Functional assessment of angiotensin II and bradykinin analogues containing the paramagnetic amino acid TOAC

Santos, Edson Lucas dos; Souza, Kely de Picoli; Sabatini, Regiane A.; Martin, Renan Paulo; Fernandes, Liliam; Nardi, Daniela Teves; Malavolta, Luciana; Shimuta, Suma I.; Nakaie, Clóvis R.; Pesquero, João Bosco

doi:10.1016/j.intimp.2007.07.029

Cited by 10 publications

(7 citation statements)

References 30 publications

(38 reference statements)

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“…In addition, we were also able to discriminate differences between ligand specificities for ACE or AT 1 by using the TOAC 3 -Ang II derivative, where TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) is a spin-labeled amino acid that was internally coupled to Ang II. 21 TOAC 3 -Ang II, which is not able to bind or activate AT 1 receptors, 11,14 could stimulate CHO-ACE cells, increasing intracellular Ca 2ϩ , again suggesting a specific calcium signaling by ACE. In addition, we could demonstrate that the effect was specific for Ang II, because other peptides related to ACE metabolism, such as BK, BK1-5, Ac-SDKP, and Ang 1-7, were inactive.…”

Section: Discussionmentioning

confidence: 97%

“…Figure S1A) and confirmed by earlier data. 11,12 After the stable transfection of these cells with an empty plasmid (CHO-mock) and a plasmid containing a DNA cassette for somatic human ACE expression (CHO-ACE), specific ACE activity could be measured in CHO-ACE cells but not in the CHO-mock cells ( Figure S1B). To exclude any possible involvement of the AT 1 receptor in this effect, we confirmed its absence in CHO-ACE cells by flow cytometry using specific AT 1 antibodies ( Figure S1C).…”

Section: Inositol Triphosphate Formation Assaymentioning

confidence: 99%

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Angiotensin II Binding to Angiotensin I–Converting Enzyme Triggers Calcium Signaling

et al. 2011

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Section: Discussionmentioning

confidence: 97%

Section: Inositol Triphosphate Formation Assaymentioning

confidence: 99%

Angiotensin II Binding to Angiotensin I–Converting Enzyme Triggers Calcium Signaling

et al. 2011

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“…Functional studies of AII (TOAC 1 -AII and TOAC 3 -AII) and BK (TOAC 0 -BK and TOAC 3 -BK) analogues showed that, while N-terminally-labeled peptides retained at least part of their biological activity, internally-labeled analogues were inactive, possibly due to the fact that the TOAC-imposed bend did not allow acquisition of the receptor-bound conformation (Nakaie et al 2002; Santos et al 2008). …”

Section: Biologically Active Peptides: Ligands and Fragments Of G Promentioning

confidence: 99%

The spin label amino acid TOAC and its uses in studies of peptides: chemical, physicochemical, spectroscopic, and conformational aspects

et al. 2012

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We review work on the paramagnetic amino acid 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, TOAC, and its applications in studies of peptides and peptide synthesis. TOAC was the first spin label probe incorporated in peptides by means of a peptide bond. In view of the rigid character of this cyclic molecule and its attachment to the peptide backbone via a peptide bond, TOAC incorporation has been very useful to analyze backbone dynamics and peptide secondary structure. Many of these studies were performed making use of EPR spectroscopy, but other physical techniques, such as X-ray crystallography, CD, fluorescence, NMR, and FT-IR, have been employed. The use of double-labeled synthetic peptides has allowed the investigation of their secondary structure. A large number of studies have focused on the interaction of peptides, both synthetic and biologically active, with membranes. In the latter case, work has been reported on ligands and fragments of GPCR, host defense peptides, phospholamban, and β-amyloid. EPR studies of macroscopically aligned samples have provided information on the orientation of peptides in membranes. More recent studies have focused on peptide–protein and peptide–nucleic acid interactions. Moreover, TOAC has been shown to be a valuable probe for paramagnetic relaxation enhancement NMR studies of the interaction of labeled peptides with proteins. The growth of the number of TOAC-related publications suggests that this unnatural amino acid will find increasing applications in the future.

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“…The peptides behavior in contraction experiments was determined in guinea pig ileum and rat uterus following standard procedures [ 24 – 25 , 31 ]. ACE is known to convert AngI to the strongly vasoactive and spasmogenic peptide AngII, which, in turn, induces contraction of muscle tissues.…”

Section: Resultsmentioning

confidence: 99%

“…Conformational studies in solution and in the presence of model membranes, making use of additional techniques such as circular dicroism (CD) and fluorescence, of AngII and another vasoactive peptide, bradykinin (BK), as well as their TOAC-labeled derivatives, have been reported [ 22 – 23 ]. The effect of TOAC’s introduction on biological activity of these peptides was also investigated [ 24 – 25 ]. Labeling of other signaling peptides [ 26 – 28 ] showed that labeled α-MSH displayed full biological activity.…”

Section: Introductionmentioning

confidence: 99%

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates

et al. 2015

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Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra.

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Functional assessment of angiotensin II and bradykinin analogues containing the paramagnetic amino acid TOAC

Cited by 10 publications

References 30 publications

Angiotensin II Binding to Angiotensin I–Converting Enzyme Triggers Calcium Signaling

Angiotensin II Binding to Angiotensin I–Converting Enzyme Triggers Calcium Signaling

The spin label amino acid TOAC and its uses in studies of peptides: chemical, physicochemical, spectroscopic, and conformational aspects

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates

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