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PubMed1988

Interaction of spin-labeled nucleotides with sarcoplasmic reticulum adenosinetriphosphatase.

Spin-labeled derivatives of AMP-PCP, ATP, and 2'-deoxy-ATP, with a nitroxide moiety attached to the ribose ring [3'-O-(1-oxy-2,2,5,5-tetramethylpyrroline-3-carbonyl)nucleotide], are used to study the nucleotide binding site stoichiometry of sarcoplasmic reticulum (SR) ATPase. With all derivatives, a maximal binding of 4.5 nmol/mg of SR protein is found, a value close to the number of phosphorylation sites obtained with ATP. The spin-labeled nucleotides cannot be utilized by the enzyme as substrates. Binding of spin-labeled nucleotides is inhibited by labeling the ATPase with fluorescein 5'-isothiocyanate, indicating that all the labeled nucleotide is located at the catalytic site. Additions of spin-labeled ATP to vesicle suspensions during steady turnover demonstrate competitive inhibition of both catalysis and the regulatory effect normally exhibited by ATP. As secondary binding of spin-labeled ATP is not detected at pertinent concentrations, it is suggested that both functions of ATP may be effected through a single site.

Adenosine Triphosphatases↗
PubMed1984

The characterization of vanadate-trapped nucleotide complexes with spin-labelled myosins.

The properties of spin-labelled myosin, prepared from rabbit skeletal and scallop adductor muscle, on forming a long-lived complex with ADP and vanadate (M.ADP.Vi), have been investigated. In the case of an iodoacetamide-based label attached to rabbit myosin or subfragment 1, M.ADP.Vi formation is characterized by a marked increase in the mobility of the probe, similar to that seen during steady-state ATPase activity. Hence, this complex appears to be a good analogue of the M**ADP.Pi state. The kinetics of M.ADP.Vi formation were determined by following the electron paramagnetic resonance (e.p.r.) signal with time and were analysed according to the scheme: (formula; see text) After correction for Vi polymerization, K'4 = 3.2 X 10(-4)M, k'-3 = 8.7 X 10(-3) s-1 and k'3 = 1.5 X 10(-4) s-1. The major effect of spin-labelling the reactive SH1 thiol is to increase k'3, so that M.ADP.Vi dissociates over a period of hours rather than days. In contrast, a maleimide-based spin-label attached to rabbit myosin does not exhibit a large change in mobility, on formation of the M.ADP.Vi complex. However, the small change observed in both the conventional and saturation transfer spectra questions the assumption that this probe is completely insensitive to librational motion during ATPase activity. The immobilized spectrum of the iodoacetamide-based spin label attached to scallop myosin is insensitive to M.ADP.Vi formation in the presence or absence of Ca2+. Under these conditions, the label appears to reflect gross head motion and hence this observation lends no support to the idea that, in the myosin-linked regulatory system, Ca2+ operates by controlling the flexibility of the subfragment 1-subfragment 2 joint.

Adenosine Diphosphate↗
PubMed1976

Spin-labelled AMP - an activator of phosphorylase.

1. A spin-labelled AMP derivative and its diamagnetic analogue activate phosphorylase b in the same way, but do not activate phosphorylase a. 2. The electron-spin-resonance spectra of the spin-labelled AMP derivative bound to phosphorylase b and a have "powderlike" characteristics indicating that the spin label is immobilised on the protein. From changes in the electron-spin-resonance spectrum of spin-labelled AMP as phosphorylase b or a is added, the dissociation constants were calculated. 3. The interactions of spin-labelled AMP and the diamagnetic analogue with phosphorylase b and a have been monitored by observing changes in the spectral properties of fluorescent and spin-label probes covalently attached to the enzyme. 4. The dissociation constants of spin-labelled AMP and phosphorylase b or a are 175 +/- 25 muM and 15 +/- 5 muM respectively. Similar dissociation constants are obtained for the diamagnetic analogue. The effect of these AMP derivatives on the covalently attached probe groups and on phosphorylase activity is compared to the effect of AMP and IMP.

Adenosine Monophosphate↗
PubMed1969

Motion of steroid spin labels in membranes.

Steroid spin labels have been found to undergo rapid and sometimes anisotropic rotational motion in membranes and phospholipid vesicle preparations. N-oxyl-4',4'-dimethyloxazolidine derivatives of 5alpha-androstan-3-one-17beta-ol, 5beta-androstan-3-one-17beta-ol, and 5alpha-androstan-3-one were prepared according to the procedure described by Keana. The paramagnetic five-membered ring is rigidly attached to the steroid nucleus at the 3-position, and thus the resonance spectrum of the nitroxide group reflects the motion of the entire steroid nucleus. This rapid motion, with rotational diffusion frequencies of the order of 10(7) to 10(8) second(-1) is considered to lend plausibility to models of membrane transport involving rotations and/or translations of carriers within the hydrophobic region of the membrane. The steroid spin labels also form a convenient class of probes for studying conformational changes in membranes, since (a) their resonance spectra often fall in the category of "intermediate immobilization," which is most sensitive to small changes, and (b) these labels are quite soluble in a wide variety of membranes.

Journal Article↗
PubMed1983

Binding of spin-labeled carboxyatractylate to mitochondrial adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier as studied by electron spin resonance.

The spin-label 2,2,5,5-tetramethyl-1-oxy-3-pyrroline-3-carboxylic acid was attached to the inhibitor carboxyatractylate of the mitochondrial ADP/ATP carrier. Being closely linked to the inhibitor, the spin-label should reflect the mobility of the carboxyatractylate. When bound to the carrier in mitochondria, spin-labeled carboxyatractylate reveals a most unusual hyperfine splitting of 72 G. A second spectral component with a hyperfine splitting of 62 G is also mainly due to carrier-bound inhibitor. A similar spectrum with somewhat reduced hyperfine splitting was observed with the detergent-solubilized protein, whereas reincorporation into phospholipid membranes yielded almost the same spectra as in mitochondria. The carrier-bound spin-label is concluded to be highly immobilized. The less immobilized spectral component is discussed in terms of strongly anisotropic label motion. In addition, the unusual splitting is interpreted to indicate the highly polar environment of the nitroxide. The interpretations are supported by the temperature dependence, which indicates a reversible progressive spin-label mobilization up to 50 degrees C. Membrane-impermeable reducing agents showed that the spin-label is easily accessible from the aqueous phase.

Animals↗
PubMed1994

The structural differences between the embryos of viable and nonviable wheat seeds as studied with the EPR spectroscopy of lipid-soluble spin labels.

Dried and hydrated embryos of wheat seeds (viable and nonviable, harvested in 1992 and 1976, respectively) were studied by the EPR method with the use of the spin-labeling technique. Spin label Tempone was used for testing the plasmalemma integrity. It has been demonstrated that the loss of seed viability correlates with the loss of external membrane integrity. Spin-labeled derivatives of stearic acids, 5-doxylstearate I(12.3) and 16-doxylstearate I(1.14), were used to monitor the changes in structural characteristics of embryo cell membranes. The EPR spectra of these spin labels represent the superpositions of at least two signals from the molecules located in domains characterized by different fluidity. The comparison of the EPR spectra from I(12.3) in embryo cells and model systems (total fraction of lipids and purified seed oil) indicates that the majority of spin label molecules is located in the lipid surroundings, while the minor portion of I(12.3) is localized in so-called lipid bodies which contain seed oil. The embryo cells of viable and nonviable seeds differ in the sizes of these 'solid' and 'fluid' intracellular domains. The environment of spin label molecules located in cell membranes of nonviable seeds is more rigid, as compared with that in the membranes of the viable cells. The study of dehydration-rehydration effects has demonstrated that the loss of water causes the restriction of spin label mobility in embryo cells from both kinds of seeds.

Electron Spin Resonance Spectroscopy↗
PubMed1980

The synthesis and properties of four spin-labeled analogs of adenosylcobalamin.

Four spin-labeled analogs of adenosylcobalamin have been synthesized to aid in the detection and identification of radical intermediates in the adenosylcobalamin-dependent enzymatic reactions and to serve as probes of the coenzyme, substrate, and effector binding sites of the protein. Three isomers of adenosylcobalamin, in which one of the propionamide side chains (b, d, or e) was hydrolyzed, and adenosylepicobalamin e-carboxylic acid were reacted with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to yield the spin-labeled adenosylcorrinoids. These spin-labeled derivatives of adenosylcobalamin function as coenzymes and/or inhibitors of dioldehydrase from Klebsiella pneumoniae and of ribonucleotide reductase from Corynebacterium nephridii. Electron spin resonance has been used to monitor the photolytic cleavage of the carbon-cobalt bond of these analogs.

Adenosine↗
PubMed2006

Feasibility of velocity selective arterial spin labeling in functional MRI.

Arterial spin labeling (ASL) magnetically inverts or saturates the spins in arterial blood and uses them as endogenous tracers. Conventionally, the tagging band is upstream or nonselective to the target slices. In the brain, ASL-based functional magnetic resonance imaging (fMRI) has been shown to detect activation better localized in gray matter than blood oxygenation level dependent contrast. More recently, velocity selective-ASL (VS-ASL) was proposed to tag spins according to their flow velocity. One desirable characteristic of VS-ASL is its capability to generate tags sufficiently close to the target slices and thereby circumvent the complication of non-zero transit delay. In this study, we investigate the feasibility of VS-ASL in fMRI by comparing it with a conventional ASL method (PICORE). The results from the visual cortex of healthy volunteers show that VS-ASL and PICORE have comparable spatial specificity in detecting the flow change induced by neuronal activity. Velocity selective-arterial spin labeling can further distinguish the contribution from different flow directions but spurious elevation of fractional signal change may occur when the VS tagging is applied off the direction of blood supply. The flow reaches the vicinity of perfusion at a cutoff velocity (Vc) of 2 cm/sec whereas the activation exclusively detected by Vc=4 cm/sec implies the arteriolar response to the neuronal activity and a respondent vessel diameter up to 240 microm. Velocity selective imaging can remove intravascular signal from the vessels where the flow velocity is above Vc.

Adult↗
PubMed1983

A spin-label study of plasma membranes of adrenal chromaffin cells.

Chromaffin-cell membranes were labeled with two nitroxide spin labels, one probing the interior of the membrane and one probing the interfacial region. Both spin labels indicate that the membrane undergoes a phase transition at about 26 degrees C. An Arrhenius plot of acetylcholinesterase activity exhibits a discontinuity at 26 degrees C, consistent with the existence of a phase transition at that temperature. Acetylcholine, which stimulates chromaffin cells to secrete catecholamines, and hexamethonium, a cholinergic blocker, do not affect the rotational correlation times of the spin labels. These results argue that cholinergic stimulation does not affect the fluidity of the chromaffin-cell membrane.

Acetylcholinesterase↗
PubMedDate not supplied

[Spin label study of the surface of lipid bilayers].

The surface of liposomes and natural membranes has been studied by the method of "spin label--spin probe". Various nitroxyl radicals have been attached to membranes either dueto covalent binding with NH2-groups of phosphatidylethanolamine or due to hydrophobic interactions. Addition of paramagnetics of different charge signs (potassium ferricyanide and dibenzenechromium iodide) to spin labeled membranes results in a decrease of the EPR line intensity without marked broadening. Since the paramagnetic is attached to the membrane surface near the label it broadens the label's EPR spectrum so that it can not be observed. In this case one can observe only the spectrum of labels which have no paramagnetics in the vicinity, the nitroxyl part of radicals being inaccessible to direct paramagnetic impacts. The constants of binding of paramagnetics to the membrane surface have been determined from these experiments. All results (including the assymetry of label rotation) can be explained most simply by assuming that the polar lipid groups are oriented parallel to the membranes surface.

Electron Spin Resonance Spectroscopy↗
PubMed1988

Localizing the nitroxide group of fatty acid and voltage-sensitive spin-labels in phospholipid bilayers.

The intramembrane locations of several spin labeled probes in small egg phosphatidylcholine (egg PC) vesicles were determined from the enhancement of the 13C nuclear spin lattice relaxation of the membrane phospholipid. Electron paramagnetic resonance (EPR) spectroscopy was also used to measure the relative environmental polarities of the spin labels in egg PC vesicles, ethanol and aqueous solution. The binding location of the spin label group was determined for a pair of hydrophobic ion spin labels, a pair of long chain amphiphiles, and three stearates containing doxyl groups at the 5, 10 and 16 positions. The nuclear relaxation results indicate that the spin label groups on the stearates are located nearer to the membrane exterior than the analogous positions of the unlabeled phospholipid acyl chains. In addition, the spin label groups of the hydrophobic ions and long chain amphiphiles are located near the acyl chain methylene immediately adjacent to the carboxyl group. The relative polarities, determined by the EPR technique, are consistent with the nuclear relaxation results. This information, when combined with information on their electrical properties, allows for an assessment of the conformation and position of these voltage sensitive probes in membranes.

Electron Spin Resonance Spectroscopy↗
PubMedDate not supplied

[Study of the nitrogenase from Azotobacter vinelandii by the method of spin labels].

The interaction of nitrogenase with spin labels of four types have been studied. Conclusion about the presence of two SH-groups in the nitrogenase active site (one in Mo-Fe-protein and one in the Fe-protein) have been drawn from the correlation between the degree of inhibition of nitrogenfixing activity by the labels derived from p-Cl-Hg-benzoate and degree of binding of these labels to the nitrogenase molecule. Anaysis of EPR spectra of spin-labeled nitrogenase at 77 degrees K and at room temperature have shown that the labels bind to the free SH-groups and interact with iron containing center (ICC) of nitrogenase through the exchange mechanism. Distance between SH-group and ICC have been found to be 12 A. Spin labels derived from isocyanide have been bound directly to ICC in amount of 6--10 labels per one nitrogenase molecule. Due to the exchange interaction between these labels they give the singlet ESR spectra both at 77 degrees and at room temperature which is characteristic for the closely disposed labels. From this fact a conclusion have been drawn about the cluster structure of ICC. The labels derived from iodoacetamide ana maleimide bind SH- and NH2-groups of nitrogenase molecules. Analysis of temperature dependence of the effective rotational frequency of this labels have revealed a conformational transition in nitrogenase molecule at 19 degrees C, that has made it possible to explain the break in the Arrenius plots of enzyme activity.

Azotobacter↗
PubMed1999

Probing iso-1-cytochrome c structure by site-directed spin labeling and electron paramagnetic resonance techniques.

A cysteine-specific methanethiosulfonate spin label was introduced into yeast iso-1-cytochrome c at three different positions. The modified forms of cytochrome c included: the wild-type protein labeled at naturally occurring C102, and two mutated proteins, S47C and L85C, labeled at positions 47 and 85, respectively (both S47C and L85C derived from the protein in which C102 had been replaced by threonine). All three spin-labeled protein derivatives were characterized using electron paramagnetic resonance (EPR) techniques. The continuous wave (CW) EPR spectrum of spin label attached to L85C differed from those recorded for spin label attached to C102 or S47C, indicating that spin label at position 85 was more immobilized and exhibited more complex tumbling than spin label at two other positions. The temperature dependence of the CW EPR spectra and CW EPR power saturation revealed further differences of spin-labeled L85C. The results were discussed in terms of application of the site-directed spin labeling technique in probing the local dynamic structure of iso-1-cytochrome c.

Base Sequence↗
PubMed1975

Spin label and lanthanide binding sites on glyceraldehyde-3-phosphate dehydrogenase.

The electron spin resonance spectrum of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl has two components. One component is due to a spin label highly immobilized on the enzyme surface and the other to a nitroxyl group able to tumble more rapidly. The spin-labelled enzyme is inactive. Selective modification of the active site cysteine residue (149) and determinations of total sulphydryl content implicate this residue as the site of the immobile spin-label. The mobile spin label is attached to another sulphydryl group. Crystallographic studies on the human muscle enzyme (Watson, H.C., Duee, E. and Mercer, W.D. (1972) Nat. New Biol., 240, 130) have located a binding site for samarium ion in the active centre. Addition of the paramagnetic gadolinium ion to spin-labelled enzyme reduces the intensity of both the spin label signals (by 72% for the mobile and by 11% for the immobile component). This indicates that the metal ion site (Kd equals 0.7 mM) is close to both types of spin label. Measurements of the effect of gadolinium-protein binding on the relaxation rate of solvent water protons enable the enzyme-bound spin label-metal ion distances to be tentatively estimated as 15 angstrom.

Animals↗
PubMed2004

The subunit b dimer of the FOF1-ATP synthase: interaction with F1-ATPase as deduced by site-specific spin-labeling.

We have used site-specific spin-labeling of single cysteine mutations within a water-soluble mutant of subunit b of the ATP synthase and employed electron spin resonance (ESR) spectroscopy to obtain information about the binding interactions of the b dimer with F1-ATPase. Interaction of b2 with a delta-depleted F1 (F1-delta) was also studied. The cysteine mutations used for spin-labeling were distributed throughout the cytosolic domain of the b subunit. In addition, each position between residues 101 and 114 of b was individually mutated to cysteine. All mutants were modified with a cysteine-reactive spin label. The room temperature ESR spectra of spin-labeled b2 in the presence of F1 or F1-delta when compared with the spectra of free b2 indicate a tight binding interaction between b2 and F1. The data suggest that b2 packs tightly to F1 between residues 80 and the C terminus but that there are segments of b2 within that region where packing interactions are quite loose. Two-dimensional gel electrophoresis confirmed binding of the modified b mutants to F1-ATPase as well as to F1-delta. Subsequent addition of delta to F1-delta.b2 complex resulted in changes in the ESR spectra, indicating different binding interactions of b to F1 in the presence or absence of delta. The data also suggest that the reconstitution of the ATP synthase is not ordered with respect to these subunits. Additional spectral components observed in b preparations that were spin-labeled between amino acid position 101 and 114 are indicative of either two populations of b subunits with different packing interactions or to helical bending within this region.

Adenosine Triphosphatases↗
PubMed1982

ESR spin-label studies of lipid-protein interactions in membranes.

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.

Animals↗
PubMed1978

Steroid modulation of human serum albumin binding properties. A spin label study.

The binding isotherm and unique electron spin resonance spectral characteristics of a monoanionic spin label (1-gamma-aminobutyrate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) and a dianionic spin label (1-glutamate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-2,4-dinitrobenzene) are used to prove the steroid modulation of serum albumin binding properties. Effects of a selected number of steroids (progesterone, testosterone, estradiol, aldosterone, estriol, corticosterone, deoxycorticosterone, hydrocortisone, and cortisone) on the binding isotherm of the monoanionic spin label binding to serum albumin have been determined. At the steroid/albumin ratio of 0.5 to 1, progesterone, testosterone, and estradiol enhance binding of the spin label at all concentrations studied. However, the remaining steroids exert an inhibitory effect at low spin label/albumin ratios and an enhancement effect at high spin label/albumin ratios. Progesterone and cortisone effects on the resonance spectra of the spin label bound to serum albumin confirm the enhancement and displacement properties of these ligands. Thus, like fatty acids, steroids may bind to either the primary or secondary bilirubin binding sites and also allosterically perturb the binding properties of serum albumin. The in vivo importance of the steroid-albumin interaction is discussed.

Cortisone↗
PubMed1979

Electron spin resonance study of human alpha 1-acid glycoprotein interaction with a spin labelled steroid.

The interaction of human alpha 1-acid glycoprotein (AAG) with a corticosteroid was studied using nitroxide labeled deoxycorticosterone and electron spin resonance (ESR) spectroscopy. The ESR spectra of the spin labeled steroid in the presence of AAG could be used to characterize the ligand-protein interaction at equilibrium without the need of a separation between bound and free species. An association constant Ka of 6.10(5) M-1 at 20 degrees C and a binding capacity of one site per mole protein were found. ESR spectra recorded at equilibrium at various temperatures allowed the calculation of enthalpy and entropy variations for the steroid-protein interaction; these thermodynamic parameters exhibited a rapid change above 45 degrees C which may be related to a protein conformational modification above this temperature, as detected by circular dichroism study. The ESR spectra width could be used to define a polar character for the spin label environment in the steroid binding site of AAG and to calculate an apparent rotational correlation time of 2.8 x 10(-8) sec for the steroid-protein complex in aqueous solution at 20 degrees C. It can be concluded that spin labeling and ESR methodology is of value in the study of steroid-protein interactions of biological significance above all because it can provide direct physico-chemical information concerning the local environment of the ligand in its binding site at equilibrium.

Binding Sites↗