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PubMed1985

Spin-label studies on the origin of the specificity of lipid-protein interactions in Na+,K+-ATPase membranes from Squalus acanthias.

The pH dependence and salt dependence of the lipid-protein interactions of phosphatidic acid, phosphatidylserine, and stearic acid with Na+,K+-ATPase membranes from Squalus acanthias have been studied with spin-label electron spin resonance spectroscopy, using lipids with nitroxide labels on the 14-position C atom of the sn-2 chain. For phosphatidic acid and stearic acid, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 6.6 and 8.0, respectively. In contrast, the pKa of stearic acid in the bulk lipid environment of the membrane is estimated from spin-label spectroscopy to be approximately equal to 6.6. The fraction of motionally restricted phosphatidylserine spin-label remains constant over the pH range 4.7-9.2. In the fully dissociated state the fractions of motionally restricted spin-labeled phosphatidic and stearic acids decrease with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M. For stearic acid the net decrease is comparable to that obtained on protonation, but for phosphatidic acid the decrease is considerably smaller (by approximately 55%) than that obtained on protonating the lipid. The fraction of motionally restricted phosphatidylserine spin-label varies relatively little with salt concentration up to 1 M NaCl. Direct electrostatic effects alone cannot account for the whole of the observed specificity of interaction of the two phospholipids with Na+,K+-ATPase membranes.

Animals↗
PubMed1983

Rotational dynamics of spin-labelled muscle proteins.

Electron paramagnetic resonance (EPR) spectroscopy on spin-labelled proteins was used to investigate the role of rotational motions in the two key ATPase systems of muscle: (a) the force-generating interaction of myosin and actin and (b) the calcium pump of sarcoplasmic reticulum (SR). The spin labels in these studies were rigidly fixed to the protein framework, so there were none of the nanosecond motions detectable by conventional EPR techniques. Thus conventional EPR was useful only in the study of static orientation in oriented systems (parallel muscle fibres or membrane bilayers), while saturation transfer EPR was required to detect protein motions in the microsecond range. Spin labels attached to myosin 'heads' were used to test the proposal that these heads form force-generating cross-bridges to actin that rotate when driven by the ATPase cycle. These probes are highly oriented and strongly immobilized when heads are bound to actin, but undergo microsecond rotations when the heads are detached from actin. During contraction, the majority of heads are mobile and disoriented, indicating detachment, while the remainder appear to be attached with the same orientation as in the absence of ATP. Thus the probed part of the myosin head does not appear to rotate during force generation. Saturation transfer EPR was used to monitor the microsecond rotational motions of the Ca2+-ATPase in SR membranes, while conventional EPR was used to monitor lipid hydrocarbon rotations in the microsecond time range. Changes in lipid fluidity and protein mobility, induced by changes in lipid/protein ratio, lipid composition, [Ca2+] and protein cross-linking, correlate well with changes in enzymic activity, suggesting the importance of molecular motions for Ca2+ transport.

Actins↗
PubMed1975

A spin label study of egg white avidin.

Avidin is a tetrametric protein (mass 68,000 daltons) that binds 4 molecules of vitamin biotin (1). The biotin binding sites, 1 per subunit, are grouped in two pairs at opposite ends of the avidin molecule (GREEN, N.M., KONIECZNY, L., TOMS, E.J., and VALENTINE, R.C. (1971) Biochem. J. 125, 781). We have studied the topography of the avidin binding sites with the aid of four spin-labeled analogs of biotin: 4-biotinamido-2,2,6,6-tetramethyl-1-piperidinyloxy (II), 3-biotinamido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (III), 3-biotinamidomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IV), 4-(biotinylglycyl)-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (V). Fluorescence and optical absorption spectroscopy indicated that II to V occupied the same binding sites on avidin as did biotin. The electron spin resonance spectrum of the 4:1 complex between II and avidin contained broad line components characteristic of a highly immobilized spin label. Dipole-dipole interactions between spin labels bound to adjacent sites split each of the three major hyperfine lines into doublets with a separation of 13.8 G. The distance between adjacent bound nitroxide groups was calculated from this splitting to be 16 A. The dissociation of the 4:1 complex between II and avidin was biphasic with approximately half of the labels dissociating at a rate (kdiss equal to 2.51 times 10- minus 4 s- minus 1) that was much faster than the remainder (kdiss equal to 1.22 times 10- minus 5 s- minus 1). The electron spin resonance spectrum of the 2:1 complex between II and avidin clearly showed that, immediately after mixing, the spin labels were distributed in a random fashion among the available binding sites but that they slowly redistributed themselves so that each label bound to a site which was adjacent to an unoccupied site. The final time-independent electron spin resonance spectrum exhibited a splitting 69 G between the low and high field hyperfine lines which is characteristic of a highly immobilized, noninteracting spin label. Spin labels III and IV interacted with avidin in a similar fashion to that described for II with the exception that their dipolar splittings were 11.9 G and 14.2 G, respectively. From these splittings it was estimated that the distance between adjacent avidin-bound nitroxides was 16.7 A for labeled III and 15.7 A for label IV. The electron spin resonance spectrum of label V bound to avidin was characteristic of a noninteracting highly immobilized nitroxide with a maximum splitting of 62 G. The spectrum of V bound to avidin was independent of both time and the amount of bound label. The rate of dissociation of V from a 4:1 complex with avidin was monophasic. A model is proposed in which the recognition site for the heterocyclic ring system of biotin is represented as a cleft located within a hydrophobic depression in the surface of avidin.

Avidin↗
PubMed1981

Membrane action of tricyclic drugs. Spectroscopic studies of a series of phenothiazines compared with tricyclic antidepressive substances in red cell membrane, using the spin labelling technique.

Previous results were extended by binding studies of drugs on red cell membrane by means of the spin labelling technique. A series of phenothiazines was investigated and compared with several tricyclic antidepressive substances. It was found that phenothiazines at concentrations below 3 mumol/l decisively increase the order parameter of spin label 618, which reports on the interface of the membrane. Thus the fluidity of the interface is decreased. Spin label 616 reports on the hydrophobic membrane interior. Within this hydrophobic phase, the order is decreased by 100 mumol to 1 mmol/l of the phenothiazines. With tricyclic antidepressive substances, a rise of order parameter of spin label 618 is observed, which, however, is less steep. At about 30 mumol/l a maximum is eventually reached. At even higher concentrations of amitriptyline, nortriptyline or imipramine with spin label 616, however, no significant change of order parameter was observed indicating that perturbation of the hydrophobic membrane interior is not sensed. These findings thus corroborate our previous results, revealing that influence of phenothiazines can be measured through the polar and apolar phases of the membrane, while the antidepressive drugs primarily act on the polar phase.

Antidepressive Agents, Tricyclic↗
PubMed2002

Dap-SL: a new site-directed nitroxide spin labeling approach for determining structure and motions in synthesized peptides and proteins.

A new approach for site-directed placement of nitroxide spin labels in chemically synthesized peptides and proteins is described. The scheme takes advantage of a novel diaminopropionic acid scaffold to independently control backbone and side chain elongation. The result is a spin-labeled side chain, referred to as Dap-SL, in which an amide bond forms a linker between the nitroxide and the peptide backbone. The method was demonstrated in a series of helical peptides. Circular dichroism and nuclear magnetic resonance showed that Dap-SL introduces only a minor perturbation in the helical structure. The electron paramagnetic resonance spectrum of the singly labeled species allowed for determination of the spin label rotational correlation time and suggests that the Dap-SL side chain is more flexible than the modified Cys side chain frequently used in site-directed spin label studies. Spectra of the doubly labeled peptides indicate a mixture of 3(10)-helix and alpha-helix, which parallels findings from previous studies. The scheme demonstrated here offers a fundamentally new approach for introducing spin labels into proteins and promises to significantly extend biophysical investigations of large proteins and receptors. In addition, the technique is readily modified for incorporation of any biophysical probe.

Electron Spin Resonance Spectroscopy↗
PubMed1974

Immune lysis of liposomes and erythrocyte ghosts loaded with spin label.

It is shown that the paramagnetic resonance of spin labels offers a convenient technique for monitoring the complement-mediated immune lysis of erythrocyte ghosts, and sensitized phospholipid liposomes. The ghosts or liposomes are loaded with a concentrated solution of a water-soluble, membrane-impermeable spin label, so that there is a strong exchange broadening of the paramagnetic resonance signal due to labels enclosed within the liposomes or ghosts. Complement-mediated lysis releases the labels into a dilute solution, where a relatively sharp and intense paramagnetic resonance signal is detected. AN UNANTICIPATED RESULT IS ALSO REPORTED: this is the augmentation of release of spin label from loaded ghosts sensitized with egg albumin, in the presence of antibody against egg albumin, complement, and egg albumin in solution, over and above the release in the absence of egg albumin in solution. Comparison with complement fixation suggests that the augmentation of lysis is effected by crosslinking of immune complexes with the cell membrane.

Animals↗
PubMedDate not supplied

[Segmental flexibility of single-, double-, and triple-stranded polyribonucleotides from the data of spin label method. Formation of the triple-stranded poly(A.A.U) polynucleotide helix].

Segmental mobility dynamic peculiarities of poly(U), poly(A) and poly(C) synthetic polymers and their complexes were investigated by spin-label method. Imidazolide spin-label was introduced into 2'-oxi-groups of polymer ribose in correlation: one spin-label on 18-20 bases. Formation of complexes was observed by ESR spectra at two pH: 4.2 and 7.2. Segmental mobility of only single strand spin-labelled polymer segment and in the complex was evaluated by measuring rotational correlation time (tau) determined by dependence of distances between outer wide extrema in ESR spectra from solvent viscosity at different temperatures. It turned out that correlation time tau of single strand structures in a high degree depend on pH and temperature. For three strand structures abrupt increase of tau because of appearance of rigidity was observed. It is possible to evaluate part of triple complexes poly(U.A.A) and poly(U.U.A) existing in dynamic equilibrium depending on pH and temperature by the form of outer wide extrema. Adding of dye to complex of poly(U).poly(A) causes an increase of rigidity of the supermolecular structure. Quantitative characteristics of formed complexes were obtained by simulation of ESR spectra on computer.

Electron Spin Resonance Spectroscopy↗
PubMed1980

Studies with a hydrophobic, spin-labeled virucidal agent.

A spin-labeled virucidal agent was synthesized, purified, and tested for its activity against the enveloped bacterial virus phi6 and herpes simplex virus. This compound, designated BPN, inactivated greater than 99% of phi6 and herpes simplex virus in vitro at concentrations as low as 0.1 mM. Detailed studies were carried out on the mechanism of inactivation of phi6 by BPN. These studies revealed that treatment of phi6 by BPN specifically removes a single envelope protein that is considered to be responsible for adsorption of the virus to the host cell. Related experiments with the phi6 host, Pseudomonas phaseolicola strain HB10Y, showed that this organism is insensitive to the effects of BPN. The basis for the differential sensitivity of phi6 to BPN, in comparison to the host cell, was investigated by electron spin resonance techniques. It was found that, for phi6, HB10Y, and their extracted phospholipids, BPN is localized in the hydrocarbon zones of the membrane bilayer. However, in the case of phi6, the rotational mobility of BPN is much reduced in comparison to that in HB10Y and the phospholipid preparations. Furthermore, an Arrhenius plot of rotational correlation time (tau(c)) showed a marked discontinuity in slope at 31 degrees C in the case of phi6, but not for the other samples studied. This suggests a strong interaction between the phi6 envelope proteins and the lipid domains in which BPN is localized. Calculations based on the known lipid and protein composition of phi6 indicate that there is an absence of "free-lipid" pools in the viral envelope. It is suggested that BPN localizes in free-lipid pools of cell membranes, where its presence is of little or no consequence, but that in phi6 the BPN perturbs the hydrophobic interactions between phospholipids and proteins in the envelope.

Antiviral Agents↗
PubMed1984

Electron paramagnetic resonance of spin-labeled aequorin.

Aequorin is a Ca-activated bioluminescent protein from jellyfish. This protein contains two sulfhydryl groups, one of which is essential for its bioluminescence. Little information concerning the structure of and relationship between the metal binding sites of aequorin and the sulfhydryl group(s) is known. Aequorin was modified by attachment of either a maleimide spin-label [studied by electron paramagnetic resonance (EPR)] or the fluorescent label Acrylodan at the essential sulfhydryl in order to gain such information. These modifications caused destabilization of the chromophore of aequorin. Both of the attached labels showed considerable freedom of motion. The spin-label was quite accessible to the solvent, and the fluorescent label was less so. In addition the metal binding properties of the spin-labeled aequorin were studied by Mn(II) EPR. One tight Mn(II) binding site per spin-labeled aequorin was found. The distance between the Mn(II) binding site and the spin-label is at least 20 A. Furthermore, the relative affinity of spin-labeled aequorin for various metal ions was found to be in the order Pr(III) greater than Mn(II) greater than Ca(II) greater than Mg(II).

2-Naphthylamine↗
PubMed1976

Toxicity, antitumor activity, and pharmacokinetics of spin-labeled thioTEPA analogs.

Two spin-labeled analogs of thioTEPA, containing an iminoxyl radical, have been shown to have significant antitumor activity in several experimental tumor systems, combined with a marked decrease in toxicity compared with the parent compound, thioTEPA. Walker 256 carcinosarcoma, Guérin carcinoma, and Schweitz erythromyelosis showed complete regression using one of these analogs. Pharmacokinetic studies have shown that these compounds possess particular affinity towards tumor tissue.

Adenocarcinoma↗
PubMed1990

Spin label probes of the environment of cysteine beta-93 in hemoglobin.

The environment of cysteine beta-93 is altered during the oxygenation of hemoglobin. Electron spin resonance was used to probe the hemoglobin conformation in this crucial region on the proximal side of the heme. Spin-labeled hemoglobins in both the R-liganded state [methemoglobin and oxyhemoglobin] and the T-unliganded state [deoxyhemoglobin as well as Ni(II) and Cu(II) substituted hemoglobins] were investigated. Included in this study are iodoacetamide and maleimide labels with different constraints at the point of reaction with the SH-group, as well as a series of pyrrolidinyloxyl maleimide labels of different chain length. From differences in the correlation time of the spin labels it was possible to identify two distinct strongly immobilized configurations in addition to the relatively mobile configuration with the label on the surface of the protein. By dipolar interactions between the spin labels and paramagnetic Cu(II) at the heme center, the relative position of the three orientations for the spin label are defined. Differences are observed between the two hemoglobin conformations with respect to the relative population of the various orientations and with respect to the potential barrier associated with the reorientation of the spin labels.

Animals↗
PubMed1985

Interaction between bound cupric ion and spin-labeled cysteine beta-93 in human and horse hemoglobins.

The location of the various copper binding sites for horse and human hemoglobin was probed using spin labels attached to the beta-93 cysteine residue. Dipole-dipole interactions between the spin label and bound copper produce a decrease in the amplitude of the spin label spectrum which was used to estimate the Cu(II) spin label distance. By comparing the results with horse and human hemoglobin at 298 and 77 K four different Cu(II) binding sites were identified. The low affinity horse hemoglobin site with the sulfhydryl blocked (site 1) was found to be located 10-13 A from the sulfhydryl spin label on the surface of the molecule. Only with a free sulfhydryl is the site (site 2) in the pocket between the F and H helices closer to the SH-group and the iron populated. It is site 2 which is responsible for the oxidation. In frozen solutions a Cu-nitroxide distance of about 17 A was determined with human hemoglobin. This distance is consistent with the previously postulated location of the "high affinity" human hemoglobin site near the amino terminus of the beta-chain. At 298 K a much shorter Cu-nitroxide distance of about 7 A was calculated for human hemoglobin. This shorter distance at higher temperature also correlated with a slightly smaller value of g11 and A11 for the Cu(II) ESR spectrum. It is postulated that in solution cross-linking between nitrogenous ligands in the region of the amino terminus of one beta-chain and the carboxyl terminus of the other beta-chain can explain this shorter distance. This cross-link could involve histidine beta-143, which is one of the ligands thought to be also involved in site 1. Binding to the "high-affinity" site in solution thus stabilizes the "low-affinity" site 2 relative to site 1 explaining the reported interaction between the "high-affinity" and "low-affinity" sites.

Animals↗
PubMed1994

Time-resolved detection of structural changes during the photocycle of spin-labeled bacteriorhodopsin.

Bacteriorhodopsin was selectively spin labeled at residues 72, 101, or 105 after replacement of the native amino acids by cysteine. Only the electron paramagnetic resonance spectrum of the label at 101 was time-dependent during the photocycle. The spectral change rose with the decay of the M intermediate and fell with recovery of the ground state. The transient signal is interpreted as the result of movement in the C-D or E-F interhelical loop, or in both, coincident with protonation changes at the key aspartate 96 residue. These results link the optically characterized intermediates with localized conformational changes in bacteriorhodopsin during the photocycle.

Bacteriorhodopsins↗
PubMed1980

Spin labeling of human spectrin. Effects of temperature, divalent cations and reassociation with erythrocyte membrane.

Spectrin extracted from human red blood cells has been spin labeled in its dimeric and tetrameric forms with five different nitroxide derivatives of increasing chain length between their maleimide binding group and their nitroxide reporter group. Three molecules of spin label are bound per spectrin dimer. Electron spin resonance spectra show the simultaneous presence of strongly and weakly immobilized spin labels. Their relative proportion depends on the label length and is suddenly modified when it reaches 12 A This indicates the presence of cavities of approximately this size in the tertiary structure of spectrin in solution at 0 degrees C. The conformation of spectrin varies greatly with temperature. Reversible changes occur between 0 and 35 degrees C. At higher temperatures, partial denaturation is observed. Divalent cations (Mg2+ and Ca2+) stabilize spectrin in a more constrained conformation and protect it against thermal denaturation. The same behavior is observed when spin-labeled spectrin is reassociated with spectrin-depleted inside-out erythrocyte vesicles. When fatty acid spin labels are incorporated in the phospholipidic structure of these vesicles, the reassociation of spectrin does not change their electron spin resonance spectra. This result confirms the fact that spectrin interacts predominantly with proteins on erythrocyte membranes.

Calcium↗
PubMed2000

Cerebral perfusion and arterial transit time changes during task activation determined with continuous arterial spin labeling.

Perfusion imaging by arterial spin labeling (ASL) can be highly sensitive to the transit time from the labeling site to the tissue. We report the results of a study designed to separate the transit time and perfusion contributions to activation in ASL images accompanying motor and visual stimulation. Fractional transit time decreases were found to be comparable to fractional perfusion increases and the transit time change was found to be the greatest contributor to ASL signal change in ASL sequences without delayed acquisition. The implications for activation imaging with ASL and the arterial control of flow are discussed.

Blood Flow Velocity↗
PubMed1992

Investigation of the active site of human placenta glutathione transferase pi by means of a spin-labelled glutathione analogue.

A spin-labelled analogue of glutathione (sl-glutathione) has been used in order to characterize the active site of human placenta glutathione transferase pi. The sl-glutathione shows a competitive inhibition towards glutathione (Ki = 14 microM). Binding of sl-glutathione to the enzyme, followed by electron paramagnetic resonance spectroscopy, gives a Kd of 3 microM and two identical binding sites for dimeric unit. Inhibition of the enzyme, by modification of the Cys-47 residue, completely prevents the binding of sl-glutathione. The same results are obtained by monitoring the binding of glutathione by means of fluorescence spectroscopy. It is concluded that integrity of the thiolate of Cys-47 is necessary to maintain an active conformation of the enzyme able to efficiently bind glutathione into the active site.

Binding Sites↗
PubMed1989

Anticancer drugs. II. Synthesis and biological evaluation of spin labeled derivatives of podophyllotoxin.

The spin labeled derivatives of podophyllotoxin, 4-[4''-(2'',2'',6'',6''-tetramethyl-l''-piperidinyloxy)amino] -4'-demethylepipodophyllotoxin(GP-7,3) and N-podophyllic acid-N''-[4-(2,2,6,6-tetramethyl-l-piperidinyloxy)] thiosemicarbazide(GP-4,5) were synthesized and tested for their anticancer activity against the mouse solid tumors S180 and HepA in vivo, and the mouse lymphocytic leukemia L1210 and human stomach carcinoma SGC-7901 cells in vitro. At equitoxic concentrations, the anticancer activity of GP-7(3) was found to be similar to that of the clinically used VP-16(2). The toxicity of GP-7(3) (LD50231.2 mg/Kg) is 3.3 times lower than that of VP-16 (LD50 69.5 mg/Kg). GP-7(3) exhibits low subchronic toxicity. The total chemical yield of GP-7 (26%) is 4 times higher than that of VP-16 (6%) (based on podophyllotoxin). Therefore, GP-7(3) seems to be a promising new entry into the podophyllotoxin class of anticancer drugs.

Animals↗
PubMed2006

Removing the effects of CSF partial voluming on fitted CBF and arterial transit times using FAIR, a pulsed arterial spin labelling technique.

FAIR, an arterial spin labelling technique, provides non-invasive, quantitative CBF values and arterial transit times deltat. This paper focuses on the negative impact of CSF partial voluming on FAIR results. To understand and solve this problem, we performed a theoretical analysis and a range of simulations. We then acquired FAIR data from a volunteer to illustrate our findings. We found that the determinant effect of CSF is a delayed zero-crossing during inversion recovery. The subtraction of magnitude inversion recovery data in FAIR generates erroneous negative data and distorted fit results: we simulated that for CSF percentages of 0-40%, CBF and deltat will be progressively overestimated by up to 50%. For higher CSF percentages the errors were found to increase steeply. We explored a straightforward solution: taking the magnitude of the FAIR data before fitting. This provided a remarkably strong antidote against the effects of CSF partial voluming: for CSF percentages of 0-40%, simulations now gave CBF values accurate within 1%, and deltat within 5%. The fit remained robust for high CSF fractions. Our analysis and simulations demonstrate that using magnitude FAIR data minimises the detrimental effects of CSF partial voluming. Data from a healthy volunteer illustrate these results.

Artifacts↗