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PubMed1984

Isaxonine base is a strong perturber of phospholipid bilayer order and fluidity--a differential scanning calorimetry and spin labeling study.

The effects of the neurotropic drug isaxonine on fully hydrated dipalmitoyl-phosphatidyl-choline (DPPC) bilayers has been studied in the temperature range 0 degree-60 degrees, using differential scanning calorimetry and electron spin resonance spectroscopy, with two stearic acid spin labels. At low concentration (1% mol/mol), isaxonine is trapped in the polar interface and enhances the phospholipid multibilayers organization in the gel state. In contrast, at high concentration (30% mol/mol), the drug disorganizes the phospholipidic structures and may induce domain formation by phase separation. The strong interactions of isaxonine at the lipid-water interface change the ionization state of the stearic acid spin labels which become totally ionized. Then isaxonine acts as a modifier of the surface pH of the bilayer. The strong membrane effects of isaxonine may explain in part its pharmacological properties in vivo.

Calorimetry, Differential Scanning↗
PubMed1997

Spin label oximetry to assess extracellular oxygen during myocardial ischemia.

We describe real-time measurement of myocardial oxygen consumption during ischemia in the intact heart. Measurement of extracellular oxygen concentration during myocardial ischemia by spin label oximetry has been limited by ischemia-induced reduction of the neutral, water-soluble nitroxide TEMPONE. We have overcome this problem by encapsulating the nitroxides. Isolated immature (7-10 d old) rabbit hearts (n = 8) were perfused aerobically within the cavity of a loop gap resonator with bicarbonate buffer containing an oxygen-sensitive, lipid-soluble nitroxide (14N-TEMPO laurate in FC-43 perfluorocarbon micelles) and a much less oxygen-sensitive and positively charged nitroxide (15N-TEMPO choline in multilamellar vesicles) as an internal standard. The ratio of the ESR signal amplitudes of these nitroxides was used as a sensitive index of oxygen concentration. Sequestration of the nitroxides decreased their reduction rate by ascorbate in comparison with nonsequestered nitroxides. Hearts were subjected to 60 min of global no-flow ischemia at 20 degrees C. Extracellular oxygen content (mean +/- SD) during aerobic perfusion was 1195 +/- 55 mumol/liter. The electron spin resonance signal from TEMPO laurate increased with the onset and progression of ischemia, consistent with a decrease in extracellular oxygen, while the signal for TEMPO choline was relatively unchanged. Extracellular oxygen content after 40 and 60 min of ischemia was reduced to 393 +/- 27 mumol/liter (p < .05) and 61 +/- 5 mumol/liter (p < .05), respectively. We conclude that spin-label oximetry can directly and precisely measure myocardial oxygen consumption at constant temperature during ischemia in the intact heart.

Animals↗
PubMed1980

Calcium translocation mechanism in sarcoplasmic reticulum vesicles, deduced from location studies of protein-bound spin labels.

Sarcoplasmic reticulum vesicles were exposed to various thiol-directed spin labels, and the position of the label on the inner or outer vesicle surface was investigated as a function of the ATPase (adenosinetriphosphatase; ATP phosphohydrolase, EC 3.6.1.3) chemical state. Previous measurements of label accessibility to externally added ascorbate had been considered to suggest an external-internal transition of protein-bound labels, coupled with ion translocation [Tonomura, Y. & Morales, M.F. (1974) Proc. Natl. Acad. Sci. USA 71, 3687-3691]. We show that these ascorbate studies do not lead to convincing conclusions. We demonstrate, on the contrary, that transition ions (nickel and ferricyanide) can be used as selective line-broadening agents for the signals arising from external labels. No significant difference in nickel- or ferricyanide-label interaction can be attributed to a different orientation of the label in any of the enzyme chemical states tested. Our results therefore contradict the current interpretation of ascorbate quenching experiments in terms of calcium ATPase rotatory motion; rather they are consistent with ion transport models involving only limited conformational rearrangements of the pump.

Biological Transport, Active↗
PubMed2005

Quantification of cerebral perfusion using arterial spin labeling: two-compartment models.

One of the advantages of arterial spin labeling (ASL) techniques over other techniques for measuring cerebral perfusion is that with ASL it is possible to achieve accurate quantification. This is particularly useful in the field of functional imaging, where accurate measurements of perfusion change can help untangle the complex physiological changes that occur following neuronal activation. However, the linearity of the perfusion estimate over a wide range of perfusion values may be more important than absolute values. For several years, single-compartment models have dominated the literature, and it has been assumed that the labeled water diffuses freely throughout the tissue voxel. However, recent work, as summarized in this review, has shown that this assumption is inaccurate and leads to an overestimation of perfusion at low perfusion rates, and an underestimation at high rates. The inclusion of restricted permeability of the capillary wall to water in a two-compartment model offers improved quantification.

Arteries↗
PubMed2000

Turbo ASL: arterial spin labeling with higher SNR and temporal resolution.

A modified pulsed arterial spin labeling (ASL) technique is introduced here that has both higher temporal resolution and higher SNR per unit time than existing ASL techniques. In this technique, the time TI between the application of the tag and image acquisition is longer than the repetition time TR, allowing for the use of greatly reduced TR values without a significant decrease in the amplitude of the ASL signal. This improves both the temporal resolution and the sensitivity of ASL for functional brain mapping.

Arteries↗
PubMedDate not supplied

Structure of ascorbic acid and its biological function: V. Transport of ascorbate and isoascorbate across artificial membranes as studied by the spin label technique.

It could be shown by the reduction of the spin label (1,14) located within DPPC vesicles, that Na-ascorbate and K-isoascorbate can permeate membranes. At physiologic pH value, these two compounds exist as electroneutral radicals with a cyclic side chain structure. Ascorbic acid and isoascorbic acid, on the contrary, can hardly permeate such an artificial membrane. Since the radical will cause lipid peroxidation, it must be modified prior to permeation. This can be done by GSH which changes the radical state but maintains the electroneutral bicyclic configuration.

Ascorbic Acid↗
PubMed1983

Spin-label study of histone H1-DNA interaction. Involvement of the central part of the molecule in reconstituted chromatin.

As shown in a previous paper [J. J. Lawrence et al. (1980) Eur. J. Biochem. 107, 263-269], covalent spin labeling of basis residues in histone H1 allows the study of the interaction of this protein with DNA. Using a step gradient dialysis procedure to reconstitute chromatin from labeled H1 and stripped chromatin, it is shown that the process of interaction of the lysine residues and DNA is the same whether histone H1 is bound to linear purified DNA or to H1-depleted chromatin. In contrast, spin labeling of the unique tyrosine of histone H1 located in the globular part of the molecule shows that this part is more involved in the interaction with chromatin than it is with linear DNA (as judged from the lengthening of the rotational correlation time). These data are interpreted as reflecting different roles for the N and C termini of the molecule of H1 and the central globular part. A model, based on these observations together with examination of the primary structures of histones H1, is proposed which accounts for the H1 involvement in the chromatosome structure.

Animals↗
PubMedDate not supplied

Residual motion of hemoglobin-bound spin labels as a probe for protein dynamics.

The residual motion of spin labels bound to cysteine beta 93 of methemoglobin and oxyhemoglobin has been analyzed as a function of temperature and hydration. The rotational diffusion of the whole protein molecule has been prevented or restricted by crystallization, lyophilization or by high viscosity of the solution. The residual motion of the labels is characterized by an angle of the limited motion cone and their rotational correlation time using computer simulations of the EPR spectra. Two types of motion can be separated due to different correlation times and different dependences on temperature and hydration. One of these motional mechanisms can be shown to be determined by protein fluctuations. Correlation times of these fluctuations decrease from 2 X 10(-8) s at T = 220 K to 10(-9) s at T = 300 K in the samples of high water concentration. Strong correlation between the properties of the hydration shell and these fluctuations are observed.

Animals↗
PubMed2002

Spin labels as a tool to identify and characterize protein-ligand interactions by NMR spectroscopy.

NMR spectroscopy based discovery and optimization of lead compounds for a given molecular target requires the development of methods with maximum sensitivity and robustness. It is shown here that organic nitroxide radicals ("spin labels") can be used to boost the sensitivity of NMR spectroscopic screening in drug discovery research. The concept of utilizing spin labels in NMR spectroscopy is summarized, examples for successful first-site and second-site NMR spectroscopic screens are given, and guidelines for linker design are presented.

Ligands↗
PubMed1998

A theoretical and experimental comparison of continuous and pulsed arterial spin labeling techniques for quantitative perfusion imaging.

Under ideal conditions, continuous arterial spin labeling (ASL) techniques are higher in SNR than pulsed ASL techniques by a factor of e. Presented here is a direct theoretical and experimental comparison of continuous ASL and pulsed ASL, using versions of both that are amenable to multislice imaging and insensitive to variations in transit times (continuous ASL with a delay before imaging, and QUIPSS II (Quantitative Imaging of Perfusion Using a Single Subtraction-second version)). Perfusion image quality for comparable imaging time was nearly identical for both single-slice and multislice imaging. The measured raw signal was approximately 25% higher with continuous ASL, but the SNR per unit time was identical.

Artifacts↗
PubMed1977

Spin-label studies on rat liver and heart plasma membranes: do probe-probe interactions interfere with the measurement of membrane properties?

The structures of purified rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(12,3). ESR spectra were recorded with a 50 gauss field sweep, and also with a new technique which "expands" the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The hyperfine splittings measured from the "expanded" spectra were significantly more precise than those obtained from the "unexpanded" spectra. Both procedures were used to study the effects of various I(12,3) probe concentrations on the spectra of liver and heart membranes, as well as the effects of temperature and CaCl2 additions on the spectra of liver membranes, and revealed the following: The polarity-corrected order parameters of liver (31 degrees) and heart (22 degrees) membranes were found to be independent of the probe concentration, if experimentally-determined low I(12,3)/lipid ratios were employed. The absence of obvious radical-interaction broadening in the unexpanded spectra indicated that "intrinsic" membrane properties may be measured at these low probe/lipid ratios. Here, "intrinsic" properties are defined as those which are measured when probe-probe interactions are negligible, and do not refer to membrane behavior in the absence of a perturbing spin label. At higher I(12,3)/lipid ratios, the order parameters of liver and heart membranes were found to substantially decrease with increasing probe concentration. The increase in the "apparent" fluidity of both membrane systems is attributed to enhanced radical interactions; however, an examination of these spectra (without reference to "low" probe concentration spectra) might incorrectly suggest that radical interactions were absent. For the membrane concentrations employed in these studies, the presence of "liquid-lines" (or "fluid components") in the unexpanded ESR spectra was a convenient marker of high probe concentrations. A thermotropic phase separation was observed in liver membranes between 19 degrees and 28 degrees. Addition of CaCl2 to liver plasma membrane [labelled with "low" I(12,3) concentrations] increased the rigidity of the membrane at 31 degrees and 37 degrees, without inducing a segregation of the probe in the bilayer. Previously reported data are discussed in relation to these results, and suggested minimal criteria for performing membrane spin label studies are included.

Animals↗
PubMedDate not supplied

Site directed spin labeling studies of structure and dynamics in bacteriorhodopsin.

Site-directed spin labeling of membrane proteins has been used to determine: (1) the topography of the polypeptide chain with respect to the membrane/solution interface, and (2) the identity and orientation of secondary structure in selected regions. These features are deduced from the collision rates of nitroxide side chains with paramagnetic reagents in solution, and the principles of the method are reviewed with reference to bacteriorhodopsin. The dynamics of the nitroxide side chains relative to the backbone reveal tertiary interactions of the labeled site, and provide a promising means of time-resolving conformational changes. This aspect is illustrated by recent studies of structural changes in bacteriorhodopsin during the photocycle. In these experiments, nitroxide side chains were introduced at residues 72, 101 and 105 after replacement of the original residues by cysteine. Upon flash photolysis, the electron paramagnetic resonance spectrum of a nitroxide at 101, but not those at 72 or 105, is time-dependent. The spectral change develops during the decay of the M-intermediate, and reverses upon return to the ground state. The results suggest a movement of the C-D or E-F interhelical loops during the protonation changes of aspartate 96.

Bacteriorhodopsins↗
PubMed2005

Development of spin-labeled probes for adenosine receptors.

Functionalized xanthine derivatives bearing a nitroxide moiety at the 3- or 8-position were synthesized as electron paramagnetic resonance (EPR) probes. The 8-cyclopentyl-1-propylxanthine derivative 4, spin-labeled at N3 by substitution with a nitroxide-bearing dihydropyrrole moiety, was a potent and selective A(1) adenosine receptor antagonist (K(i) for A(1) 5.5 nM, 1600-fold selectivity vs A(2A), >200-fold vs A(2B), and 310-fold vs A(3) adenosine receptors). 8-(1-Oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)-1,3-dipropylxanthine 10 (K(i) for A(1) 8.2 nM) was similarly potent and selective, while 8-(1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl)-1,3-dipropylxanthine 11 (K(i) for A(1) 160 nM) exhibited significantly lower affinity for A(1) adenosine receptors. 8-[4-(((1-Oxyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)-2-oxoethoxy)phenyl]-1-propylxanthine14, a 3-unsubstituted xanthine derivative, was found to be a potent A(2B) adenosine receptor antagonist (K(i) for A(2B) 48 nM) but also exhibited high affinity for A(1) receptors (K(i) for A(1) 15.7 nM). An X-ray structure of compound 10 was obtained, confirming the proposed structure. The novel spin-labeled A(1)-selective or A(1)/A(2B)-nonselective adenosine receptor antagonists may become useful probes for biophysicochemical investigations of adenosine receptors in their membrane environment.

Adenosine A1 Receptor Antagonists↗
PubMedDate not supplied

Complex formation of spin-labeled 9-aminoacridine with DNA and polynucleotides.

Complex formation between spin-labeled 9-aminoacridine and DNA or polynucleotides has been studied by differential spectrophotometry and ESR. The differential spectra of the strong type 9-aminoacridine-DNA complex showed characteristic absorption bands at 270 and 290 nm, and the intensity ratio of these bands varied according to the degree of DNA denaturation. The ESR spectra of this complex were characterized by slow rotation of the radical; as the macromolecule became increasingly denatured and in the polynucleotide complex, a rapid signal appeared in the ESR spectrum. The temperature at which DNA undergoes a structural transition in the premelting region could be determined from the temperature dependence of the ESR spectral form of the dye-DNA complex. The spectral characteristics of the complexes give additional information about structural disturbances in DNA.

Animals↗
PubMed2005

A signal processing model for arterial spin labeling functional MRI.

A model of the signal path in arterial spin labeling (ASL)-based functional magnetic resonance imaging (fMRI) is presented. Three subtraction-based methods for forming a perfusion estimate are considered and shown to be specific cases of a generalized estimate consisting of a modulator followed by a low pass filter. The performance of the methods is evaluated using the signal model. Contamination of the perfusion estimate by blood oxygenation level dependent contrast (BOLD) is minimized by using either sinc subtraction or surround subtraction for block design experiments and by using pair-wise subtraction for randomized event-related experiments. The subtraction methods all tend to decorrelate the 1/f type low frequency noise often observed in fMRI experiments. Sinc subtraction provides the flattest noise power spectrum at low frequencies, while pair-wise subtraction yields the narrowest autocorrelation function. The formation of BOLD estimates from the ASL data is also considered and perfusion weighting of the estimates is examined using the signal model.

Artifacts↗
PubMed1997

Two modes of ligand binding in maltose-binding protein of Escherichia coli. Electron paramagnetic resonance study of ligand-induced global conformational changes by site-directed spin labeling.

Binding of ligands to the maltose-binding protein (MBP) of Escherichia coli often causes a global conformational change involving the closure of its two lobes. We have introduced a cysteine residue onto each of these lobes by site-directed mutagenesis and modified these residues with spin labels. Using EPR spectroscopy, we examined the changes, caused by the ligand binding, in distance between the two spin labels, hence between the two lobes. The binding of both maltose and maltotetraose induced a considerable closure of the N- and C-terminal lobes of MBP. Little closure occurred upon the binding of maltotetraitol or beta-cyclodextrin. Previous study by fluorescence and UV differential absorbance spectroscopy (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609) showed that maltose and a large portion of maltotetraose bound to MBP via one mode (R mode or "end-on" mode), which is physiologically active and leads to the subsequent transport of the ligands across the cytoplasmic membrane. In contrast, maltotetraitol and beta-cyclodextrin bound to MBP via a different mode (B mode or "middle" mode), which is physiologically inactive. The present work suggests that the B mode is nonproductive because ligands binding in this manner prevent the closure of the two domains of MBP, and, as a result, the resulting ligand-MBP complex is incapable of interacting properly with the inner membrane-associated transporter complex.

ATP-Binding Cassette Transporters↗
PubMed2000

Insights into ATP synthase structure and function using affinity and site-specific spin labeling.

A variety of different approaches has been used during the last couple of decades to investigate structure and function relationships within the catalytic portion of the F0F1-ATP synthase and of its interactions with the proton-translocator F0. In our group, we employ ESR spectroscopy with the use of stable organic radicals, so-called spin labels, as reporter groups. The radicals are either attached to substrates/ligands or specifically inserted into the protein structure by "site-specific spin labeling." Both approaches bear intrinsic advantages for their special uses and result in the specific information that is available through ESR, e.g., structural changes due to binding of effector molecules (e.g., Mg2+ ions), conformational transitions during catalytic turnover, distance information on radicals bound at 20 A or less, and information on the binding characteristics of labeled substrates. This review summarizes the results of a variety of different approaches we have used during the last years to study, with the help of ESR spectroscopy, the structure of the nucleotide binding sites of F1-ATPases of different origins as well as interactions with F0 subunits.

Affinity Labels↗
PubMed2006

An automated image-processing strategy to analyze dynamic arterial spin labeling perfusion studies. Application to human skeletal muscle under stress.

Arterial spin labeling (ASL) perfusion measurements allow the follow-up of muscle perfusion with high temporal resolution during a stress test. Automated image processing is proposed to estimate perfusion maps from ASL images. It is based on two successive analyses: at first, automated rejection of the image pairs between which a large displacement is detected is performed, followed by factor analysis of the dynamic data and cluster analysis to classify pixels with large signal variation characteristic of vessels. Then, after masking these "vascular" pixels, factor analysis and cluster analysis are further applied to separate the different muscles between low or high perfusion increase, yielding a functional map of the leg. Data from 10 subjects (five normal volunteers and five elite sportsmen) had been analyzed. Resulting time perfusion curves from a region of interest (ROI) in active muscles show a good accordance whether extracted with automated processing or with manual processing. This method of functional segmentation allows automated suppression of vessels and fast visualization of muscles with high, medium or low perfusion, without any a priori knowledge.

Adult↗