticandidal activity and we associate that increased biological activity to a preferred structural and spatial organization of the C-terminal region favored by amidation. Finally, the data show that the most active peptides (Hb33-61a and Hb40-61a) are characterized by a central hinge joining the C-terminal region that presents, containing a beta-turn, followed by and a helical element, to the N-terminal region that presents only a beta-turn. We hypothesize that these two structured regions, by fluctuating independently in the lipid environment, may act in a coordinated fashion disrupting the yeast plasma membrane.


Subject(s)
Hemoglobins/chemistry , Hemoglobins/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Amino Acid Sequence , Animals , Antifungal Agents/chemical synthesis , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Candida albicans/drug effects , Cattle , Drug Design , Hemoglobins/genetics , Hemolysis/drug effects , Humans , Hydrophobic and Hydrophilic Interactions , In Vitro Techniques , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/chemical synthesis , Peptide Fragments/toxicity , Protein Structure, Secondary , Static Electricity
7.
Biochim Biophys Acta ; 1768(1): 52-8, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17027634

ABSTRACT

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC(1)]-Gm; [TOAC(1), Ser(2,6,11,15)]-Gm; [Trp(7)]-Gm; [Ser(2,6,11,15), Trp(7)]-Gm; [Trp(9)]-Gm; and [Ser(2,6,11,15), Trp(9)]-Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a beta-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC(1)]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.


Subject(s)
Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Cell Membrane/drug effects , Circular Dichroism , Electron Spin Resonance Spectroscopy , Spectrometry, Fluorescence , Anti-Infective Agents/chemical synthesis , Anti-Infective Agents/chemistry , Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/chemistry , Bacillus megaterium/drug effects , Bacillus megaterium/growth & development , Candida albicans/drug effects , Candida albicans/growth & development , Cell Membrane/chemistry , Cyclic N-Oxides , Escherichia coli/drug effects , Escherichia coli/growth & development , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers/chemistry , Membrane Lipids/chemistry , Micelles , Microbial Sensitivity Tests , Protein Conformation , Spin Labels , Structure-Activity Relationship , Tryptophan/chemistry , Water/chemistry
8.
Biochem Biophys Res Commun ; 352(4): 953-9, 2007 Jan 26.
Article in English | MEDLINE | ID: mdl-17157805

ABSTRACT

We have isolated a 417Da antibacterial molecule, named mygalin, from the hemocytes of the spider Acanthoscurria gomesiana. The structure of mygalin was elucidated by tandem mass spectrometry (MS/MS) and by two spectroscopic techniques, nuclear magnetic resonance (NMR) and ultraviolet (UV) spectroscopy. Mygalin was identified as bis-acylpolyamine N1,N8-bis(2,5-dihydroxybenzoyl)spermidine, in which the primary amino groups of the spermidine are acylated with the carboxyl group of the 2,5-dihydroxybenzoic acid. Mygalin was active against Escherichia coli at 85muM, being this activity inhibited completely by catalase. Therefore, the antibacterial activity of mygalin was attributed to its production of hydrogen peroxide (H(2)O(2)). The putative mechanisms of formation of H(2)O(2) from mygalin are discussed. To our knowledge this is the first report of one bis-acylpolyamine with antibacterial activity purified from animal source.


Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Hemocytes/chemistry , Spermidine/analogs & derivatives , Spiders/chemistry , Animals , Anti-Bacterial Agents/isolation & purification , Candida albicans/drug effects , Chromatography, Gel , Chromatography, High Pressure Liquid , Escherichia coli/drug effects , Isomerism , Magnetic Resonance Spectroscopy , Mass Spectrometry , Micrococcus luteus/drug effects , Molecular Structure , Spermidine/chemistry , Spermidine/isolation & purification , Spermidine/pharmacology
9.
Biopolymers ; 88(3): 386-400, 2007.
Article in English | MEDLINE | ID: mdl-17183513

ABSTRACT

Gomesin (Gm) is a potent antimicrobial peptide isolated from the spider Acanthoscurria gomesiana. The two disulfide bridges Cys(2,15) and Cys(6,11) facilitate the folding of the molecule in a beta-hairpin structure, conferring on the peptide a high stability in human plasma. We report herein biological and structural features of new linear Gm analogues, obtained by combining the removal of both disulfide bridges and the incorporation of a D- or L-proline. Regarding their biological properties, two analogues, namely, [D-Thr(2,6,11,15), Pro(9)]-D-Gm and [Thr(2,6,11,15), D-Pro(9)]-Gm, are as potent as Gm against Candida albicans and only fourfold less against Staphylococcus aureus and Escherichia coli. In addition, at 100 microM they are approximately threefold less hemolytic than Gm. The best therapeutic indices were found for [D-Thr(2,6,11,15), Pro(9)]-D-Gm and for [(Des-pGlu(1), -Thr(2), -Arg(3)), Thr(6,11,15), D-Pro(9)]-Gm with a 32-fold increase of their activity against bacteria, and from 128- to 512-fold against yeast when compared with Gm. Regarding the stability, [D-Thr(2,6,11,15), Pro(9)]-D-Gm appeared to be the most resistant in human serum, along with [D-Thr(2,6,11,15), Pro(8)]-D-Gm and [Thr(2,6,11,15), D-Arg(4,16), D-Pro(9)]-Gm. When evaluating their conformation by CD spectroscopy in sodium dodecyl sulfate (SDS), most linear analogues display beta-conformation characteristics. Moreover, considering its high therapeutic index and stability in serum, [D-Thr(2,6,11,15), Pro(9)]-D-Gm was further analyzed by NMR spectroscopy. (1)H NMR experiments in SDS micelles demonstrated that [D-Thr(2,6,11,15), Pro(9)]-D-Gm presents a conformation very similar to that of Gm. In our search for Gm analogues with enhanced potential for drug development, we demonstrated that designing cysteine-free analogues can improve the therapeutic index of Gm derivatives.


Subject(s)
Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Amino Acid Sequence , Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/toxicity , Candida albicans/drug effects , Circular Dichroism , Disulfides/chemistry , Drug Design , Drug Stability , Escherichia coli/drug effects , Hemolysis/drug effects , Humans , In Vitro Techniques , Micelles , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Proline/chemistry , Protein Conformation , Staphylococcus aureus/drug effects , Static Electricity
10.
Biopolymers ; 84(2): 205-18, 2006.
Article in English | MEDLINE | ID: mdl-16235231

ABSTRACT

Gomesin is an antimicrobial peptide isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana that contains two disulfide bridges Cys(2-15)/Cys(6-11) and presents a beta-hairpin structure. To investigate the role of the disulfide bridges on gomesin conformation, bioactivities, and serum stability, structure-activity relationship (SAR) studies were conducted. Initially, gomesin and variants lacking one or both disulfide bridges were synthesized. CD studies showed that the gomesin structure is very rigid independently of the solvent environment. On the other hand, the linearized analogues adopted secondary structures according to the environment, while the monocyclic disulfide-bridged peptides had a tendency to adopt a turn structure. The absence of one or both bridges resulted in a decrease in the antimicrobial and hemolytic activities. In addition, serum stability studies revealed that, contrasting to gomesin that was stable even after 48 h of incubation, the linearized analogues were rapidly degraded. The replacement of the disulfide bounds by lactam bridges led to monocyclic and bicyclic compounds. SAR studies indicated that the monocyclic lactam-bridged analogues tend to assume a alpha-helical structure being less potent, hemolytic, and serum stable than the wild-type gomesin. On the other hand, the bicyclic lactam/disulfide-bridged analogues displayed a similar conformation and degradation kinetics identical to gomesin. However, the antimicrobial activity appeared to be dependent on the lactam bridge position and size. These findings indicated that (i) the secondary structure plays a pivotal role for the full activity of gomesin; (ii) the antimicrobial and hemolytic activities of gomesin are correlated events; (iii) while at least one of the disulfide bridges is needed for the maintenance of a significant antimicrobial activity of gomesin, both bridges are required for high serum stability and optimal conformation; and finally (iv) the best analogue obtained was the bicyclo (2-15,6-11)[Glu2, Cys(6,11), Lys15]-Gm since it is as stable and potent as gomesin.


Subject(s)
Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/metabolism , Disulfides/chemistry , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/isolation & purification , Antimicrobial Cationic Peptides/pharmacology , Blood Proteins/chemistry , Candida albicans/drug effects , Chromatography, High Pressure Liquid , Circular Dichroism , Cysteine/chemistry , Escherichia coli/drug effects , Hemolysis/drug effects , Humans , Kinetics , Lactams/chemistry , Molecular Weight , Protein Conformation , Protein Structure, Secondary , Solutions , Spectrometry, Mass, Electrospray Ionization , Spiders/chemistry , Staphylococcus aureus/drug effects , Structure-Activity Relationship , Water/chemistry
11.
Biochemistry ; 44(17): 6440-51, 2005 May 03.
Article in English | MEDLINE | ID: mdl-15850378

ABSTRACT

Hemoglobin is known to be a source of peptides involved in several functions. The peptide FLSFPTTKTYFPHFDLSHGSAQVKGHGAK (Hb33-61) is a proteolytic product of the bovine hemoglobin alpha-chain found in the gut content of the cattle tick, Boophilus microplus, and it possesses antimicrobial activity. Since in the past we showed that the amidated form of Hb33-61, Hb33-61a, is active against a few Gram-positive bacteria and fungi strains at micromolar concentration [Fogaca et al. (1999) J. Biol. Chem. 274, 25330-25334], we have been prompted to shed more light on its functional and structural features. Here we show that the peptide is able to disrupt the bacterial membrane ofMicrococcus luteus A270. As for its structure, it has a random conformation in water, and it does not interact with zwitterionic micelles. On the other hand, it binds to negatively charged micelles acquiring a finite structural organization. The 3D structure of Hb33-61a bound to SDS micelles exhibits a nonconventional conformation for an antimicrobial peptide. The backbone is characterized by the presence of a beta-turn in the N-terminus and by a beta-turn followed by a alpha-helical stretch in the C-terminus. A hinge, whose spatial organization is stabilized by side-chain-side-chain interactions, joins these two regions. Interestingly, it preserves structural features present in the corresponding segment of the bovine hemoglobin alpha-chain. Hb33-61a does not possess a well-defined amphipathic nature, and H/D exchange experiments show that while the C-terminal region is embedded in the SDS micelle, one face of the N-terminal half is partly exposed to the solvent.


Subject(s)
Antimicrobial Cationic Peptides/chemistry , Hemoglobin A/chemistry , Ixodidae/chemistry , Micelles , Peptide Fragments/chemistry , Protein Subunits/chemistry , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/isolation & purification , Antimicrobial Cationic Peptides/pharmacology , Binding Sites , Cattle , Circular Dichroism/methods , Deuterium Exchange Measurement , Hemoglobin A/isolation & purification , Hemoglobin A/pharmacology , Magnetic Resonance Spectroscopy/methods , Micrococcus luteus/drug effects , Micrococcus luteus/growth & development , Models, Molecular , Molecular Sequence Data , Peptide Fragments/isolation & purification , Peptide Fragments/pharmacology , Protein Structure, Secondary , Protein Subunits/isolation & purification , Protein Subunits/pharmacology , Structure-Activity Relationship
12.
Regul Pept ; 127(1-3): 123-32, 2005 Apr 15.
Article in English | MEDLINE | ID: mdl-15680478

ABSTRACT

Leptin presents an important role in energy balance and neuroendocrine control in mammals. In an attempt to identify regions of the leptin molecule responsible for its bioactivity, we have synthesized six peptides based on the protein three-dimensional structure. Fragments were synthesized by the solid-phase methodology, purified by reverse-phase high-performance liquid chromatography (RP-HPLC), and characterized by liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS). They were injected intravenously and their ability to induce Fos immunoreactivity (Fos-ir) in rat hypothalamus was compared with that of the recombinant human leptin and saline. Fragment Ac-[Ser117]Lep116-140-NH2 (V) induced Fos-ir in hypothalamic nuclei that express leptin receptor long form. No similar ability was observed for the other five fragments. To investigate whether Fos-ir was induced in the same neuronal group activated by leptin, we proceeded with a dual-label immunohistochemistry for cocaine- and amphetamine-regulated transcript (CART), a neuropeptide related to leptin action in rat hypothalamus. We found that Ac-[Ser117]Lep116-140-NH2 (V) differentially activates CART neurons through the rostrocaudal extension of the arcuate nucleus. These results suggest that this fragment acts in the same group of neurons that mediate leptin response. This approach may offer the basis for the development of leptin-related compounds, having potential application in human or veterinary medicine.


Subject(s)
Hypothalamus/drug effects , Leptin/pharmacology , Peptide Fragments/pharmacology , Proto-Oncogene Proteins c-fos/metabolism , Animals , Humans , Hypothalamus/cytology , Hypothalamus/metabolism , Immunohistochemistry , Leptin/administration & dosage , Leptin/chemistry , Male , Nerve Tissue Proteins/metabolism , Neurons/cytology , Neurons/metabolism , Peptide Fragments/administration & dosage , Peptide Fragments/chemical synthesis , Peptide Fragments/chemistry , Rats , Rats, Wistar
13.
Bioorg Med Chem ; 11(22): 4779-83, 2003 Nov 03.
Article in English | MEDLINE | ID: mdl-14556793

ABSTRACT

The synthesis of mutual prodrugs of nitrofurazone with primaquine, using specific and nonspecific spacer groups, has been previously attempted seeking selective antichagasic agents. The intermediate reaction product, hydroxymethylnitrofurazone (NFOH-121), was isolated and tested in LLC-MK(2) culture cells infected with trypomastigotes forms of Trypanosoma cruzi showing higher trypanocidal activity than nitrofurazone and benznidazol in all stages. The mutagenicity tests showed that the prodrug was less toxic than the parent drug. Degradation assays were carried out in pH 1.2 and 7.4.


Subject(s)
Nitrofurazone/analogs & derivatives , Nitrofurazone/chemical synthesis , Nitrofurazone/pharmacology , Prodrugs/chemical synthesis , Prodrugs/pharmacology , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/pharmacology , Animals , Cell Line , Chagas Disease , Macaca mulatta , Mutagenicity Tests/methods , Nitrofurazone/metabolism , Prodrugs/chemistry , Prodrugs/metabolism , Spectrophotometry, Ultraviolet , Trypanocidal Agents/metabolism , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/physiology
14.
J Med Chem ; 46(12): 2325-33, 2003 Jun 05.
Article in English | MEDLINE | ID: mdl-12773037

ABSTRACT

Fibroblast growth factors (FGFs) are regulatory proteins associated with a number of physiological and pathological states. On the basis of data suggesting a functional role for specific regions of human acidic FGF (aFGF), a linear peptide encompassing residues 99-108 (peptide1) and its cyclic analogue (peptide 2) were synthesized and their functional and structural features were investigated. While peptide 1 is inactive on Balb/c 3T3 fibroblasts, peptide 2 is mitogenic with ED(50) of approximately 50 microM. Moreover, peptide 1 is not able to inhibit the binding of human aFGF to cellular receptors whereas peptide 2 exhibits significant inhibitory activity. The NMR-derived solution conformers indicated the presence, only in peptide 2, of structural elements that we believe are related to its ability to emulate the biological activity of the native protein. These results suggest that the expression of mitogenic activity in short peptides, besides the presence of specific amino acids, requires the existence of stable structural features. In addition, they indicate that the introduction of chemical restraints in peptides can provide novel possibilities for the development of receptor agonists or antagonists.


Subject(s)
Fibroblast Growth Factor 1/chemistry , Fibroblast Growth Factors/chemical synthesis , Mitogens/chemical synthesis , Oligopeptides/chemical synthesis , Peptide Fragments/chemical synthesis , Peptides, Cyclic/chemical synthesis , 3T3 Cells , Animals , Circular Dichroism , Fibroblast Growth Factors/chemistry , Fibroblast Growth Factors/pharmacology , Humans , Magnetic Resonance Spectroscopy , Mice , Mitogens/chemistry , Mitogens/pharmacology , Models, Molecular , Oligopeptides/chemistry , Oligopeptides/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Protein Conformation , Thymidine/metabolism
15.
Arch Biochem Biophys ; 409(2): 349-56, 2003 Jan 15.
Article in English | MEDLINE | ID: mdl-12504902

ABSTRACT

5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has been implicated in iron-mediated oxidative damage to biomolecules and cell structures. From previous observations of ferritin iron release by ALA, we investigated the ability of ALA to cause oxidative damage to ferritin apoprotein. Incubation of horse spleen ferritin (HoSF) with ALA caused alterations in the ferritin circular dichroism spectrum (loss of a alpha-helix content) and altered electrophoretic behavior. Incubation of human liver, spleen, and heart ferritins with ALA substantially decreased antibody recognition (51, 60, and 28% for liver, spleen, and heart, respectively). Incubation of apoferritin with 1-10mM ALA produced dose-dependent decreases in tryptophan fluorescence (11-35% after 5h), and a partial depletion of protein thiols (18% after 24h) despite substantial removal of catalytic iron. The loss of tryptophan fluorescence was inhibited 35% by 50mM mannitol, suggesting participation of hydroxyl radicals. The damage to apoferritin had no effect on ferroxidase activity, but produced a 61% decrease in iron uptake ability. The results suggest a local autocatalytic interaction among ALA, ferritin, and oxygen, catalyzed by endogenous iron and phosphate, that causes site-specific damage to the ferritin protein and impaired iron sequestration. These data together with previous findings that ALA overload causes iron mobilization in brain and liver of rats may help explain organ-specific toxicities and carcinogenicity of ALA in experimental animals and patients with porphyria.


Subject(s)
Aminolevulinic Acid/pharmacology , Ferritins/drug effects , Oxidative Stress , Aminolevulinic Acid/metabolism , Animals , Apoferritins/chemistry , Apoferritins/drug effects , Circular Dichroism , Dose-Response Relationship, Drug , Electrophoresis, Gel, Two-Dimensional , Ferritins/chemistry , Horses , Humans , In Vitro Techniques , Iron/metabolism , Liver/chemistry , Mannitol/pharmacology , Myocardium/chemistry , Oxidation-Reduction , Oxygen/metabolism , Spectrometry, Fluorescence , Spleen/chemistry , Sulfhydryl Compounds/analysis , Superoxides/metabolism , Tryptophan/drug effects
16.
J Inorg Biochem ; 91(2): 363-70, 2002 Aug 15.
Article in English | MEDLINE | ID: mdl-12161306

ABSTRACT

This work reports Eu(III) and Tb(III) luminescence titrations in which the lanthanide ions were used as spectroscopic probes for Ca(II) ions to determine the metal binding ability of Ac-NESVKEEGGW-NH(2) and Ac-NESVKEDGGW-NH(2). These decapeptides correspond to the putative calcium binding region of the plant antifungal proteins SI-alpha1 from Sorghum bicolor and of Zeathionin from Zea mays, respectively. The luminescence spectra for the Eu(III)-decapeptide system (red emission) with the excitation at the Trp band at 280 nm showed an enhancement of the intensities of the 5D(0)-->7F(J) transitions (where J=0-4) with increments of Eu(III) ion concentration. The photoluminescence titration data of the terbium ion (green emission) in the decapeptide solutions showed intensification of the 5D(4)-->7F(J) transitions (J=0-6), similar to that observed for the Eu(III) ion. Thus, energy transfer from Ac-NESVKEEGGW-NH(2) and Ac-NESVKEDGGW-NH(2) to the trivalent lanthanide ions revealed that these peptides are capable of binding to these metal ions with association constants of the order of 10(5) M(-1). The amino acid derivative Ac-Trp-OEt also transferred energy to Tb(III) and Eu(III) ions as judged from the quenching of tryptophan luminescence. However, the energy transfers were significantly lower. Taken together the luminescence titration data indicated that Ac-NESVKEEGGW-NH(2) and Ac-NESVKEDGGW-NH(2) bind efficiently to both trivalent lanthanide ions and that these ions may be used as probes to distinguish an anionic peptide from a neutral amino acid derivative.


Subject(s)
Europium/metabolism , Peptides/metabolism , Plant Proteins/metabolism , Plants/metabolism , Terbium/metabolism , Amino Acid Sequence , Luminescent Measurements , Plant Proteins/chemistry
17.
J Biol Chem ; 277(42): 39574-84, 2002 Oct 18.
Article in English | MEDLINE | ID: mdl-12167616

ABSTRACT

Tropomyosin is a flexible 410 A coiled-coil protein in which the relative stabilities of specific regions may be important for its proper function in the control of muscle contraction. In addition, tropomyosin can be used as a simple model of natural occurrence to understand the inter- and intramolecular interactions that govern the stability of coiled-coils. We have produced eight recombinant tropomyosin fragments (Tm(143-284(5OHW),) Tm(189-284(5OHW)), Tm(189-284), Tm(220-284(5OHW)), Tm(220-284), Tm(143-235), Tm(167-260), and Tm(143-260)) and one synthetic peptide (Ac-Tm(215-235)) to investigate the relative conformational stability of different regions derived from the C-terminal region of the protein, which is known to interact with the troponin complex. Analytical ultracentrifugation experiments show that the fragments that include the last 24 residues of the molecule (Tm(143-284(5OHW)), Tm(189-284(5OHW)), Tm(220-284(5OHW)), Tm(220-284)) are completely dimerized at 10 microm dimer (50 mm phosphate, 100 mm NaCl, 1.0 mm dithiothreitol, and 0.5 mm EDTA, 10 degrees C), whereas fragments that lack the native C terminus (Tm(143-235),Tm(167-260), and Tm(143-260)) are in a monomer-dimer equilibrium under these conditions. The presence of trifluoroethanol resulted in a reduction in the [theta](222)/[theta](208) circular dichroism ratio in all of the fragments and induced stable trimer formation only in those containing residues 261-284. Urea denaturation monitored by circular dichroism and fluorescence revealed that residues 261-284 of tropomyosin are very important for the stability of the C-terminal half of the molecule as a whole. Furthermore, the absence of this region greatly increases the cooperativity of urea-induced unfolding. Temperature and urea denaturation experiments show that Tm(143-235) is less stable than other fragments of the same size. We have identified a number of factors that may contribute to this particular instability, including an interhelix repulsion between g and e' positions of the heptad repeat, a charged residue at the hydrophobic coiled-coil interface, and a greater fraction of beta-branched residues located at d positions.


Subject(s)
Tropomyosin/chemistry , Animals , Chickens , Circular Dichroism , Cross-Linking Reagents/pharmacology , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Glutaral/pharmacology , Peptide Biosynthesis , Plasmids/metabolism , Protein Binding , Protein Folding , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Temperature , Thermodynamics , Ultracentrifugation , Ultraviolet Rays , Urea/pharmacology
18.
Ciênc. cult. (Säo Paulo) ; 48(5/6): 359-60, Sept.-Dec. 1996. tab
Article in English | LILACS | ID: lil-186336

ABSTRACT

The synthetic peptide Ac-WFVGLKKNGSSKRGPRT-NH2,related to the human FGF-1 sequence, was shown to be mitogenic upon Balb/c 3T3 fibroblasts in culture (ED50=10-20 muM) and to compete with the growth factor for cellular binding (ID50 = 30-50 muM). The results described suggest that the mitogenic activity of the peptide is dependent on the presence of the residues 122-127 (WFVGLK) in its structure. Also, its affinity for the cellular receptors seems to be dependent on the presence of residues that are important for FGF-heparin binding such as K127, K133 and R137.


Subject(s)
Animals , Mice , Fibroblast Growth Factors/metabolism , Mitosis , Peptides/metabolism , Amino Acid Sequence , Binding Sites , Fibroblast Growth Factors/antagonists & inhibitors , Fibroblast Growth Factors/genetics , Heparin , Mice, Inbred BALB C , Peptides/pharmacology
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