Somatostatin-detergent interaction

The effect of cationic, anionic and nonionic detergents on the EPR spectrum of spin-labeled somatostatin has been studied. At detergent concentrations well above the critical micelle concentration, nonionic detergents do not alter the EPR spectrum. Sodium dodecyl sulfate markedly alters both the line height ratio and the hyperfine splitting constant, whilst dodecyltrimethylammonium bromide alters only slightly the hyperfine splitting constant and line height ratio. The somatostatin-sodium dodecyl sulfate complex appeared monodisperse by sedimentation equilibrium with about 17 g bound detergent per g peptide. Circular dichroic and difference spectra of the dodecyl sulfate-somatostatin complex show that the tryptophanyl residue is buried in a nonpolar environment and that the secondary and tertiary structure of the peptide is markedly altered. Sedimentation equilibrium studies suggest that two types of dodecyltrimethylammonium-somatostatin complex exist. One type resembles the dodecyl sulfate-peptide complex, whilst the other appears to include several peptide units with only about one gram bound detergent per gram peptide.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.     

Chronic exposure to inhaled anesthetics increases cholesterol content in Acholeplasma laidlawii

Acholeplasma laidlawii cells were grown in cholesterol-enriched medium and exposed continuously to either air (control), 4.0 vol.% halothane in air at 1 atm pressure (4% atm halothane), or 80% cyclopropane in oxygen for 24 h at 37°C. Cells grown in the presence of 4% atm halothane or 80% cyclopropane had approximately twice as much membrane cholesterol content/mg protein as the control cells. Cells grown in an anesthetic environment also tended to have a higher membrane cholesterol/phospholipid molar ratio compared to control cells. Membranes isolated from halothane-exposed cells grown in a cholesterol-enriched medium were more ordered at 37°C (measurements were made with no anesthetic present) than membranes from control cells grown in an identically enriched medium. This difference in membrane physical state between control and anesthetic-exposed cells decreased as the temperature decreased, and disappeared at approx. 23°C. Continuous exposure of A. laidlawii to 4% atm halothane or 80% cyclopropane for 24 h did not markedly affect membrane fatty acid composition, either in cells grown on an unsupplemented medium or in cells grown in a medium enriched in myristic, palmitic or stearic acids. These results further support the hypothesis that an increased membrane cholesterol content may play a role in the tolerance or dependence that develops after chronic exposure to anesthetic agents.     

Procedure using voltage-sensitive spin-labels to monitor dipole potential changes in phospholipid vesicles: The estimation of phloretin-induced conductance changes in vesicles

Summary Voltage-sensitive membrane potential probes were used to monitor currents resulting from positive or negative charge movement across small and large unilamellar phosphatidylcholine (PC) vesicles. Positive currents were measured for the paramagnetic phosphonium ion or for K⁺-valinomycin. Negative currents were indirectly measured for the anionic proton carriers CCCP and DNP by monitoring transmembrane proton currents. Phloretin, a compound that is believed to decrease dipole fields in planar bilayers, increases positive currents and decreases negative currents when added to egg PC vesicles. In these vesicles, positive currents are increased by phloretin addition to a much larger degree than CCCP currents are reduced. This asymmetry, with respect to the sign of the charge carrier, is apparently not the result of changes in the membrane dielectric constant. It is most easily explained by deeper binding minima at the membrane-solution interface for the CCCP anion, when compared to the phosphonium. The measured asymmetry and the magnitudes of the current changes are consistent with the predictions of a point dipole model. The use of potential-sensitive probes to estimate positive and negative currents, provides a methodology to monitor changes in the membrane dipole potential in vesicle systems.     

Comparison of the electron spin polarized spectrum found in plant photosystem I and in iron-depleted bacterial reaction centers with time-resolved K-band EPR; evidence that the photosystem I acceptor A1 is a quinone

The suggestion that the electron acceptor A₁ in plant photosystem I (PSI) is a quinone molecule is tested by comparisons with the bacterial photosystem. The electron spin polarized (ESP) EPR signal due to the oxidized donor and reduced quinone acceptor (P ₈₇₀ ⁺ Q^-) in iron-depleted bacterial reaction centers has similar spectral characteristics as the ESP EPR signal in PSI which is believed to be due to P ₇₀₀ ⁺ A ₁ ^- , the oxidized PSI donor and reduced A₁. This is also true for better resolved spectra obtained at K-band (24 GHz). These same spectral characteristics can be simulated using a powder spectrum based on the known g-anisotropy of reduced quinones and with the same parameter set for Q^- and A₁ ^-. The best resolution of the ESP EPR signal has been obtained for deuterated PSI particles at K-band. Simulation of the A₁ ^- contribution based on g-anisotropy yields the same parameters as for bacterial Q^- (except for an overall shift in the anisotropic g-factors, which have previously been determined for Q^-). These results provide evidence that A₁ is a quinone molecule. The electron spin polarized signal of P₇₀₀ ⁺ is part of the better resolved spectrum from the deuterated PSI particles. The nature of the P₇₀₀ ⁺ ESP is not clear; however, it appears that it does not exhibit the polarization pattern required by mechanisms which have been used so far to explain the ESP in PSI.Abbreviations hf hyperfine - A₀ A₀ acceptor of photosystem I - A₁ A₁ acceptor of photosystem I - Brij-58 polyoxyethylene 20 cetyl ether - CP1 photosystem I particles which lack ferridoxin acceptors - ESP electron spin polarized - EPR electron paramagnetic resonance - I intermediary electron acceptor, bacteriopheophytin - LDAO lauryldimethylamine - N-oxide, P₇₀₀ primary electron donor of photosystem I - PSI photosystem I - P₇₀₀ ^T triplet state of primary donor of photosystem I - P₈₇₀ primary donor in R. sphaeroides reaction center - Q quinore-acceptor in photosynthetic bacteria - RC reaction center     

Characterization of specific fluorenylmethyloxycarbonyl-containing calmodulin adducts by spectroscopy and phosphodiesterase stimulation

A reagent (I, N⁴-(9-fluorenylmethyloxycarbonyl-4-amino-1-oxyl-4-succinimidyloxycarbonyl-2,2,6,6-tetramethylpiperidine)) that acylates calmodulin specifically at lysines 75 and 148 was recently described (Jackson and Puett, 1984). Chromatographic procedures are described that permit purification to apparent homogeneity of a 1 : 1 and a 2 : 1 adduct characterized by modification at just Lys 75 or at Lys 75 and Lys 148, respectively. These adducts are suitable for detailed characterization in an effort to provide information on calmodulin structure-function relationships. The adducts were incapable of, or exhibited low potency (e.g., 0.1% that of calmodulin) in, stimulating the activity of an activatable bovine brain cyclic nucleotide phosphodiesterase (3,5-cyclic AMP 5-nucleotidehydrolase, EC 3.1.4.17) preparation. Electron paramagnetic resonance (EPR) spectroscopy of the adducts yielded rotational correlation times of approximately 3–6 nsec, in agreement with the expected value for a hydrated protein of this molecular weight (5–7 nsec). Thus, the nitroxide reporter group appears to monitor closely the motion of the protein, and there is no evidence of a major conformational change in the derivative relative to calmodulin. Interestingly, removal of the fluorenylmethyloxycarbonyl portion from the 1 : 1 adduct to give a deprotected 1 : 1 adduct resulted in apparent greater mobility of the probe, since the rotational correlation coefficient was found to be 1 nsec. Circular dichroic spectra were obtained over the wavelength interval 200–250 nm on the two adducts and on the deprotected 1 : 1 adduct. These derivatives, like calmodulin, exhibited a Ca²⁺-mediated increase in helicity, and the spectra of the adducts in the presence of a chelating agent and in the presence of saturating Ca²⁺ were similar to those obtained for calmodulin. Thus, the adducts have secondary structures similar to the native protein. Proton nuclear magnetic resonance spectra were determined in the aromatic region (6–8 ppm) for the deprotected 1 : 1 adduct before and after reduction of the nitroxide with ascorbate. The nitroxide had little effect on the chemical shifts of the two tyrosines and the single histidine relative to calmodulin, although the histidine C4 resonance was markedly altered by the addition of ascorbate. In order to explore in greater detail the tertiary structure of the 1 : 1 adduct, a reagent similar to I, but not paramagnetic, was synthesized. This compound II, -N-(9-fluorenylmethyloxycarbonyl)alanine N-hydroxysuccinimide ester, like I, forms a 1 : 1 adduct at Lys 75 and a 2 : 1 adduct at Lys 75 and Lys 148. Proton NMR spectra of adducts with II were not complicated by the relaxation effects arising from adducts with I; thus more definitive assignments could be made to the upfield resonances, including the fluorene protons. Again, it was possible to conclude that adduct formation had no major effect on the tertiary structure of the protein as monitored by chemical shifts associated with various residues. We conclude that modification of just Lys 75, a residue in the long connecting helix of calmodulin, does not lead to major changes in protein conformation but does interfere with the ability of calmodulin to stimulate an activatable form of bovine brain cyclic nucleotide phosphodiesterase.     

Superoxide scavenging activity of BG-104 before and after sonication-determined by ESR spin trapping method

BG-104 is a compound of Chinese herbs which were treated with infrared ray-heating and brewed with microorganisms resulting in activation of actual principles contained in the herbs, and its effectiveness has been reported on various inflammatory disorders such as Behcet's disease, rheumatoid arthritis, bacterial infections and so on. In addition, it is noted as an effective agent to retard cancer progress. This report shows a superoxide scavenging activity of BG-104 using an ESR spin trapping method. For the measurement of superoxide scavenging activity, recombinant human superoxide dismutase is used as a standard. Superoxide scavenging activity of BG-104 increases in a dose-dependent manner and activity is significantly augmented by its sonication.     

Cu~(2+),Cu~(2+)/Vit C对亚油酸过氧化起动机理的ESR研究

本文直接用亚油酸为材料,利用ESR自旋捕集技术和紫外分光技术,分别检测了脂质过氧化反应过程中产生的中间自由基产物L·和共轭二烯结构,对Cu~(2+),Cu~(2+)/Vit C起动脂质过氧化反应的机理进行了研究。结果表明:Vit C自由基,Cu~(2+),Cu~+都不能起动纯的亚油酸的过氧化;Cu~(2+),Cu~+可以使含有部分过氧化成份的亚油酸继续氧化。     

The involvement of ethanol in the free radical reaction of 6-hydroxydopamine

ESR spin trapping technique was used to detect and analyze free radical formation. When 6-hydroxydomine (6-OHDA) was incubated alone or in the presence of a free radical generating system (H₂O₂ and FeSO₄), hydroxyl free radicals were observed in a concentration-dependent manner. Glutathione was found to be the most effective scavenger of the ESR signal when compared with vitamin E or Mannitol. The addition of ethanol resulted in the formation of the pure hydroxyethyl free radicals. The amount of hydroxyethyl free radicals in the system was dependent upon the concentration of ethanol and the formation of hydroxyethyl free radicals correlated well with the extent of lipid peroxidation and the loss of enzymic activity of the membrane-bound (Na⁺, K⁺)-ATPase. We suggest that in the biological system ethanol may potentiate the neurotoxicity of 6-OHDA with the formation of hydroxyethyl free radicals, which are longer-lived and far more damaging to membranes that the hydroxyl radicals. These data lead us to further hypothesize that the neuronal degeneration caused by 6-OHDA and other compounds that generate free radicals could be potentiated in the presence of ethanol.