Differential mechanisms of activation of the Ang peptide receptors AT1, AT2, and MAS: using in silico techniques to differentiate the three receptors.

The renin-angiotensin system is involved in multiple conditions ranging from cardiovascular disorders to cancer. Components of the pathway, including ACE, renin and angiotensin receptors are targets for disease treatment. This study addresses three receptors of the pathway: AT1, AT2, and MAS and how the receptors are similar and differ in activation by angiotensin peptides. Combining biochemical and amino acid variation data with multiple species sequence alignments, structural models, and docking site predictions allows for visualization of how angiotensin peptides may bind and activate the receptors; allowing identification of conserved and variant mechanisms in the receptors. MAS differs from AT1 favoring Ang-(1-7) and not Ang II binding, while AT2 recently has been suggested to preferentially bind Ang III. A new model of Ang peptide binding to AT1 and AT2 is proposed that correlates data from site directed mutagenesis and photolabled experiments that were previously considered conflicting. Ang II binds AT1 and AT2 through a conserved initial binding mode involving amino acids 111 (consensus 325) of AT1 (Asn) interacting with Tyr (4) of Ang II and 199 and 256 (consensus 512 and 621, a Lys and His respectively) interacting with Phe (8) of Ang II. In MAS these sites are not conserved, leading to differential binding and activation by Ang-(1-7). In both AT1 and AT2, the Ang II peptide may internalize through Phe (8) of Ang II propagating through the receptors' conserved aromatic amino acids to the final photolabled positioning relative to either AT1 (amino acid 294, Asn, consensus 725) or AT2 (138, Leu, consensus 336). Understanding receptor activation provides valuable information for drug design and identification of other receptors that can potentially bind Ang peptides.

TOF-SIMS with argon gas cluster ion beams: a comparison with C60+.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an established technique for the characterization of solid sample surfaces. The introduction of polyatomic ion beams, such as C(60), has provided the associated ability to perform molecular depth-profiling and 3D molecular imaging. However, not all samples perform equally under C(60) bombardment, and it is probably naïve to think that there will be an ion beam that will be applicable in all situations. It is therefore important to explore the potential of other candidates. A systematic study of the suitability of argon gas cluster ion beams (Ar-GCIBs) of general composition Ar(n)(+), where n = 60-3000, as primary particles in TOF-SIMS analysis has been performed. We have assessed the potential of the Ar-GCIBs for molecular depth-profiling in terms of damage accumulation and sputter rate and also as analysis beams where spectral quality and secondary ion yields are considered. We present results with direct comparison with C(60) ions on the same sample in the same instrument on polymer, polymer additive, and biomolecular samples, including lipids and small peptides. Large argon clusters show reduced damage accumulation compared with C(60) with an approximately constant sputter rate as a function of Ar cluster size. Further, on some samples, large argon clusters produce changes in the mass spectra indicative of a more gentle ejection mechanism. However, there also appears to be a reduction in the ionization of secondary species as the size of the Ar cluster increases.

The effect of the secondary structure on dissociation of peptide radical cations: fragmentation of angiotensin III and its analogues.

Fragmentation of protonated RVYIHPF and RVYIHPF-OMe and the corresponding radical cations was studied using time- and collision energy-resolved surface-induced dissociation (SID) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) specially equipped to perform SID experiments. Peptide radical cations were produced by gas-phase fragmentation of Co (III)(salen)-peptide complexes. Both the energetics and the mechanisms of dissociation of even-electron and odd-electron angiotensin III ions are quite different. Protonated molecules are much more stable toward fragmentation than the corresponding radical cations. RRKM modeling of the experimental data suggests that this stability is largely attributed to differences in threshold energies for dissociation, while activation entropies are very similar. Detailed analysis of the experimental data obtained for radical cations demonstrated the presence of two distinct structures separated by a high free-energy barrier. The two families of structures were ascribed to the canonical and zwitterionic forms of the radical cations produced in our experiments.

Hydrolysis of various bioactive peptides by goat brain dipeptidylpeptidase-III homologue.

DPP-III from goat brain was purified to apparent electrophoretic homogeneity which showed several characteristics similar to other reported DPP-IIIs although it possesses dissimilar molecular weight and different inhibition behavior. Thin layer chromatographic studies with goat brain DPP-III revealed that it hydrolyses Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) at the Gly-Gly bond producing Tyr-Gly and Gly-Phe-Leu with no further degradation of liberated tripeptide. (Ala)(4) is hydrolyzed to dialanine whereas trialanine is not cleaved. ACTH, angiotensin II and III were also hydrolyzed whereas angiotensin I was not. It was concluded that the enzyme requires at least a tetrapeptide to act and that it removes a dipeptidyl moiety from the NH(2)-terminus of the studied peptides. Goat brain DPP-III may be involved in the metabolism of very important bioactive peptides such as enkephalins and angiotensins.

Formation of anionic peptide radicals in vacuo.

In this paper, we demonstrate for the first time the formation of radical anionic peptides [M - 2H]*- through a one-electron transfer mechanism upon low-energy collision-induced dissociation (CID) of gas-phase singly charged [Mn(III)(salen)(M - 2H)]*- complex ions [where salen is N,N'-ethylenebis(salicylideneiminato) and M is an angiotensin III derivative]. The types of fragment ions formed from [M - 2H]*- share some similarities with those from the cationic radical peptides M*+ and [M + H]*2+, but differ significantly from those of the corresponding deprotonated peptides [M - H]-. Fragmentation of [M - 2H]*- radical anionic angiotensin III derivatives leads preferentially to product ions of side-chain cleavage of amino acid residues, z-type and minor x-type fragment ions, most of which are types rarely observed in low-energy CID spectra of deprotonated analogs. The degree of competitive dissociation of the complexes is highly dependent on the nature of the substituted salen derivatives. The yields of anionic peptide radicals were enhanced to the greatest extent when electron withdrawing groups were positioned at the 5 and 5' positions, but the effect was rather modest when such groups resided at the 3 and 3' positions. Substituting a cyclohexyl unit of a salen with phenyl or naphthyl moieties at the 8 and 8' positions also facilitated electron-transfer pathways.

A mechanistic investigation of the enhanced cleavage at histidine in the gas-phase dissociation of protonated peptides.

Enhanced gas-phase cleavage of peptides adjacent to histidine was investigated. The peptides examined were angiotensins III (RVYIHPF) and IV (VYIHPF) as well as synthetic peptide analogues with altered key residues ((R)VYI-X-Z-F; X = F or H and Z = A, P, or Sar) or a fixed charge M3P(+)CH(2)C(O)-VYIHPF. While all singly protonated peptide ions containing both histidine and arginine fragment nonselectively, the doubly protonated peptide ions with arginine and histidine, and the singly protonated peptides containing histidine but not arginine, cleave in a selective manner. In particular, dominant complementary b+/y+ product ions resulting from cleavage between the HP amide bond are observed. For the fixed-charge derivative, selective cleavage occurs only if a proton is added to produce a doubly charged precursor. The results are consistent with involvement of a protonated histidine in the selective cleavage. The ratio of b+/y+ is determined by the identity of the residue C-terminal to histidine and by the ability of protonated histidine to transfer a proton to the C-terminal leaving fragment. This was probed further by systematically changing the residue C-terminal to histidine and by alkylating histidine. The results indicate that while b+/y+ complementary ion pairs dominate in doubly protonated RVYIHPF, b5(2+) and b6(2+) product ions dominate the spectra of doubly protonated RVYIHAF. Also, dominant b5(2+) product ions are observed when the histidine side chain is alkylated (H) in doubly protonated RVYIHPF. Based on all of the results, a selective fragmentation mechanism for enhanced cleavage at histidine involving an atypical b ion structure is proposed.

Determination of endogenous peptides with in vitro ACE inhibitory activity in normotensive human plasma by the fluorometric HPLC method.

An in vitro degradation test of angiotensin (ANG) II or III in normotensive supine human plasma from 9 healthy male subjects confirmed the production of smaller ANG metabolites with angiotensin I-converting enzyme inhibitory activity. These metabolites were identified as ANG (3-8), ANG (5-8), and ANG (3-4), whose respective peptide concentrations were determined by our proposed naphthalene-2,3-dialdehyde (NDA)-HPLC method to be 64 +/- 9, 39 +/- 5, 176 +/- 22, and 197 +/- 35 fmol/ml of plasma.

Conformational analysis of neuropeptide Y-[18-36] analogs in hydrophobic environments.

The interactive and conformational behavior of a series of neuropeptide Y-[18-36] (NPY-[18-36]) analogs in hydrophobic environments have been investigated using reversed-phase high-performance liquid chromatography (RP-HPLC) and circular dichroism (CD) spectroscopy. The peptides studied comprised a series of 16 analogs of NPY-[18-36], each containing a single D-amino acid substitution. The influence of these single L-->D substitutions on the alpha-helical conformation of the NPY-[18-36] analogs in different solvent environments was determined by CD spectroscopy. Retention parameters related to the hydrophobic contact area and the affinity of interaction were determined with an n-octadecyl (C18) adsorbent. Structural transitions for all peptides were manifested as significant changes in the hydrophobic binding domain and surface affinity between 4 degrees C and 37 degrees C. The results indicated that the central region of NPY-[18-36] (residues 23-33) is important for maintenance of the alpha-helical conformation. Moreover, L-->D amino acid residue substitutions within the N- and C-terminal regions, as well as Asn29 and Leu30, do not appear to affect the secondary structure of the peptide. These studies demonstrate that RP-HPLC provides a powerful adjunct for investigations into the induction of stabilized secondary structure in peptides upon their interaction with hydrophobic surfaces.

Angiotensin II receptors in Xenopus oocytes.

Electrical recordings were used to study the sensitivity of native Xenopus oocytes to the octapeptide angiotensin II (AII). AII elicited oscillatory currents associated with an increase in membrane conductance to Cl-. Responsiveness to AII varied greatly between oocytes taken from different frogs, and to a lesser extent between oocytes from the same ovary. Oocytes from frogs showing high sensitivity had response thresholds between 0.5-1.0 nM AII, and at a holding potential of -60 mV, responded to 1 microM AII with currents greater than 3 microA. In contrast, oocytes from some frogs gave no response, even to 10 microM AII. A total of 618 oocytes from 79 frogs were tested for sensitivity to AII, and oocytes from 85% of frogs gave detectable electrical responses. Oscillatory Cl- currents elicited by AII were largely independent of extracellular Ca2+, were abolished by chelation of intracellular Ca2+ using EGTA and were mimicked by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3). In addition to oscillatory Cl- currents, AII also evoked an influx of extracellular Ca2+, giving rise to a transient inward Cl- current on membrane hyperpolarizing steps. These experiments all suggested that AII responses were elicited through activation of an intracellular messenger pathway triggered by hydrolysis of inositolphospholipids, mobilization of intracellular Ca2+ by inositol polyphosphates, and activation of Ca(2+)-gated Cl- channels. The effect of manual or enzymic defolliculation on AII responses was studied in nine separate experiments recording from 70 defolliculated oocytes. Efficacy of defolliculation procedures was assayed using scanning electron microscopy, which confirmed removal of 90 to greater than 98% of follicular cells.(ABSTRACT TRUNCATED AT 250 WORDS)