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1.
Dent Mater ; 39(8): 708, 2023 08.
Article in English | MEDLINE | ID: mdl-37394390

ABSTRACT

OBJECTIVES: Evaluate molecularly the role of P11-4 self-assembly peptide in dentin remineralization and its interaction with collagen I. METHODS: The calcium-responsive P11-4 peptide was analyzed by intrinsic fluorescence emission spectrum, circular dichroism spectrum (CD), and atomic force microscope (AFM). Differential light scattering was used to monitor the nucleation growth rate of calcium phosphate nanocrystals in the absence or in the presence of P11-4. AFM was used to analyze the radial size (nm) of calcium phosphate nanocrystals formed in the absence or in the presence of P11-4, as well as to verify the spatial structure of P11-4 in the absence or in the presence of Ca2+. RESULTS: The interaction of Ca2+ with the P11-4 (KD = 0.58 ± 0.06 mM) promotes the formation of ß-sheet antiparallel structure, leads to its precipitation in saturated solutions of Ca/P = 1.67 and induces the formation of parallel large fibrils (0.6 - 1.5 µm). P11-4 organized the HAP nucleation by reducing both the growth rate and size variability of nanocrystals, analyzed by the F test (p < 0.0001, N = 30). P11-4 interacts (KD = 0.75 ± 0.06 µM) with the KGHRGFSGL motif present at the C-terminal collagen telopeptide domain. P11-4 also increased the amount of HAP and collagen in the MDPC-23 cells. SIGNIFICANCE: The presented data propose a mechanism that will help future clinical and/or basic research to better understand a molecule able to inhibit structural collagen loss and help the impaired tissue to remineralize.


Subject(s)
Calcium , Collagen Type I , Peptides , Collagen , Calcium Phosphates/pharmacology , Ions
2.
Dent Mater ; 36(6): e184-e193, 2020 06.
Article in English | MEDLINE | ID: mdl-32305153

ABSTRACT

OBJECTIVES: This study evaluated the cell viability and expression of different major genes involved in mineralization in odontoblast-like cells exposed to sodium trimetaphosphate (STMP). It was also investigated the influence of STMP on the rate of calcium phosphate crystal growth, its anti-proteolytic action against the enzymatic degradation of type I collagen, the binding mechanism of STMP to collagen fibrils, and the potential mechanism to induce collagen stabilization. METHODS: Immortalized rat odontoblast MDPC-23 cells were cultured. Cell viability was assessed by trypan blue staining, and the changes in gene expression balance induced by STMP were assessed by quantitative reverse transcription (qRT) PCR assays. Crystalline particle formation was monitored by light-scattering detectors to estimate pH variation and the radial size of the crystalline particles as a function of reaction time (pH 7.4, 25°C) in the presence of STMP in supersaturated calcium phosphate solution (Ca/P=1.67). Images were obtained under atomic force microscopy (AFM) to measure the particle size in the presence of STMP. A three-point bending test was used to obtain the elastic modulus of fully demineralized dentin beams after immersion in STMP solution. The binding mechanism of STMP to collagen fibrils and potential stabilization mechanism was assessed with circular dichroism spectrometry (CD). The data were analyzed statistically (α=0.05). RESULTS: STMP had no significant influence on the cell viability and gene expression of the MDPC-23 cells. STMP greatly increased the rate of crystal growth, significantly increasing the average radial crystal size. AFM corroborated the significant increase of STPM-treated crystal size. Mineralized collagen I fibrils exhibited less collagenase degradation with lower STMP concentration. CD analysis demonstrated changes in the conformational stability after STMP binding to type I collagen. SIGNIFICANCE: The increased resistance of collagen against the proteolytic activity of collagenases appears to be related to the conformational change induced by STMP binding in collagen I and the STMP capacity for promoting biomimetic mineralization in type I collagen fibrils.


Subject(s)
Collagen Type I , Dentin , Animals , Collagen , Collagenases , Polyphosphates , Rats
3.
Parasite Immunol ; 42(5): e12705, 2020 05.
Article in English | MEDLINE | ID: mdl-32096238

ABSTRACT

Although antibodies are considered critical for malaria protection, little is known about the mechanisms/factors that maintain humoral immunity, especially regarding the induction and maintenance of memory B cells over time. In Brazilian endemic areas, this is the first time that the profile of antibody responses and the occurrence of antigen-specific memory B cells (MBC) against P vivax were investigated during acute malaria and up to six months after parasite clearance. For this, we selected two peptides, PvAMA-1(S290-K307) and PvMSP-9(E795-A808) , which represent the apical membrane antigen-1 and merozoite surface protein-9 of P vivax, respectively. Both peptides were previously described as containing linear B-cell epitopes. Our findings were as follows: 1-both peptides were recognized by IgG antibodies at a high frequency (between 24% and 81%) in all study groups; 2-in the absence of infection, the IgG levels remained stable throughout 6 months of follow-up; and 3-PvAMA-1(S290-K307) and PvMSP-9(E795-A808) -specific MBCs were detected in all individual groups in the absence of reinfection throughout the follow-up period, suggesting long-lived MBC. However, no positive association was observed between malaria-specific antibody levels and frequency of MBCs over time. Taken together, these results suggest that peptides can be, in the future, an alternative strategy to polypeptidic vaccine formulation.


Subject(s)
Antibodies, Protozoan/immunology , Epitopes, B-Lymphocyte/immunology , Malaria, Vivax/immunology , Plasmodium vivax/growth & development , Plasmodium vivax/immunology , Protozoan Proteins/immunology , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , B-Lymphocytes/immunology , Brazil , Epitopes, B-Lymphocyte/genetics , Humans , Immunity, Humoral , Immunoglobulin G/immunology , Immunologic Memory , Malaria, Vivax/genetics , Malaria, Vivax/parasitology , Peptides/immunology , Plasmodium vivax/genetics , Protozoan Proteins/genetics
4.
Inflamm Res ; 68(10): 845-855, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31218444

ABSTRACT

INTRODUCTION: Carboxypeptidase M (CPM) is a glycosylphosphatidylinositol anchored enzyme that plays an important role in the kallikrein-kinin system (KKS). CPM catalytic domain hydrolyzes Arg from C-terminal peptides (i.e., bradykinin and kallidin), generating des-Arg-kinins, the agonists of B1 receptor (B1R). It is known that CPM and kinin B1R are co-localized in the plasma membrane microdomains, where they interact with each other, facilitating receptor signaling. AIMS: We hypothesized here that this CPM-B1R interaction could also affect the activity of the enzyme. METHODS: Thus, in this work, we evaluated the impact of B1R presence or absence on CPM activity and expression, using primary culture of microvascular endothelial cells from wild-type, kinin B1R knockout mice (B 1 -/- ), and transgenic rats overexpressing B1 receptor exclusively in the endothelium. In addition, HEK293T cells, as wells as B 1 -/- primary culture of endothelial cells, both transfected with B1R, were also used. RESULTS: CPM expression and activity were downregulated in cells of knockout mice compared to control and this reduction was rescued after B1R transfection. Cells overexpressing B1R presented higher levels of CPM mRNA, protein, and activity. This profile was reverted by pre-incubation with the B1R antagonist, R715, in highly expressing receptor cells. CONCLUSIONS: Our data show that kinin B1R positively modulates both CPM expression and activity, suggesting that CPM-B1R interaction in membrane microdomains might affect enzyme activity, beyond interfering in receptors signaling. This work highlights the interactions among different components of KKS and contributes to a better understanding of its patho-physiological role.


Subject(s)
Endothelial Cells/metabolism , Metalloendopeptidases/metabolism , Receptor, Bradykinin B1/metabolism , Animals , Cells, Cultured , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Humans , Lung/cytology , Metalloendopeptidases/genetics , Mice, Inbred C57BL , Mice, Knockout , Rats, Sprague-Dawley , Rats, Transgenic , Receptor, Bradykinin B1/genetics
5.
Biomed Chromatogr ; 31(3)2017 Mar.
Article in English | MEDLINE | ID: mdl-27557455

ABSTRACT

We have developed and validated a fast and sensitive ultra high-performance liquid chromatography with positive ion electrospray ionization tandem mass spectrometry method for determining N-butylscopolamine levels in human plasma using propranolol as an internal standard. The acquisition was set up in the multiple reaction monitoring mode with the transitions m/z 360.3 → 138.0 for N-butylscopolamine and m/z 260.2 → 116.1 for IS. This method uses a liquid-liquid extraction process with dichloromethane. The analyte and IS were chromatographed on a C18 , 50 × 2.1 mm, 1.7 µm column through isocratic elution with acetonitrile-5 mm ammonium acetate (adjusted to pH 3.0 with formic acid). The method was linear in the 1-1000 pg/mL range for N-butylscopolamine and was selective, precise, accurate and robust. The validated method was successfully applied to perform a bioequivalence study of the reference (Buscopan® , from Boehringer Ingelheim) and the test sample coated-tablet formulations (from Foundation for Popular Remedy), both containing 10 mg of N-butylscopolamine bromide administered as a single dose. Using 58 healthy volunteers and accounting for the high intra-individual variability confirmed by statistical calculations (38%), the two formulations were considered bioequivalent because the rate and extent of absorption (within 80-125% interval), satisfying international requirements.


Subject(s)
Butylscopolammonium Bromide/blood , Chromatography, High Pressure Liquid/methods , Tandem Mass Spectrometry/methods , Humans
6.
Biol Pharm Bull ; 36(4): 664-75, 2013.
Article in English | MEDLINE | ID: mdl-23546297

ABSTRACT

The present work aimed at evaluating the radiolysis effect upon a set of peptides, most of them involved in physiological functions. To generate reactive radical species, a Co(60) source (up to 15 kGy) was used for controlled gamma irradiation of some peptide solutions including derivatives attaching the stable free radical Toac (2,2,6,6-tetramethypiperidine-1-oxyl-4-amino-4-carboxylic acid). Regardless of the peptide sequence, a nonlinear and progressive degradation of a total of nine peptides was detected. The results were interpreted in the light of the half-life dose (D(1/2)) parameter which represents the dose necessary for 50% peptide structure degradation. The vasoactive angiotensin II (AngII)'s analogue Ang-(1-7) showed greater stability towards gamma ray radiation than bradykinin (BK), Toac(0)-BK, Pro(4)-BK (D(1/2) around 4 and 2 kGy, respectively) which decreased to about 0.5-1.0 kGy in the case of acetyl-α-melanocyte-stimulating hormone (Ac-α-MSH) and substance P (SP). In terms of peptide structural modifications, the data acquired from different analytical methods suggested a Phe to Tyr (or its ortho and/or meta isomers) transformation as a consequence of the hydroxyl moiety insertion. Noteworthy, this effect seemed to be position-dependent as only Phe located at or near the C-terminal portion seemed to display this transformation. In contrast, Met is comparatively more easily oxidized, thus allowing to conclude that gamma irradiation may induce a complex position and/or sequence-dependent effect on peptides. As previously applied for BK, some irradiated peptides were submitted to their by-products purification, indeed a complementary target of the present approach for development of uncommon analogues for further structure-function investigation.


Subject(s)
Gamma Rays , Peptides/radiation effects , Animals , Cyclic N-Oxides/chemistry , Gastric Fundus/drug effects , Gastric Fundus/physiology , Mice , Mice, Inbred C57BL , Molecular Structure , Muscle Contraction/drug effects , Peptides/chemistry , Peptides/pharmacology
7.
J Colloid Interface Sci ; 373(1): 102-9, 2012 May 01.
Article in English | MEDLINE | ID: mdl-21999959

ABSTRACT

We report the effects of a synthetic peptide designed to act as a nuclear localization signal on the treatment of tuberculosis. The peptide contains 21 amino acid residues with the following specific domains: nuclear localization signal from SV 40T, cationic shuttle sequence, and cysteamide group at the C-terminus. The peptide was complexed with the plasmid DNAhsp65 and incorporated into cationic liposomes, forming a pseudo-ternary complex. The same cationic liposomes, composed of egg chicken L-α-phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-dioleoyl-3-trimethylammonium-propane (2:1:1M), were previously evaluated as a gene carrier for tuberculosis immunization protocols with DNAhsp65. The pseudo-ternary complex presented a controlled size (250 nm), spherical-like shape, and various lamellae in liposomes as evaluated by transmission electron microscopy. An assay of fluorescence probe accessibility confirmed insertion of the peptide/DNA into the liposome structure. Peptide addition conferred no cytotoxicity in vitro, and similar therapeutic effects against tuberculosis were seen with four times less DNA compared with naked DNA treatment. Taken together, the results indicate that the pseudo-ternary complex is a promising gene vaccine for tuberculosis treatment. This work contributes to the development of multifunctional nanostructures in the search for strategies for in vivo DNA delivery.


Subject(s)
DNA/therapeutic use , Liposomes/therapeutic use , Peptides/therapeutic use , Tuberculosis/drug therapy , Animals , Cations/chemistry , Cations/therapeutic use , DNA/chemistry , Genetic Therapy , Liposomes/chemistry , Mice , Mycobacterium tuberculosis/isolation & purification , Peptides/chemical synthesis , Peptides/chemistry
8.
J Mol Med (Berl) ; 89(1): 65-74, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20941593

ABSTRACT

Fabry disease is a multisystem X-linked disorder resulting from α-galactosidase A (α-GalA) gene mutations leading to the accumulation of globotriaosylceramide mainly in endothelium compromising heart, kidney, and brain. In Fabry patients, progressive renal failure is frequently treated with angiotensin I-converting enzyme (ACE) inhibitors. We were interested in the possible interactions between ACE inhibitors therapy and the only causative therapy for Fabry disease, the enzyme replacement therapy (ERT) using recombinant human α-GalA (rhα-GalA). Our results suggest that ACE activity was significantly inhibited in plasma of Fabry patients and the blood pressure level decreased just after ERT (at the end of the rhα-GalA infusion). Interestingly, 2 weeks later, ACE activity was significantly upregulated and the plasma levels of angiotensin II increased in the patients treated with rhα-GalA following the elevations of ACE activity. The same inhibitory effect on ACE activity was also observed in rats after rhα-GalA infusion. Furthermore, ACE activity in CHO cells transfected with the human ACE was inhibited dose and time-dependently by rhα-GalA. In vitro, the incubation of plasma from healthy volunteers with rhα-GalA significantly reduced ACE activity. Finally, rhα-GalA also inhibited ACE activity and released galactose residues from purified rabbit lung ACE dose-dependently. In summary, our results suggest that rhα-GalA interacts with ACE and inhibits its activity, possibly by removing the galactose residues from the enzyme. This modulation might have profound impact on the clinical outcome of Fabry patients treated with rhα-GalA.


Subject(s)
Blood Pressure/drug effects , Fabry Disease/enzymology , Gene Expression Regulation, Enzymologic/drug effects , Peptidyl-Dipeptidase A/metabolism , alpha-Galactosidase/pharmacology , Adolescent , Adult , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Angiotensins/blood , Animals , CHO Cells , Cricetinae , Cricetulus , Fabry Disease/drug therapy , Female , Humans , Male , Middle Aged , Models, Animal , Peptidyl-Dipeptidase A/blood , Rabbits , Rats , Rats, Wistar , Recombinant Proteins/pharmacology , Young Adult , alpha-Galactosidase/therapeutic use
9.
Biol Chem ; 391(10): 1189-95, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20707602

ABSTRACT

Previous research showed that disruption of the Cys(18)-Cys(274) bond in the angiotensin II (AngII) AT1 receptor mutant (C18S), expressed in CHO cells, causes an increase in the basal activity and attenuation of the maximum response to AngII. In addition, this mutant was mostly intracellularly distributed. Our aim was to investigate whether the intracellular presence of the mutant was due to a constitutive internalization or to a defective maturation of the receptor. The first hypothesis was assessed by pretreating the cells with losartan or [Sar¹Leu8]-AngII, specific AT1 receptor antagonists, a maneuver to revert the receptor internalization. The second hypothesis was tested using calnexin, an endoplasmic reticulum marker. We found that treatment with AT1 receptor antagonists causes an increase in the binding ability of the mutant to AngII. Furthermore, whereas the maximum effect is increased, it reduces the enhanced basal levels of IP3. The hypothesis for a lack of maturation of the mutant receptor was ruled out because calnexin was poorly colocalized with the intracellular C18S receptor. Our results suggest that the mutation of the AT1 receptor leads to a conformational structure similar to that of the active mode of the AT1 receptor, favoring its internalization in the absence of the agonist.


Subject(s)
Cysteine/metabolism , Disulfides/metabolism , Receptor, Angiotensin, Type 1/chemistry , Receptor, Angiotensin, Type 1/metabolism , Animals , Binding, Competitive , Calcium Signaling , Cell Membrane/metabolism , Cells, Cultured , Cysteine/chemistry , Disulfides/chemistry , Endoplasmic Reticulum/metabolism , Gene Expression Regulation , Inositol Phosphates/metabolism , Models, Molecular , Mutation , Protein Conformation , Protein Stability , Protein Transport , Receptor, Angiotensin, Type 1/genetics
10.
J Mol Med (Berl) ; 88(9): 899-907, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20614101

ABSTRACT

Leptin is a hormone related to metabolism. It also influences blood pressure, but the mechanisms triggered in this process are not yet elucidated. Angiotensin-I converting enzyme (ACE) regulates cardiovascular functions and recently has been associated with metabolism control and obesity. Here, we used ob/ob mice, a model lacking leptin, to answer the question whether ACE and leptin could interact to influence blood pressure, thereby linking the renin-angiotensin system and obesity. These mice are obese and diabetic but have normal 24 h mean arterial pressure. Our results show that plasma and lung ACE activities as well as ACE mRNA expression were significantly decreased in ob/ob mice. In agreement with these findings, the hypotensive effect produced by enalapril administration was attenuated in the obese mice. Plasma renin, angiotensinogen, angiotensin I, bradykinin, and angiotensin 1-7 were increased, whereas plasma angiotensin II concentration was unchanged in obese mice. Chronic infusion of leptin increased renin activity and angiotensin II concentration in both groups and increased ACE activity in ob/ob mice. Acute leptin infusion restored ACE activity in leptin-deficient mice. Moreover, the effect of an ACE inhibitor on blood pressure was not changed in ob/+ mice during leptin treatment but increased four times in obese mice. In summary, our findings show that the renin-angiotensin system is altered in ob/ob mice, with markedly reduced ACE activity, which suggests a possible connection between the renin-angiotensin system and leptin. These results point to an important interplay between the angiotensinergic and the leptinergic systems, which may play a role in the pathogenesis of obesity, hypertension, and metabolic syndrome.


Subject(s)
Leptin/metabolism , Peptidyl-Dipeptidase A/metabolism , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Blood Pressure/physiology , Enalapril/pharmacology , Male , Mice , Mice, Obese , Mice, Transgenic , Obesity/metabolism , Peptidyl-Dipeptidase A/blood , Peptidyl-Dipeptidase A/genetics , RNA, Messenger/metabolism , Renin-Angiotensin System/physiology
11.
Neuropeptides ; 44(2): 115-8, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20006383

ABSTRACT

INTRODUCTION: Angiotensin II (AII) is the main active product of the renin angiotensin system. Better known effects of AII are via AT1 receptor (AT1R). Expression of AT1R mutants (L265D and L262D) in CHO cells increased cAMP formation when compared to CHO cells expressing the wild type (WT) AT1R. Morphological transformation of CHO cells transfected with mutants correlated with their increased cAMP formation. DNA synthesis was inhibited in these cells too, indicating that cAMP promotes inhibitory effects on transfected CHO cells growth and causes their morphological change from a tumorigenic phenotype to a non-tumorigenic one. OBJECTIVES: To assess the importance of leucine 262 and 265 in determining AT1R structure by means of a comparative structural analysis of two mutant peptides and of a wild-type fragment. METHODOLOGY: Three peptides had their conformation compared by circular dichroism (CD): L262D(259-272), L265D(259-272) (mutants) and WT(260-277). RESULTS: Secondary structures were: beta-turn for WT and L262D and random coil for L265D. CONCLUSIONS: Strong correlation was found in the results of biochemical, cellular and structural approaches used to compare WT AT1R to mutant types. Random coil structure of the L265D mutant may be a key point to explain those changes observed in biochemical (binding and signal transduction) and proliferation assays (Correa et al., 2005). beta-Turn formation is an important step during early protein folding and this secondary simple structure is present in L262D and WT, but not in L265D. Therefore, leucine 265 seems to play a crucial role in determining an entirely functional AT1R.


Subject(s)
Peptide Fragments/chemistry , Receptor, Angiotensin, Type 1/chemistry , Amino Acid Sequence , Animals , CHO Cells , Chromatography, High Pressure Liquid , Circular Dichroism , Cricetinae , Cricetulus , Mass Spectrometry , Mutation/genetics , Peptide Fragments/genetics , Protein Structure, Secondary/genetics , Receptor, Angiotensin, Type 1/genetics
12.
J Sep Sci ; 32(4): 637-43, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19212975

ABSTRACT

A new automated SPE-LC-ESI-MS/MS method was developed and validated to quantify venlafaxine in human plasma using fluoxetine as an internal standard. The analytes were automatically extracted from plasma by C18 SPE cartridges, separated on a C8 RP column and analyzed by MS in the multiple reaction-monitoring (MRM) mode. The method has a chromatographic run time of 4.0 min and a linear calibration curve over the range of 0.25-200 ng/mL (r >0.997). The between-run precisions, based on the percent RSD for replicate quality controls (0.75; 80, and 200 ng/mL), were < 8.5% for all concentrations. The between-run accuracies, based on the percent relative error, were < 4.0%. This method was successfully employed in a bioequivalence study of two venlafaxine capsule formulations (test formulation from Eurofarma (Brazil) and Efexor XR, reference formulation, from Wyeth-Whitehall, Brazil) in 48 healthy volunteers of both sexes who received a single 150 mg dose of each formulation. More than 3000 samples were analyzed eliminating the analyst's exposure to hazardous organic solvents normally employed in off-line liquid-liquid extractions. The 90% confidence interval (CI) of the individual ratio geometric mean for Test/Reference was 91.6-103.4% for AUC(0-48 h) and 102.2-112.6% for C(max). Since both 90% CI for AUC(0-48 h) and C(max) were included in the 80-125% interval proposed by the US Food and Drug Administration (FDA) and the Brazilian National Health Surveillance Agency (ANVISA), the test formulation was considered bioequivalent to Efexor XR according to both the rate and extent of absorption.


Subject(s)
Cyclohexanols/blood , Cyclohexanols/chemistry , Solid Phase Extraction/instrumentation , Solid Phase Extraction/methods , Tandem Mass Spectrometry/instrumentation , Tandem Mass Spectrometry/methods , Calibration , Humans , Molecular Structure , Venlafaxine Hydrochloride
13.
Arch Biochem Biophys ; 479(1): 20-7, 2008 Nov 01.
Article in English | MEDLINE | ID: mdl-18755139

ABSTRACT

In this work, we show that alternagin-C (ALT-C) and ALT-C PEP, a peptide derived from its sequence, were able to induce angiogenesis in wounded rat skin. A spherical cutaneous excision was made in the back of each animal and treated with three different concentrations of ALT-C or ALT-C PEP. After that, the skin was removed and analyzed to verify the presence of new vessels and the expression of growth factors. ALT-C and ALT-C PEP induced the formation of new vessels and modulated the expression of growth factors, mainly VEGF and FGF1. The expression of VEGF increased and it could be detected up to 7 days after injury. FGF1 also significantly increased, but at a lesser extent than VEGF. In conclusion, the present study shows for the first time the stimulation of angiogenesis in an injured tissue by a disintegrin-like protein and that ALT-C may exert this effect by modulating the expression of growth factors.


Subject(s)
Intercellular Signaling Peptides and Proteins/metabolism , Models, Biological , Neovascularization, Physiologic/physiology , Skin/drug effects , Snake Venoms/pharmacology , Animals , Disintegrins , Dose-Response Relationship, Drug , Male , Prostaglandins F/metabolism , Protein Structure, Tertiary , Random Allocation , Rats , Rats, Wistar , Skin/blood supply , Skin/metabolism , Snake Venoms/chemistry , Snake Venoms/metabolism , Time Factors , Vascular Endothelial Growth Factors/metabolism
14.
FEBS Lett ; 581(13): 2411-5, 2007 May 29.
Article in English | MEDLINE | ID: mdl-17485083

ABSTRACT

The angiotensin I-converting enzyme (ACE) converts the decapeptide angiotensin I (Ang I) into angiotensin II by releasing the C-terminal dipeptide. A novel approach combining enzymatic and electron paramagnetic resonance (EPR) studies was developed to determine the enzyme effect on Ang I containing the paramagnetic 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) at positions 1, 3, 8, and 9. Biological assays indicated that TOAC(1)-Ang I maintained partly the Ang I activity, and that only this derivative and the TOAC(3)-Ang I were cleaved by ACE. Quenching of Tyr(4) fluorescence by TOAC decreased with increasing distance between both residues, suggesting an overall partially extended structure. However, the local bend known to be imposed by the substituted diglycine TOAC is probably responsible for steric hindrance, not allowing the analogues containing TOAC at positions 8 and 9 to act as substrates. In some cases, although substrates and products differ by only two residues, the difference between their EPR spectral lineshapes allows monitoring the enzymatic reaction as a function of time.


Subject(s)
Cyclic N-Oxides/metabolism , Peptidyl-Dipeptidase A/metabolism , Animals , Cyclic N-Oxides/chemistry , Electron Spin Resonance Spectroscopy , Female , Guinea Pigs , Ileum/enzymology , Kinetics , Mass Spectrometry , Peptides/chemistry , Peptides/metabolism , Rats , Spectrometry, Fluorescence , Substrate Specificity , Uterus/enzymology
15.
Chem Pharm Bull (Tokyo) ; 55(3): 468-70, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17329894

ABSTRACT

Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the Nalpha-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the Nalpha-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.


Subject(s)
Amino Acids/chemistry , Fluorenes/chemistry , Oligopeptides/chemical synthesis , Protein Conformation
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