Role of cholesterol as a structural and functional effector of the nicotinic acetylcholine receptor.
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Allosteric Regulation↗
Biomedical subjects
J Castresana
Publications and source records attributed to J Castresana.
Familial cutaneous melanoma and two-mutational-event modeling.
BACKGROUND: According to the Knudson two-mutational-event theory, two mutations at a genetic locus may be required for the development of some cancers. Persons who have inherited a defect in one chromosome and therefore require only one more mutation for cancer development are at a higher risk of manifesting cancer at a younger age than persons without an inherited mutation, who need two acquired "hits." This difference allows one to distinguish familial and sporadic types of the same malignancy by evaluating age of disease onset. METHODS: To study the role of inheritance in the etiology of familial cutaneous melanoma, characteristics of patients with familial versus nonfamilial melanoma were analyzed according to the Knudson two-mutational-event model. RESULTS: The familial versus nonfamilial graphs, based on age of diagnosis, did not support this model. However, there was a statistically significant earlier age of diagnosis for patients with familial melanoma. Melanoma thickness was less (i.e., earlier cancer at possibly younger age) for patients with a positive versus a negative family history. Conversely, linear regression, after adjusting for tumor thickness, showed that patients with hereditary melanoma still manifested earlier ages of diagnosis of melanoma compared with sporadic patients. CONCLUSIONS: Genetic patterns other than the two-step model, additional family-related factors, patient-physician sensitization due to a family history, or a combination of these factors might explain this age difference. More complex multistep modeling of the data may be helpful in better characterizing the genetic patterns of cutaneous melanoma.
Age Factors↗
Quantitative studies of the structure of proteins in solution by Fourier-transform infrared spectroscopy.
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Biophysical Phenomena↗
Protein structural effects of agonist binding to the nicotinic acetylcholine receptor.
The effects on the protein structure produced by binding of cholinergic agonists to purified acetylcholine receptor (AcChR) reconstituted into lipid vesicles, has been studied by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Spectral changes in the conformationally sensitive amide I infrared band indicates that the exposure of the AcChR to the agonist carbamylcholine, under conditions which drive the AcChR into the desensitized state, produces alterations in the protein secondary structure. Quantitative estimation of these agonist-induced alterations by band-fitting analysis of the amide I spectral band reveals no appreciable changes in the percent of alpha-helix, but a decrease in beta-sheet structure, concomitant with an increase in less ordered structures. Additionally, agonist binding results in a concentration-dependent increase in the protein thermal stability, as indicated by the temperature dependence of the protein infrared spectrum and by calorimetric analysis, which further suggest that AcChR desensitization induced by the cholinergic agonist implies significant rearrangements in the protein structure.
Animals↗
Protein stability and interaction of the nicotinic acetylcholine receptor with cholinergic ligands studied by Fourier-transform infrared spectroscopy.
Based on the conformational dependence of the amide-I i.r. band, this paper explores the use of Fourier-transform i.r. spectroscopy methods to probe structural features of proteins present in native membranes from Torpedo highly enriched in acetylcholine receptor (AcChR). The interference of water absorbance on the amide-I spectral region has been eliminated through isotopic exchange by freeze-drying the membranes in the presence of trehalose to avoid protein denaturation induced by drying, followed by resuspension in deuterated water. AcChR-rich membrane samples prepared in such a way maintained an ability to undergo affinity-state transitions and to promote cation translocation in response to cholinergic agonists, which are functional characteristics of native untreated samples. The temperature-dependence of the i.r. spectrum indicates a massive loss of ordered protein structure, occurring at temperatures similar to those reported for thermal denaturation of the AcChR by differential scanning calorimetry and by thermal inactivation of alpha-bungarotoxin-binding sites on the AcChR [Artigues, Villar, Ferragut & Gonzalez-Ros (1987) Arch. Biochem. Biophys. 258, 33-41], thus suggesting that the observed i.r. spectral changes correspond to alterations in the structure of the AcChR protein. Furthermore, the presence of detergents as well as cholinergic agonists and antagonists produces spectral changes that are also consistent with the alterations in AcChR protein structure expected from previous calorimetric studies. In contrast with the information obtained by calorimetry, i.r. spectroscopy allows the contribution of secondary structural changes to be distinguished from the overall change in protein structure. Thus prolonged exposure to cholinergic agonists, which drives the AcChR protein into the desensitized state, produces only negligible alterations in the amide-I band shape, but increases substantially the thermal stability of the protein. This suggests that rearrangements in the tertiary or quaternary structure of the protein are more likely to occur than extensive changes in secondary structure as a consequence of AcChR desensitization.
Amides↗
The physical state of ubiquinone-10, in pure form and incorporated into phospholipid bilayers. A Fourier-transform infrared spectroscopic study.
Long-chain quinones are essential components of both bacterial and eukaryotic respiratory chains, and some of the main unsolved questions on energy transduction in membranes are complicated by the lack of consistent information on the physical state of the quinones in membrane bilayers. We have recorded, at various temperatures and under different conditions, the infrared spectra of ubiquinone-10 (the main species in mitochondria) and several analogues. The C = O stretching vibration band located at 1663-1670 cm-1 has been identified as the most sensitive one to phase and environmental changes. Three distinct phases have been characterized in which pure ubiquinone-10 may exist: crystalline (LC1), isotropic liquid (IL) and liquid crystalline (Lc). The only allowed thermotropic transitions are LC1----IL, IL----Lc and Lc----LC1. Our investigations with pure quinones provide a simpler and more detailed description of their phase changes than any of the previous studies and shed light on their behaviour in membranes. When incorporated into phospholipid bilayers, ubiquinone-10 appears to be removed from the aqueous environment and is found to exist, in the 4-70 degrees C range, in an isotropic liquid phase, in the form of small aggregates.
Fourier Analysis↗
Partial dehydration of phosphatidylethanolamine phosphate groups during hexagonal phase formation, as seen by i.r. spectroscopy.
The gel-to-fluid and lamellar-to-HII-hexagonal thermotropic phase transitions of egg-yolk phosphatidylethanolamine have been examined by Fourier-transform infrared spectroscopy under a variety of conditions, namely excess water at pH 5.0, excess water at pH 9.5 and low hydration. The various lamellar and hexagonal phases have been characterized by X-ray diffraction. At pH 5.0, gel-fluid and lamellar-hexagonal transitions were detected at 10 and 32 degrees C respectively, in accordance with previous data. At pH 9.5, only the first of these two transitions was detected. In the partially hydrated sample a single phenomenon was observed, probably encompassing both transitions, so that, in practice, a gel-HII-hexagonal transition appears to occur. The region of the i.r. spectrum corresponding to the phospholipid phosphate group reveals that the lamellar-hexagonal, but not the gel-fluid, transition is accompanied by a weakening in the shell of hydrogen-bonded water, thus providing direct evidence that, in a pure lipid/water system, hexagonal phase formation requires partial dehydration of the phospholipid phosphate group. X-ray diffraction data support this conclusion, since, at least in the low-hydration system, the average surface area per lipid polar group decreases with the thermotropic lamellar-hexagonal transition.
Fourier Analysis↗
An infrared spectroscopic study of specifically deuterated fatty-acyl methyl groups in phosphatidylcholine liposomes.
The region of the infrared spectrum corresponding to C-2H stretching vibrations (2050-2250 cm-1) has been examined for liposomes composed of dimyristoylphosphatidylcholine deuterated specifically at the methyl ends of either one (sn-2) or both the fatty acyl chains. This label is intended to provide information on lipid dynamics in the contact region between monolayers. The two most prominent bands observed correspond, respectively, to antisymmetric (2212 cm-1) and symmetric (2075 cm-1) C-2H stretching vibration. The antisymmetric band consists of two overlapping peaks, whose positions vary with the gel or liquid-crystalline state of the lipid. The separation between the peaks making up the antisymmetric band increases with temperature, and is maximum above the Tc transition temperature; this rules out the previously proposed assignment of these two peaks to different rotational modes of the methyl group relative to the adjacent methylene. The position and width of the symmetric band at 2075 cm-1 are also sensitive to the physical state of the lipid. The presence of cholesterol at an equimolar ratio with the phospholipid abolishes all the phase-dependent changes observed. The intrinsic polypeptide gramicidin A, at a 5:1 lipid/peptide mol ratio, is seen to enlarge the lipid thermotropic transition, with small effects above Tc. Cytochrome c, an extrinsic protein, at a 10:1 mole ratio, does not modify the phase-dependent behaviour of the terminal methyl groups, but consistently shifts all the observed bands to lower-frequency positions, which suggests a long-range effect of the protein along the phospholipid fatty acyl chains.
Cell Membrane↗
Solubilization of sarcoplasmic reticulum membranes by sodium dodecylsulphate. A Fourier-transform infrared spectroscopic study.
In order to improve our understanding of membrane protein solubilization by sodium dodecylsulphate, sarcoplasmic reticulum vesicles have been treated with this surfactant at different detergent: protein mole ratios. Effects on Ca2(+)-ATPase activity, membrane protein solubilization, and protein conformation have been independently monitored, and correlations among the various parameters have been observed. The thermal denaturation of sarcoplasmic reticulum proteins in the presence of sodium dodecylsulphate has also been characterized spectroscopically.
Animals↗
The lamellar to hexagonal phase transition in phosphatidylethanolamine liposomes: a fluorescence anisotropy study.
The steady-state anisotropy of trimethylammonium diphenylhexatriene fluorescence has been used to monitor the thermotropic lamellar to HII hexagonal phase transition in an unsaturated phosphatidylethanolamine. The transition is observed in lipid aggregates when they are heated above the transition temperature Th, as well as in diluted liposomes after aggregation above Th. Changes in fluorescence anisotropy are not observed with Ca(++)-induced fusion of phosphatidylserine vesicles, a process not involving hexagonal phase formation.
Diphenylhexatriene↗
The structure of proteins in aqueous solutions: an assessment of triose phosphate isomerase structure by Fourier-transform infrared spectroscopy.
Recent resolution enhancement and curve-fitting techniques have been applied to infrared spectra from triose phosphate isomerase in aqueous solution, in order to obtain quantitative information on its secondary structure. From our results, 57% alpha-helix, 25% beta-parallel and 10% beta-turns are predicted, in close agreement with the X-ray crystallographic data. On the other hand, measurements of band intensities, both in original and deconvolved spectra are shown to be unreliable for the quantification of secondary structures. The presence of beta-edge structure interacting with the alpha-helical barrel is described and discussed.
Carbohydrate Epimerases↗