Biomedical subjects

T Mavromoustakos

Publications and source records attributed to T Mavromoustakos.

At least 55 records · Page 3Linked to original sources

PubMed1999

Design and synthesis of a potent cyclic analogue of the myelin basic protein epitope MBP72-85: importance of the Ala81 carboxyl group and of a cyclic conformation for induction of experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is induced in susceptible animals by immunodominant determinants of myelin basic protein (MBP), such as guinea pig sequence MBP72-85. Two linear and one cyclic analogues based on MBP72-85 have been synthesized and evaluated for EAE induction in Lewis rats. The linear peptide Gln1-Lys2-Ser3-Gln4-Arg5-Ser6-Gln7-+ ++Asp8-Glu9-Asn10-Pro11-Val12 (1) was found to induce EAE, while substitution of the Asp residue at position 8 with Ala resulted in an analogue (2) which suppressed the induction of EAE by its parent peptide. Nuclear magnetic resonance studies of analogue 1 in dimethyl sulfoxide (DMSO) using TOCSY/ROESY techniques revealed a head-to-tail intramolecular interaction (ROE connectivity between betaVal12-gammaGln1), indicating a pseudocyclic conformation for the immunogenic peptide 1. A conformational model was developed using NMR constraints and molecular dynamics. Based on this model, a novel amide-linked cyclic analogue has been designed and synthesized by connecting the side-chain amino and carboxyl groups of Lys and Glu at positions 2 and 9, respectively, of linear analogue 1. The cyclic analogue (3) had similar activity to the linear peptide 1, and the EAE effects induced by cyclic analogue 3 were completely suppressed by co-injection with the Ala81-substituted analogue 2 in Lewis rats. The similar potencies of analogues 1 and 3 support the proposed cyclic comformation suggested for analogue 1 from NMR studies and computer modeling and provides the basis for designing more potent molecules with improved properties such as increased resistance to degradation.15 The present findings suggest that a cyclic conformation for the MBP72-85 epitope positions the carboxyl group of Asp81 correctly and presumably other side groups of the peptide such as Arg78 in a manner which enables functional binding of the trimolecular complex MHC-peptide-T cell receptor resulting in EAE.

Alanine↗

PubMed1999

Structure elucidation and conformational properties of synthetic cannabinoids (-)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethyl-1-hydroxy-6H-dibe nzo [b,d]pyranyl)-2-hexyl-1,3-dithiolane and its methylated analog.

The synthetic cannabinoid (-)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethyl-1-hydroxy-6H-dibenzo[b,d]+ ++pyranyl)-2-hexyl-1,3-dithiolane (AMG-3) is a cannabimimetic molecular probe with one of the highest binding affinities reported to date. Therefore, due to its potential pharmacological importance, its structure was sought to be elucidated and its conformational properties were studied using a combination of 1D, 2D NMR spectroscopy and molecular modelling. The structure of its methylated analog (-)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H dibenzo [b,d]pyranyl-1-methoxy)-2-hexyl-1,3 dithiolane (AMG-18), was also studied and its conformational properties were compared with AMG-3. AMG-18 lacks of the phenolic hydroxyl group a strict requirement for cannabimimetic activity and is almost devoid of any biological activity. The conformational analysis studies showed that 1',1' dithiolane ring restricted the orientation preferences of alkyl chain. This may account for the high binding affinity of AMG-3 to cananbinoid receptors. Grid scan search studies showed different preferences of possible adopting dihedral values of phenolic hydroxyl group and its methyl ether. These observations may account for their differences in biological activity.

Cannabinoids↗

PubMed1999

Design and synthesis of thrombin receptor-derived nonpeptide mimetics utilizing a piperazine scaffold.

Focal thrombus formation and vasoconstriction serve to defend vessels when vascular damage occurs, but may be detrimental when an atherosclerotic plaque is disrupted. Recently, the identification of the platelet thrombin receptor opened a new area in the development of agents that may selectively inhibit the effects of thrombin on cells, without affecting fibrin formation. In this regard, we have synthesized a number of 1,4-disubstituted piperazines which are designed to be analogues of thrombin receptor activating peptides (TRAP) and carry the pharmacophoric features of Phe and Arg residues present in the active pentapeptide SFLLR. These compounds were tested in the rat aorta relaxation assay and in platelet aggregation studies and their biological activity was consistent with a direct action on thrombin receptor. Furthermore, the structure activity relationships confirmed the importance of Phe and Arg for receptor activation and the molecular modeling revealed an intriguing relationship between their amphipathic similarity with SFLLR and their biological activity.

Animals↗

PubMed1998

A comparative SAR study of thrombin receptor derived non peptide mimetics: importance of phenyl/guanidino proximity for activity.

Thrombin, the most potent physiological platelet agonist interacts with cells through a specific G protein-coupled receptor which has been cloned and sequenced. Synthetic thrombin receptor peptides (TRAPs) comprising the first 5 amino acids (SFLLR and SFLLR-NH2) of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity were found to cause full platelet aggregation. During the screening of novel thrombin receptor derived non-peptide mimetics in the platelet aggregation assay we found that 1-phenylacetyl-4-(6-guanidohexanoyl)-piperazine (1) and 1-(6-guanidohexanoyl)-4-(phenylacetylamidomethyl)-piperidine (2) exerted in vitro antagonist activities (56% and 40% correspondingly) as it is depicted by the platelet aggregation assay. Using Molecular Modeling, the synthetic compounds were overlayed with SFFLR. All three superimposed low energy structures had Phe and Arg amino acids in spatial close proximity. The superimposition results revealed that 1 resembled more the stereoelectronic environment of SFLLR than 2. This difference may be related to their different antagonist efficacy.

Arginine↗

PubMed1998

Thermal properties of adamantanol derivatives and their beta-cyclodextrin complexes in phosphatidylcholine bilayers.

Differential Scanning Calorimetry (DSC) has been applied to study the thermal properties of the membrane perturbing antibacterial octyl- and dodecyl-bromide salts of quaternary dimethylamino adamantanol (ADM-8 and ADM-12 correspondingly) incorporated in free or complexed form with beta-cyclodextrin (beta-CD) into dipalmitoylphosphatidylcholine (DPPC) containing bilayers. The DSC results showed that the studied compounds exert pronounced thermotropic changes in DPPC bilayers when inserted as free molecules. These effects are reduced when are present in a complex form with beta-CD. Since the studied compounds exert destructive effects in membrane bilayers their insertion in membrane bilayers as complexes with cyclodextrin may result in differentiation of their activity. The obtained results suggest that their complexation with beta-CD may improve their biological profile. It also increases their aqueous solubility, a limited factor for their use as drugs.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1998

Nuclear magnetic resonance spectral analysis and conformational properties of 11-benzoyl-9,9a,10,11-tetrahydro-4H-indolo[4,3-ab]carbazole.

The structure of 11-benzoyl-9,9a,10,11-tetrahydro-4H-indolo [4,3-ab] carbazole, a candidate molecule to possess significant antitumor or antimicrobial activity, was elucidated using a combination of one-dimensional and two-dimensional nuclear magnetic resonance (NMR) techniques. Its conformational properties were studied using a combination of two-dimensional NOESY spectroscopy and molecular modeling. Such information will be of aid to synthetic chemists who aim to develop derivatives of this structure. It may also provide information about the stereoelectronic requirements that govern their activities.

Anti-Infective Agents↗

PubMed1998

Structural elucidation and conformational properties of the immunomodulator linomide.

Linomide is a new synthetic immunomodulator which exerts prominent anti-autoimmune effects in various experimental models. Recently, it was tested in clinical trials to patients suffering from multiple sclerosis and showed to inhibit the activity of the disease. Therefore, due to its pharmacological importance, we attempted elucidate its structure using one-dimensional and two-dimensional nuclear magnetic resonance (NMR) techniques and study its conformational properties using a combination of two-dimensional NMR spectroscopy and molecular modeling. The conformational analysis of linomide was based on the measurement of interproton nuclear Overhauser enhancement (NOE) values obtained from a two-dimensional NMR spectrum and a number of molecular modeling techniques used to calculate the low energy conformers of this compound. This information will serve as an aid to synthetic chemists whom their research activity is focused on developing linomide analogs with better biological profiles.

Adjuvants, Immunologic↗

PubMed1997

The use of high-resolution solid-state NMR spectroscopy and differential scanning calorimetry to study interactions of anaesthetic steroids with membrane.

We have used a combination of high-resolution solid-state 13C-NMR and DSC (differential scanning calorimetry) to study the distinctively different thermotropic and dynamic properties of the anaesthetic steroid alphaxalone and its inactive congener delta16-alphaxalone in dipalmitoylphosphatidylcholine (DPPC) model membranes. In the solid-state 13C-NMR, the techniques included cross polarization (CP) and/or magic angle spinning (MAS). The observed data revealed the following important results. (a) DSC as a bulk method showed that the active steroid lowers the main phase transition temperature and broadens the pretransition more significantly than the inactive congener. The 13C-CP/MAS experiments allowed us to detect the pretransition temperature in the alphaxalone-containing preparation, which was not discernible in DSC. (b) The chemical shift values varied with temperature, indicating different degrees of trans-gauche isomerization in the lipid acyl chains when the bilayer is in the liquid crystalline phase. (c) Only specific additional peaks appeared in the 13C-CP/MAS spectra when each of the steroids was present in the preparation. delta16-alphaxalone gives rise to more additional peaks than alphaxalone, indicating a different mobility of the corresponding molecular moiety in the phospholipid bilayer environment. (d) The relative intensities of these peaks also confirmed that alphaxalone is fully incorporated in the bilayer, whereas delta16-alphaxalone is only partially so. These results suggest that the differential effects of these two analogues in the membrane may, at least in part, explain the reason for their different biological activities.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1997

Conformational analysis of the thrombin receptor agonist peptides SFLLR and SFLLR-NH2 by NMR: evidence for a cyclic bioactive conformation.

The conformational properties of the pentapeptide Ser-Phe-Leu-Leu-Arg (P5), a human thrombin receptor-derived sequence forming part of a tethered ligand which activates the thrombin receptor, and its more active amide derivative Ser-Phe-Leu-Leu-Arg-NH2 (P5-NH2), have been studied by proton NMR spectroscopy in dimethylsulfoxide. Measurements of nuclear Overhauser effects, performed using two-dimensional rotating frame nuclear Overhauser (ROESY) and one-dimensional nuclear Overhauser enhancement (NOE) spectroscopy, revealed that P5 exists mainly in an extended conformation. However, proton-proton 1D-NOEs between Phe C alpha H and Ser C alpha H Leu3 C alpha H and Leu3 NH, and Leu4 C alpha H and Leu4 NH, as well as between the Ser and Arg sidechains, also implicated a minor conformer for P5 having a curved backbone and a near-cyclic structure. In contrast to P5, measurements of NOEs and ROEs for P5-NH2 revealed a more stabilized cyclic structure which may account for its higher biological potency. Thus strong interresidue sequential NH (i)-NH (i + 1) interactions, as well as C-terminal carboxamide to N-terminal side-chain interactions, i.e., Arg CONH2 to Phe ring and Arg CONH2 to Ser C alpha/C beta beta', observed at lower levels of the ROESY spectrum, supported a curved backbone structure for SFLLR-NH2. Since the higher potency P5-NH2 analogue adopts predominantly a cyclic structure, a cyclic bioactive conformation for thrombin receptor agonist peptides is suggested.

Amino Acid Sequence↗

PubMed1996

The conformational properties of the antineoplastic ether lipid 1-thiohexadecyl-2-O-methyl-S-glycero-3-phosphocholine.

Thio analogs of platelet activating factor (PAF) are of great interest because they exhibit antineoplastic properties both in vitro and in vivo. In contrast to most known anticancer agents, these lipids appear not to act through the synthesis and function of DNA and, therefore, offer a new avenue of approaching cancer chemotherapy. We have examined the conformational properties of 1-thiohexadecyl-2-O-methyl-S-glycero-3-phosphocholine (ET-S-16-OCH3) in organic solvents and in micelles. The conformational analysis was based on a combination of 1D, 2D NMR spectroscopy and molecular graphics. 1H and 13C spin lattice relaxation time (T1) experiments were also performed to study the dynamic properties of this molecule. The picture emerging from these studies is as follows. The alkyl chain of ET-S-16-OCH3 is the most mobile part of the molecule both in CDCl3/CD3OD and in micelles and exists as a mixture of interconverting conformers including an extended all trans and several low energy conformers with one or more gauche segments. This creates a twisting of the chain and facilitates a spatial communication between the alkyl chain and the glycerol backbone as well as between the alkyl chain and the headgroup. The methylene groups of the thioglycerol backbone and the headgroup are the least mobile while the methine group of the thioglycerol backbone appears to have an intermediate mobility. The conformation of the thioether lipid in the two media may be of relevance during its interaction with its site of action, the cellular membrane. Such a conformation may also play an important role in determining the selectivity of this interaction with different cell membranes.

Antineoplastic Agents↗

PubMed1996

Synthesis and contractile activities of cyclic thrombin receptor-derived peptide analogues with a Phe-Leu-Leu-Arg motif: importance of the Phe/Arg relative conformation and the primary amino group for activity.

Based on the minimal peptide sequence (Phe-Leu-Leu-Arg) that has been found to exhibit biological activity in a gastric smooth muscle contractile assay for thrombin receptor-activating peptides, the cyclic peptide analogues cyclo(Phe-Leu-Leu-Arg-Acp) (1), cyclo(Phe-Leu-Leu-Arg-epsilon Lys) (2), and cyclo(Phe-Leu-Leu-Arg-Gly) (3) have been synthesized by the solid-phase method using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluoroborate or 2-(1H-benzo-triazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate as cyclization reagents. The contractile activities of compounds 1-3 have been compared with that of the linear thrombin receptor-activating peptide (TRAP) Ser-Phe-Leu-Leu-Arg-NH2 (compound 4) using a gastric smooth muscle strip assay. Compound 2, wherein the epsilon-amino group of lysine was coupled to the alpha-carboxyl of arginine, exhibited a contractile activity comparable to that of the linear TRAP, compound 4. However compound 1, wherein the aminocaproic linker group yielded a ring size the same as for compound 2 but without a primary amino group, exhibited a contractile activity 600-1000-fold lower than compounds 2 and 4. Compound 3, which exhibited partial agonist activity, was about 100-fold less potent than either compound 2 or 4. NMR spectroscopy of compound 2 revealed a proximity of the Phe and Arg side chains, leading to a molecular model generated by distance geometry and molecular dynamics, wherein the Phe and Arg residues are shown in proximity on the same side of the peptide ring. We conclude that the Phe and Arg side chains along with the primary amino group form an active recognition motif that is augmented by the presence of a primary amino group in the cyclic peptide. We suggest that a comparable cyclic conformation may be responsible for the interaction of linear TRAPs with the thrombin receptor.

Amino Acid Sequence↗

PubMed1996

Studies on the thermotropic effects of cannabinoids on phosphatidylcholine bilayers using differential scanning calorimetry and small angle X-ray diffraction.

We have studied the thermotropic properties of a wide variety of cannabinoids in DPPC bilayers. The molecules under study were divided into four classes: (a) classical cannabinoids possessing a phenolic hydroxyl group; (b) delta9-THC metabolites with an additional hydroxyl group on the C ring; (c) non-classical cannabinoids, and (d) cannabinoids with a protected phenolic hydroxyl group. The results showed that the first three groups have similar effects on the thermotropic properties of DPPC bilayers up to x = 0.05 (molar ratio) and that these effects do not parallel their biological activity. For concentrations less than x = 0.01, cannabinoids affect mainly the pretransition temperature in a progressive manner until its final abolishment. At x = 0.05, they further affect the main phase transition by lowering its phase transition temperature and broadening its half width. At high concentrations the thermograms have multiple components, indicating that membranes are no longer homogeneous but rather consist of different domains. At these concentrations cannabinoids with more hydroxyl groups give simpler thermograms. Low concentrations of cannabinoids in group d affect significantly the pretransition temperature, while high concentrations affect only marginally the main phase transition by slightly lowering its temperature and broadening its half width. These results point out the importance of the phenolic hydroxyl group in inducing membrane perturbations. The d-spacing data from our small angle X-ray diffraction experiments show that delta8-THC produces significant structural changes in the lipid bilayer, including the gel-phase tilting angle, the intermolecular cooperativity and the gauche:trans conformer ratio. Conversely, the inactive analog Me-delta8-THC does not cause drastic changes to the bilayer structure.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1996

Topography and thermotropic properties of cannabinoids in brain sphingomyelin bilayers.

In our previous publications we compared the locations of the biologically active (-)-delta 8-tetrahydrocannabinol (delta 8-THC) with that of its inactive analog O-methyl-(-)-delta 8-tetrahydrocannabinol (Me-delta 8-THC) in the liquid crystalline phase of partially hydrated dimyristoylphosphatidylcholine (DMPC) bilayers (Mavromoustakos et al. (1990) Biophys. Acta 1024, 336-344; Yang et al. (1993) Life Sci. 53, 117-122). delta 8-THC was shown to localize itself preferentially in the vicinity of the membrane interface with its phenolic hydroxyl group anchored near the carbonyl groups of DMPC while the more lipophilic Me-delta 8-THC is located deeper towards the center of the bilayer. In the present publication we studied and compared the topography of the two analogs in the gel phase of brain sphingomyelin bilayers. Again we found that delta 8-THC is located near the membrane interface approximately 15 A from the center of the bilayer while its inactive analog localizes deeper in the bilayer at an average site only 8 A from the center of the membrane bilayer. It thus, appears that both analogs preferentially localize in distinct sites within the membrane bilayer which are independent of the mesomorphic state and the nature of the phospholipid. Our results suggest that in the more complex environment of biological membrane which is composed of different phospholipids and proteins the two analogs are expected to prefer different average locations within the bilayer, a property which may in part explain the observed differences in their biological activities.

Brain↗

PubMed1995

Differences in backbone structure between angiotensin II agonists and type I antagonists.

Type I angiotensin II antagonists with O-methyl-L-homoserine [HSer(gamma-OMe)] and delta-methoxy-L-norvaline [Nva(delta-OMe)] at position 8 have been prepared by the solid-phase method, purified by reverse-phase HPLC, and bioassayed in the rat uterus, and their backbone conformational properties were investigated by nuclear Overhauser effect (NOE) spectroscopy. [Sar1,HSer-(gamma-OMe)8]ANGII, [HSer(gamma-OMe)8]ANGII, [Des1,HSer(gamma-OMe)8]ANGII, [Sar1,Nva(delta-OMe)8]-ANGII, and [Des1,Nva(delta-OMe)8]ANGII had, respectively, the following antagonist activities, pA2: 7.6, 7.5, < 6.0, 7.1, and 6.9. Analogs of [Sar1]ANGII with delta-hydroxy-L-norvaline [Nva(delta-OH)], delta-methoxy-L-norvaline [Nva(delta-OMe)], 4'-carboxyphenylalanine [Phe(4'-COOH)], and 4'-(trifluoromethyl)phenylalanine [Phe(4'-CF3)] at position 4 were also prepared by solid phase and bioassayed in the rat uterus. [Sar1,Nva(delta-OH)4]ANGII, [Aib1,Nva(delta-OMe)4]ANGII, [Sar1,DL-Phe(4'-COOH)4]ANGII, and [Sar1,DL-Phe(4'-CF3)4]ANGII had, respectively, agonist activities as follows: 4%, 1.5%, 3%, < 0.1%, and < 0.1%. These data emphasize that replacement of Ile8 in Sarilesin with the higher homologs HSer(gamma-OMe) and Nva(delta-OMe) does not greatly alter the structural requirements necessary for expression of type I antagonist activity, while replacement of the tyrosine hydroxyl in [Sar1]ANGII by the carboxylate or the trifluoromethyl group abolishes activity, suggesting that the tyrosinate pharmacophore cannot be replaced by any negatively charged or electronegative group. Conformational investigation of the ANGII type I antagonists [HSer(gamma-OMe)8]ANGII and [Sar1Nva(delta-OMe)8]ANGII in DMSO by 1D-NOE spectroscopy revealed that the Tyr-Ile-His bend, a conformational property found in ANGII and [Sar1]ANGII (J. Biol. Chem. 1994, 269, 5303) is not present in type I antagonists, providing for the first time an important conformational difference between angiotensin II agonists and type I antagonists.

Amino Acid Sequence↗

PubMed1995

Effects of the anesthetic steroid alphaxalone and its inactive delta 16-analog on the thermotropic properties of membrane bilayers. A model for membrane perturbation.

We have studied in detail the effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on the thermotropic properties of model membranes using differential scanning calorimetry (DSC). The results obtained showed that, for model membranes from hydrated dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and egg sphingomyelin, the biologically active analog significantly broadened the phase transition, in contrast to the inactive one which produced only marginal effects. Also, alphaxalone abolished the pretransition in these preparations whereas its delta 16-analog only broadened it. However, in DPPE bilayers almost no differences were observed in the effects produced by the two analogs. These results suggest that the ability of the two steroids to perturb membranes is lipid dependent. Comparisons between the effects of the two steroids on lipid/cholesterol model membranes revealed that delta 16-alphaxalone excluded cholesterol from lipid/cholesterol/delta 16-alphaxalone ternary systems whereas alphaxalone enhanced the effects of cholesterol and reduced the cooperativity in the binary phospholipid/cholesterol system. In an attempt to determine whether the different thermotropic effects of the two steroids on model membranes were due to (a) differences in their ability to perturb the bilayers; (b) different extents of incorporation into the bilayer, solid state 2H-NMR was applied using specifically deuterated steroids. The 2H-NMR data showed that alphaxalone incorporated fully into the membrane bilayer up to a molar concentration of 20%, while its inactive analog did only up to a concentration of 1%. To compare the abilities of the two steroids to perturb membrane preparations when both analogs were present in equal amounts in the membrane, the effects of very low steroid concentrations on DPPC bilayers were studied using DSC. The experiment showed that alphaxalone perturbed the membrane bilayers more effectively than its inactive analog. These results strongly suggest that the small structural differences between the two steroids are responsible for the observed differences in their abilities to perturb membranes, possibly because of differences in the packing of these two molecules within the bilayers.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1995

Small angle X-ray diffraction and differential scanning calorimetric studies on O-methyl-(-)-delta 8-tetrahydrocannabinol and its 5' iodinated derivative in membrane bilayers.

We have previously studied and compared the location of (-)-delta 8-tetrahydrocannabinol (delta 8-THC) with that of O-methyl-delta 8-THC (Me delta 8-THC) in the membrane using partially hydrated dimyristoylphosphatidylcholine (DMPC) bilayers ((Mavromoustakos et al. (1990) Biophys. Acta 1024, 336-344; Yang et al. (1993) Life Sci. 53, 117-122). delta 8-THC was found to be located near the membrane interface with its phenolic hydroxyl group anchored near the carbonyl groups of DMPC while the more lipophilic Me-delta 8-THC is located deeper in the membrane bilayer. Parallel experiments using Me-delta 8-THC and its 5'-iodo analog (5'-I-Me-delta 8-THC) allowed us to determine the topography of these two molecules in the bilayer. Our results from small angle X-ray diffraction and differential scanning calorimetry (DSC) combined with previous data on the orientation of Me-delta 8-THC in model membranes, led us to the conclusion that these molecules intercalate between contiguous acyl chains in the lipophilic moiety of the membrane bilayer. The terminal iodo group in 5'-I-Me-delta 8-THC was found to reside in a region extending approx. +/- 5 A from the center of the bilayers. The location of Me-delta 8-THC in the membranes as well as its orientation may explain its inability to effectively perturb the bilayer lipid chains.

Calorimetry, Differential Scanning↗

PubMed1994

Topography of alphaxalone and delta 16-alphaxalone in membrane bilayers containing cholesterol.

We have used small-angle X-ray diffraction and differential scanning calorimetry (DSC) to study the topographies of alphaxalone and its biologically inactive analog delta 16-alphaxalone in dimyristoylphosphatidylcholine (DMPC) and DMPC/cholesterol model membranes. Diffraction patterns were obtained and analyzed for preparations of bilayers without and with the steroids. Temperature dependence of the total period repeat distance (d-spacing) allowed us to identify equivalent temperatures at which the preparations had similar d-spacing and were in the same mesomorphic state. The combination of X-ray and DSC data showed that the anesthetic steroid alphaxalone broadens the membrane phase transition and increases the ratio of gauche: trans conformers in the membranes in contrast to the inactive steroid delta 16-alphaxalone which affects the membranes only marginally. In model DMPC membranes alphaxalone and delta 16-alphaxalone are located near the bilayer interface. This location is maintained by alphaxalone when cholesterol is incorporated in the bilayer as evidenced by the X-ray measurements. However, when delta 16-alphaxalone is incorporated in cholesterol containing bilayers, a decrease in the electron density profile of the preparation is observed. This can be explained by invoking the formation of a delta 16-alphaxalone-cholesterol complex. The delta 16-alphaxalone complex shows no periodicity and is therefore, not detected in the X-ray diffraction experiment. Presumably, this complex forms aggregates either on the surface or inside the bilayer. This explanation corroborates DSC results which show that delta 16-alphaxalone sharpens the phase transition of DMPC/cholesterol preparations, an indication that some cholesterol is excluded from the bilayer preparation after the addition of the biologically inactive steroid.

Anesthetics↗

PubMed1994

Role of the NH2-terminal domain of angiotensin II (ANG II) and [Sar1]angiotensin II on conformation and activity. NMR evidence for aromatic ring clustering and peptide backbone folding compared with [des-1,2,3]angiotensin II.

The role of the NH2 termini of angiotensin II (ANG II) and [Sar1]ANG II on conformation and activity were examined by proton NMR two-dimensional-J-correlated spectroscopy and one-dimensional nuclear Overhauser effect studies in the relatively nonpolar "receptor-simulating" environment provided by dimethyl sulfoxide-d6, using the biologically inactive COOH-terminal pentapeptide [des1,2,3]ANG II as control. Irradiation of C alpha H, C2H, and C4H proton resonances in ANG II and [Sar1]ANG II resulted in enhancements of Tyr and Phe ring proton resonances, indicating that the three aromatic rings cluster together. Very strong enhancements (17-22%) of the C alpha Y proton resonance in ANG II and [Sar1]ANG II upon irradiation of the C alpha H proton resonance, and vice versa, revealed that a Tyr-Ile-His bend is a predominant feature of the conformation of the two agonists. In contrast, saturation of the C alpha H and C alpha Y proton resonances in the control pentapeptide [des-1,2,3]ANG II did not produce, respectively, any C alpha Y or C alpha H proton nuclear Overhauser effect enhancement, illustrating the absence of a Tyr-Ile-His bend in the truncated ANG II peptide. The present findings indicate that the NH2-terminal domain of ANG II appears to have an essential role in generating the biologically active charge relay conformation of the hormone.

Algorithms↗