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PubMed1983

Effect of thiol reagents and ionizing radiation on the permeability of erythrocyte membrane for spin-labeled non-electrolytes.

Four different thiol reagents: p-chloromercuribenzoic acid (pCMB), mercuric chloride (HgCl2), N-ethylmaleimide (NEM), and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) were employed as agents modifying the transport of a hydrophilic and hydrophobic non-electrolyte spin labels: 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) into bovine erythrocytes. Gamma-irradiation of erythrocytes amplified the effects of pCMB, HgCl2 and NEM of inhibition of TEMPOL transport and attenuated them in the case of TEMPO transport. These results suggest that the transport of TEMPOL across the erythrocyte membrane is controlled by both superficially and more deeply located membrane -SH groups while only superficial -SH groups control the transport of TEMPO. The lower extent of inhibition of TEMPO transport indicates a higher contribution of diffusion through the lipid phase to the transport of TEMPO across the erythrocyte membrane as compared with TEMPOL.

Animals↗
PubMed1975

Spin-labelled phosphofructokinase. A simple and direct approach to the study of allosteric equilibria under near-physiological conditions.

Rabbit muscle phosphofructokinase, spin-labelled at its most reactive thiol group, has an electron spin resonance spectrum which is very sensitive to the binding of substrates and allosteric effectors. The spectral changes have been interpreted in terms of a concerted allosteric transition between two conformational states with non-exclusive binding of effectors. On this basis MgATP, fructose 6-phosphate plus ATP, and NH+4ions behave as potent positive effectors, inorganic phosphate, sulphate, AMP, fructose 6-phosphate and fructose 1,6-bisphosphate are less potent activators, and free ATP and H+ions are negative effectors, in agreement with the kinetic behaviour, but citrate behaves anomalously. In addition, the allosteric equilibrium can be displaced towards the inhibited state by selectively modifying two further thiol groups. Strong positive cooperativity occurs under suitable conditions with ATP, metal-ATP and fructose 6-phosphate. Biphasic changes of conformation, attributed to binding at the catalytic and inhibitory sites, have been observed in titrations with ATP. The differentiation of the two ATP binding sites arises in the presence of fructose 6-phosphate because of a distinct concerted effect on conformation between the two substrates at the active site. A similar effect occurs between ATP and citrate. Other heterotropic effects are more consistent with simple models; phosphates favour the binding, and reduce the cooperativity, of fructose 6-phosphate and metal-ATP, whereas excess ATP and H+ ions antagonise the binding and increase the cooperativity of fructose 6-phosphate. The observations are related to existing kinetic and binding studies where possible. Anomalous features of the behaviour suggest that the model should be regarded only as a first approximation.

Adenosine Triphosphate↗
PubMed1992

Resolved conformational states of spin-labeled Ca-ATPase during the enzymatic cycle.

We have used frequency- and time-resolved electron paramagnetic resonance (EPR) to study the effects of substrate on the nanosecond conformational dynamics of the Ca-ATPase of sarcoplasmic reticulum, as detected by an iodoacetamide spin label (IASL) attached covalently to the enzyme. We confirm previous results [Coan, C. (1983) Biochemistry 22, 5826] showing that this probe is less rotationally mobile following the addition of nucleotides (ADP, AMPPNP, ATP) and that the shape of the spectrum suggests the presence of two components. We used two approaches to enhance EPR resolution in order to resolve the spectral components and their corresponding conformational states. First, to improve resolution in the frequency (spectral) domain, we used perdeuterated IASL, which results in narrower line widths. Digital spectral analysis resolves the EPR spectrum into two components, one that is indistinguishable from the spectrum observed in the absence of ligands and another that indicates more restricted probe motions, suggesting a distinct conformation of the labeled protein. Additions of substrate ligands appear to change only the mole fractions of the two components. The mole fraction of the restricted component (fR) was 0 in the absence of ligands, but increased to about 0.5 in the presence of saturating concentrations of AMPPNP and Ca2+. In general, ATP and its analogs increase fR, with larger effects observed in the presence of Ca. However, calcium has no effect by itself (fR = 0). Both monovanadate and decavanadate increase fR, but the formation of a covalent phosphoenzyme from inorganic phosphate (E2-P) had no effect (fR = 0).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed2003

Sickle cell disease: continuous arterial spin-labeling perfusion MR imaging in children.

Cerebral blood flow (CBF) was measured with continuous arterial spin-labeling perfusion magnetic resonance (MR) imaging in 14 children with sickle cell disease and seven control subjects. Mean CBF values were higher in patients (P <.005) than in control subjects in all cerebral artery territories. Three patients had decreased CBF in right anterior and middle cerebral artery territories compared with CBF on the left, and one patient had a profound decrease in CBF in all three territories in the right hemisphere. Baseline CBF was significantly decreased in territories seen as unaffected on conventional MR images and MR angiograms in four children with sickle cell disease.

Anemia, Sickle Cell↗
PubMed2005

Dynamic spin labeling angiography in extracranial carotid artery stenosis.

BACKGROUND AND PURPOSE: Similar to digital subtraction angiography, dynamic spin labeling angiography (DSLA) provides time-resolved measurements of the influx of blood into the cerebral vascular tree. We determined whether DSLA may help in assessing the degree of stenosis and whether it provides information about intracerebral collateralization and allows us to monitor the hemodynamic effects of vascular interventions. METHODS: We developed a segmented DSLA sequence that allowed the formation of images representing inflow delays in 41-ms increments. Thirty patients with unilateral carotid artery stenosis and 10 control subjects underwent DSLA. Arrival times of the labeled arterial blood bolus were measured in the carotid siphon (CS) and the middle cerebral artery (MCA) on both sides, and the corresponding side-to-side arrival time differences (ATDs) were calculated. ATDs before and after carotid endarterectomy or percutaneous angioplasty were studied in 10 patients. RESULTS: The degree of stenosis was significantly correlated with ATD in the cerebral vessels. Receiver operating characteristic analysis yielded a cutoff CS ATD of 110 ms to separate stenoses <70% from those > or =70%, with a sensitivity of 90% and a specificity of 67%. In one third of patients, ATD was higher in the MCA than in the CS; this finding suggested an absence of collateralization. Most patients had reduced ATD in the MCA. The degree of ATD reduction was regarded as a quantitative measure of collateralization. Successful intervention resulted in normalized ATDs. CONCLUSION: DSLA is a promising method that allowed us to noninvasively quantify the hemodynamic effect of extracranial carotid stenosis and the resulting intracranial collateralization.

Aged↗
PubMed2001

Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: a site-directed spin labeling study.

The GNRA (N: any nucleotide; R: purine) tetraloop/receptor interaction is believed to be one of the most frequently occurring tertiary interaction motifs in RNAs, but an isolated tetraloop/receptor complex has not been identified in solution. In the present work, site-directed spin labeling is applied to detect tetraloop/receptor complex formation and estimate the free energy of interaction. For this purpose, the GAAA tetraloop/receptor interaction was chosen as a model system. A method was developed to place nitroxide labels at specific backbone locations in an RNA hairpin containing the GAAA tetraloop. Formation of the tetraloop/receptor complex was monitored through changes in the rotational correlation time of the tetraloop and the attached nitroxide. Results show that a hairpin containing the GAAA tetraloop forms a complex with an RNA containing the 11-nucleotide GAAA tetraloop receptor motif with an apparent Kd that is strongly dependent on Mg2+. At 125 mM MgCl2, Kd = 0.40 +/- 0.05 mM. The corresponding standard free energy of complex formation is -4.6 kcal/mol, representing the energetics of the tetraloop/receptor interaction in the absence of other tertiary constraints. The experimental strategy presented here should have broad utility in quantifying weak interactions that would otherwise be undetectable, for both nucleic acids and nucleic acid-protein complexes.

Animals↗
PubMed1996

Spin label and 2H-NMR studies on the interaction of melanotropic peptides with lipid bilayers.

The interaction of the cationic tridecapeptide alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more active analog [Nle4, DPhe7]-alpha-MSH with lipid membranes was investigated by means of ESR of spin probes incorporated in the bilayer, and NMR of deuterated lipids. All spin labels used here, stearic acid and phospholipid derivatives labeled at the 5th and 12th position of the hydrocarbon chain, and the cholestane label, incorporated into anionic vesicles of DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) in the liquid-crystalline phase, indicated that both peptides decrease the motional freedom of the acyl chains. No peptide effect was detected with neutral lipid bilayers. Changes in the alpha-deuteron quadrupolar splittings and spin lattice relaxation time of DMPG deuterated at the glycerol headgroup paralleled the results obtained with ESR, showing that the peptides cause a better packing both at the headgroup and at the acyl chain bilayer regions. The stronger effect caused by the more potent analog in the membrane structure, when compared to the native hormone, is discussed in terms of its larger lipid association constant and/or its deeper penetration into the bilayer.

Amino Acid Sequence↗
PubMed1986

PDN-1: a spin-labeled analog of cis-diamminedichloroplatinum(II). Biophysical studies in aqueous, lipophilic, and biological media.

Biophysical and biochemical studies on a biradical-labeled analog of Cisplatin, PDN-1, in aqueous, biological, and lipophilic media are presented. The ESR spectra from PDN-1 in aqueous meida show five-line spectra typical of biradicals in which intramolecular spin exchange occurs. The spectrum of PDN-1 in 1-octanol is characterized by three broad lines overlying a fourth line that is approximately three times as wide as the others. Although the characteristics of the aqueous spectra differ substantially from those of the octanol spectra, both the midfield peak height and the integral of the absorption spectrum can be used as a linear measure of PDN-1 concentration in either media. The smallest concentration used in these linear calibrations was equivalent to the detection of 60 femptomoles of PDN-1 at a signal-to-noise ratio of 30. PDN-1 is shown to "decay" in tissue culture medium which involves either the reduction of a nitroxyl moiety, the labilization of an amino bond, or a combination of both. Spectra of intracellular PDN-1 show that both a mobile ("free") fraction and slowly tumbling ("bound") fraction of the compound can be detected within CHO cells. The spin-labeled cisplatin method is at least as sensitive as conventional chromatographic and spectroscopic methods, yet has the added advantage of offering information about the molecular environment of the complex.

Animals↗
PubMed1985

A comparison of the spin labels MAL-3 and TEMPAMINE for measuring the internal microviscosity of human erythrocytes.

In a recent paper, Daveloose et al. (Daveloose, D., Fabre, G., Berleur, F., Testylier, G. and Letterrier, F. (1983) Biochim. Biophys. Acta 763, 41-49) described a technique to measure the internal microviscosity of erythrocytes using the spin label MAL-3 (2,2,5,5-tetramethyl-3-malimidopyrrolidinyl-N-oxyl) to supercede the use of TEMPAMINE (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amine)ferricyanide. They cite the long time (12 h) required for TEMPAMINE to enter the erythrocyte interior and the 'unphysiological' nature of the potassium ferricyanide required to isolate the TEMPAMINE signal inside the erythrocytes as reasons for developing this technique. In the present work, the penetration of TEMPAMINE into the erythrocyte interior is found to be pH-dependent and on the order of seconds, not hours. The slow penetration of TEMPAMINE described by Daveloose et al. probably is a result of their using trimethylTEMPAMINE which is membrane-impermeable. High concentrations of ferricyanide do affect red cell morphology in a reproducible fashion. The internal microviscosity as measured by TEMPAMINE reflects this. MAL-3 does not. Sample preparation time using TEMPAMINE-ferricyanide is about 1 min compared to about 40 min for the MAL-3 technique. In the presence of ferricyanide or ascorbate, MAL-3-labeled cells show slow loss of signal intensity which indicates that MAL-3 leaks out of the cells. Both the TEMPAMINE-ferricyanide and MAL-3 techniques have advantages and disadvantages.

Cyclic N-Oxides↗
PubMed1989

Spin-labeling proton NMR study on aromatic amino acid residues in the guanine nucleotide binding site of human c-Ha-ras(1-171) protein.

A truncated human c-Ha-ras gene product, ras(1-171) protein, was prepared and chemically modified with maleimide spin-label (MSL). By trypsin digestion of the MSL-labeled ras(1-171) protein, MSL-labeled peptide fragments were isolated and sequenced. The cysteine residue in position 118 of the protein, but not the other cysteine residues, Cys-51 or Cys-80, was found to be specifically labeled by MSL. The ESR spectrum of the MSL-labeled ras(1-171) protein indicates that the MSL group attached to Cys-118 is strongly immobilized. Proton NMR spectra at 400-MHz were measured for this MSL-labeled ras(1-171) protein and also for a control sample of a labeled ras(1-171) protein whose MSL was reduced by sodium ascorbate. In the difference spectra for these two proteins, resonances of protons in the vicinity of the MSL group attached to Cys-118 of the ras(1-171) protein were observed. Thus, the MSL group was found to be in the vicinity of the protein-bound GDP. A phenylalanine residue and two histidine residues, which were characterized by 2D HOHAHA and DQF-COSY spectra, were also found to be in the vicinity of MSL. NOE and pH titration analyses indicate that this phenylalanine residue is close to the bound GDP and one of the two histidine residues. By carboxypeptidase digestion, the two histidine residues near MSL were identified as His-27 and His-94.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed2002

Electron paramagnetic resonance backbone dynamics studies on spin-labelled neuropeptide Y analogues.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammalians. NPY acts by binding to at least five G-protein coupled receptors (GPCRs) which have been named Y1, Y2, Y4, Y5 and Y6. Three spin-labelled NPY analogues containing the nitroxide group of the amino acid TOAC (2.2.6.6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) as a paramagnetic probe were synthesized by solid-phase peptide synthesis. Synthetic problems owing to the sensitivity of nitroxide towards acidic and reducing conditions have been overcome by using a cleavage cocktail that contains anisole and cresol scavengers. Concerning the receptor binding preferences, the analogues [TOAC34]-pNPY and [Ala31, TOAC32]-pNPY showed a marked selectivity for the Y5 receptor, while [TOAC2]-pNPY maintained a significant binding also to the Y2 receptor subtype. The modifications of the native peptide structure caused by the introduction of TOAC were examined by circular dichroism. In order to determine the rotational correlation time of the spin probes, electron paramagnetic resonance measurements were performed in solution and in the presence of liposomes. This allowed us to evaluate the backbone dynamics of the different parts of the NPY molecule in the free and membrane bound states. The results of these studies showed that NPY Interacts with liposomes by using the C-terminal alpha-helix while the N-terminal tail retains a flexibility that is comparable to that of the peptide in solution as already shown by NMR studies on DPC micelles. Furthermore, we demonstrated that TOAC-labelllng is a valuable tool to investigate changes in the backbone conformation and dynamics. This may be of major importance for peptides and small proteins when they bind to cell membranes.

Amino Acid Sequence↗
PubMed1985

GABA inhibits inhalation anaesthetic-induced membrane fluidization: a spin label study in synaptic and phospholipid membranes.

The hydrophobic interaction of GABA (10(-3)-10(-9) M) with the inhalation anaesthetic enflurane was studied in synaptic plasma membranes of whole rat brain or of the striatum, synaptic mitochondrial membranes and dipalmitoyl lecithin (DPL) vesicles. Stearic acid spin labels, probing either the C-5 level (hydrophilic end) or the C-12 level (hydrophobic end) of the lipid bilayer, were used as indicators of fluidity. Inhalation anaesthetic at 2 mM fluidized the C-5 and the C-12 level of the membranes. A concentration of 0.4 mM produced consistently an increased order (stabilization) at the C-5 level in synaptic plasma membranes. GABA, added prior to the anaesthetic, caused a dose-related inhibition of the anaesthetic-induced fluidization. This restorative effect, which also occurred in DPL vesicles, was not prevented by the GABAA antagonists bicuculline or picrotoxin and was therefore probably not related to receptor mechanisms. When GABA was added after the anaesthetic (2 mM) the inhibition was seen only with 10(-3) M GABA. GABA did not influence (potentiate) the fluidity changes caused by 0.4 mM inhalation anaesthetic in synaptic membranes. Glutamate had a fluidizing effect, but no interaction with enflurane, at the C-5 level of DPL only at a high concentration (10(-3) M). The observed fluidization by enflurane and the restorative effect of GABA, probably through non-receptor-hydrophobic interaction, may be involved in the mechanisms of toxic CNS effects.

Animals↗
PubMed2000

Myocardial perfusion and intracapillary blood volume in rats at rest and with coronary dilatation: MR imaging in vivo with use of a spin-labeling technique.

PURPOSE: To validate a magnetic resonance (MR) imaging technique that is not first pass and that reveals perfusion and regional blood volume (RBV) in the intact rat. MATERIALS AND METHODS: Measurement of perfusion was based on the perfusion-sensitive T1 relaxation after magnetic spin labeling of water protons. RBV was determined from steady-state measurements of T1 before and after administration of an intravascular contrast agent. The colored microsphere technique was used as a reference method for perfusion measurement. RBV and perfusion maps were obtained with the rats at rest and during administration of 3 mg of adenosine phosphate per kilogram of body weight per minute. RESULTS: At MR imaging, perfusion during resting conditions was 3.5 mL/g/min +/- 0.1 (SEM), and RBV was 11.6% +/- 0.6 (SEM). Adenosine phosphate significantly increased perfusion to 4.5 mL/g/min +/- 0.3 (SEM) and decreased mean arterial pressure from 120 mm Hg to 65 mm Hg, which implies a reduction of coronary resistance to 40% of baseline. RBV increased consistently to 23.8% +/- 0.6 (SEM). CONCLUSION: The study results show that quantitative mapping of perfusion and RBV may be performed noninvasively by means of MR imaging in the intact animal. The presented method allows determination of vasodilative and perfusion reserve, which reflects the in vivo regulation of coronary microcirculation for a given stimulus.

Adenosine↗
PubMed1989

Site-directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation.

Colicin E1 is an E. coli plasmid-encoded water-soluble protein that spontaneously inserts into lipid membranes to form a voltage-gated ion channel. We have employed a novel approach in which site-directed mutagenesis is used to provide highly specific attachment points for nitroxide spin labels. A series of colicin mutants, differing only by the position of a single cysteine residue, were prepared and selectively labeled at that cysteine. A hydrophilic sequence (398-406) within the C-terminal domain of the water-soluble form of the protein was investigated and exhibited an electron paramagnetic resonance (EPR) spectral periodicity strongly suggesting an amphiphilic alpha-helix. After removal of the N-terminus of the protein with trypsin, the spectra for this sequence indicate increased label mobility and a more flexible structure.

Amino Acid Sequence↗
PubMed1987

A phospholipid spin label used as a liposome-associated MRI contrast agent.

Given current clinical use of phospholipid bilayer structures (liposomes/vesicles) as nontoxic drug delivery vehicles, we have addressed the possibility of employing the phospholipids themselves as MRI contrast agents. To this end we have synthesized phosphatidylcholine with a nitroxide spin label replacing one methyl residue of the choline headgroup. This material was mixed with natural phosphatidylcholine in mole ratios from 1:50 to 1:1 and used to prepare sonicated unilamellar vesicles in saline. Expected structural features of these vesicles were verified by freeze-fracture electron microscopy. Proton T1 values of saline were readily decreased to less than 0.3 s by such preparations, yielding a net relaxivity of 0.6 M-1 s-1. The approach seems to be a realistic way of firmly associating a contrast agent of minimal toxicity with ordinary liposomes/vesicles in a manner that is not subject to leakage.

Contrast Media↗
PubMed1982

A comparison of resolution-enhancement methods in saturation-transfer EPR. 15N isotopically substituted spin labels and 35 GHz high-frequency operation.

The use of (15)N isotopic substitution and 35 GHz (Q-band) operation as resolution enhancement methods in saturation-transfer electron paramagnetic resonance (ST-EPR) are compared. We find that both methods offer roughly comparable enhancements in ST-EPR resolution. The most powerful approach to resolving complex ST-EPR spectral behavior, however, will probably be the combined use of multiple-frequency X- and Q-band operation with (15)N isotopically substituted spin labels.

Electron Spin Resonance Spectroscopy↗
PubMed1998

Identification of protein folding patterns using site-directed spin labeling. Structural characterization of a beta-sheet and putative substrate binding regions in the conserved domain of alpha A-crystallin.

The folding pattern of the segment of alphaA-crystallin encoded by exon 2 and containing putative substrate binding sites was explored using site-directed spin labeling (SDSL). For this purpose, a nitroxide scan was carried out between residues 60 and 108. At each site, structural constraints describing the local environment and topography were obtained from analysis of the nitroxide mobility and its solvent accessibility. Periodic patterns in the sequence-specific variation of these parameters were used to assign the secondary structure along the sequence. Geometric constraints describing the packing of secondary structure were deduced from patterns of proximities in 20 nitroxide pairs, specifically designed to differentiate between supersecondary structural motifs. Our data, in conjunction with those of Berengian et al. [Berengian, A. R., Bova, M. P., and Mchaourab, H. S. (1997) Biochemistry 36, 9951-9957], reveal that the fold of the segment between residues 84 and 120 consists of an antiparallel beta-sheet of three strands arranged in consecutive beta-hairpins. The boundaries of the sheet are defined at one end by a surface of isologous association and on the other end by an unstructured, charged interdomain segment. One of the putative substrate binding segments overlaps a buried loop, suggesting that the structural origin of the thermal activation of binding is the transient exposure of this site. This paper describes and implements a general strategy for experimental fold recognition using SDSL. The results of its application to alphaA-crystallin provide the first experimental insight into the folding pattern of the subunit and establish the structural context necessary to understand molecular recognition and substrate binding.

Amino Acid Sequence↗
PubMed1993

A spin label study of the membrane effect of various psychoactive drugs in human erythrocytes.

The effect of psychoactive agents with different clinical actions: three sedative neuroleptics (trifluoperazine, alimemazine tartrate, chlorpromazine), an anticholinergic agent (trihexyphenidyl hydrochloride), two tricyclic antidepressants (imipramine, desipramine) and lithium carbonate on the rotational correlation frequency (V+) of the spin label 16NS has been comparatively investigated in whole human erythrocytes. V+ was about 40% increased by the three neuroleptics, the anticholinergic agent and the antidepressant molecules at 0.2 mM. By contrast, lithium did not induce any significant change in V+ at the same concentrations. It can be suggested that the increase in "membrane fluidity", observed with a wide variety of drugs, is a non specific effect, unrelated to the psychotropic action, that can be ascribed to the amphiphilic properties of the tested drugs.

Electron Spin Resonance Spectroscopy↗