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PubMed1986

Calorimetric and fatty acid spin label study of subgel and interdigitated gel phases formed by asymmetric phosphatidylcholines.

Several saturated asymmetric and symmetric phosphatidylcholines were studied by ESR spectroscopy and differential scanning calorimetry in order to determine the behavior of a fatty acid spin labeled near its terminal methyl, 16-doxylstearate, in the mixed interdigitated gel phase and the Lc subgel phase and other properties of these lipids. This spin label was motionally restricted in the mixed interdigitated gel phases of 18:10PC and 18:12PC. The motional restriction was similar to that reported earlier for fully interdigitated phases. This spin label was motionally restricted almost to the same degree in 10:18PC suggesting that this asymmetric lipid may also form a mixed interdigitated bilayer. In contrast the spin label had more motion in the gel phase of 18:14PC than in symmetric forms of PC, consistent with conclusions from X-ray diffraction studies that this less asymmetric lipid does not form a mixed interdigitated phase. The spin label was partially frozen out of the Lc subgel phases of symmetric forms of PC and 18:14PC formed by storage at low temperature. The phase behavior of the other asymmetric lipids also depended on the sample history. Storage at low temperature caused 10:18PC and 18:12PC to go into ordered phases. The enthalpy of the transition of these ordered phases to the liquid-crystalline phase was 2-2.4-times greater than that of the transition of the gel phase formed on cooling back from the liquid-crystalline phase. The temperature of this high enthalpy transition was 0.8 K below that of the lower enthalpy gel to liquid-crystalline phase transition for 18:12PC, but 4.6 K higher for 10:18PC. The spin label was frozen out of these ordered phases, as it was out of the Lc subgel phases, suggesting that 18:12PC and 10:18PC may also form an Lc phase. 18:10PC was not observed to form an ordered phase although storage of the sample at low temperatures did affect the temperature of its transition from the liquid-crystalline phase back to the gel phase upon cycling through its phase transition.

Calorimetry, Differential Scanning↗
PubMed1988

Selective spin-labelling of an N-acetylneuraminic acid residue in the Fab-region oligosaccharide of immunoglobulin M.

A method is proposed for spin-labelling using 2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl, the N-acetylneuraminic acid residue within the Fab-region oligosaccharide of immunoglobulin M (IgM). The selectivity of spin-labelling was achieved by the controlled periodate oxidation of the oligosaccharide groups of the IgM prior to the spin-labelling. The spin-labelled IgM prepared in this way may be useful for the studies of dynamics of the IgM structure, particularly upon antigen binding.

Cyclic N-Oxides↗
PubMed2001

Spin-label electron paramagnetic resonance studies on the interaction of avidin with dimyristoyl-phosphatidylglycerol membranes.

The interaction of avidin--a basic protein from hen egg-white--with dimyristoyl-phosphatidylglycerol membranes was investigated by spin-label electron paramagnetic resonance spectroscopy. Phosphatidylcholines, bearing the nitroxide spin label at different positions along the sn-2 acyl chain of the lipid were used to investigate the effect of protein binding on the lipid chain-melting phase transition and acyl chain dynamics. Binding of the protein at saturating levels results in abolition of the chain-melting phase transition of the lipid and accompanying perturbation of the lipid acyl chain mobility. In the fluid phase region, the outer hyperfine splitting increases for all phosphatidylcholine spin-label positional isomers, indicating that the chain mobility is decreased by binding avidin. However, there was no evidence for direct interaction of the protein with the lipid acyl chains, clearly indicating that the protein does not penetrate the hydrophobic interior of the membrane. Selectivity experiments with different spin-labelled lipid probes indicate that avidin exhibits a preference for negatively charged lipid species, although all spin-labelled lipid species indirectly sense the protein binding. The interaction with negatively charged lipids is relevant to the use of avidin in applications such as the ultrastructural localization of biotinylated lipids in histochemical studies.

Avidin↗
PubMed1978

Temperature-induced changes of spin-labelled radioactive lipids in isolated guinea pig liver microsomal membranes before and in mitochondrial membranes after their translocation.

Lipids of isolated guinea pig liver microsomal membranes were labelled biosynthetically with isomeric doxyl stearic acid and temperature-induced changes of these membranes were studied by electron spin resonance. A noticeable discontinuity was detected at 10--12 degree C with 12- or 16-doxyl stearic acid containing membrane lipids which was attributed to the spin-labelled lipid--microsomal membrane protein interactions since no such discontinuity was detected in liposomes prepared from total lipid extracts of microsomal membranes. When microsomal membranes containing radioactive isomeric spin-labelled lipids were incubated with unlabelled mitochondria, reisolated mitochondrial membranes contained translocated radioactive isomeric spin-labelled lipids. Temperature-induced changes in these membranes showed no discontinuity with either isomeric doxyl stearic acid derivative, establishing a difference in the environment of translocated lipids in the membrane donor compared with that in the membrane acceptor. Microsomal membranes recovered from translocation experiments showed the same behaviour as the original membranes and exhibited the same discontinuity at 10--12 degree C, establishing that the translocation incubation itself did not alter the spin-labelled lipid interaction within these membranes. Studies of the loss of paramagnetism of spin-labelled lipids in microsomal membranes before and in mitochondrial membranes after their translocation showed a significant difference and suggested that both the outer and the inner mitochondrial membranes might have been involved.

Animals↗
PubMed1977

Spin-labeled acyl atractyloside as a probe of the mitochondrial adenosine diphosphate carrier. Asymmetry of the carrier and direct lipid environment.

A number of spin-labeled acyl derivatives of atractyloside, (m,n)acyl-ATR (general formula: CH3- (CH2)mCX(CH2)nCOO-ATR, where X is an o-azolidine ring containing a nitroxide), have been synthesized. As shown by electron spin resonance (ESR) spectra of spin-labeled acyl-ATR, the nitroxide placed on the acyl chain interacts with the diterpene residue of the atractyloside moiety when incorporated in liposomes. Spin-labeled acyl-ATRs were used to probe the ADP carrier in heart mitochondria. They inhibit ADP transport with the same efficiency as unlabeled acyl-ATRs. The inhibition is a mixed competitive and noncompetitive inhibition. The inhibitor constant is close to 10(-7) M. The long chain acyl-ATRs (10,3)- (7,6)-, (7,8)-, and (5,10)acyl-ATRs) and also the short chain (0,2)acyl-ATR, when added at low concentrations to heart mitochondria, give rise to more immobilized ESR spectra than when added to liposomes. Immobilization is stronger for the first three molecules of the series. The (1,14)acyl-ATR, which possesses a nitroxide almost at the end of the acyl chain near the terminal methyl, gives rise to a spectrum corresponding to a high degree of fluidity. Upon addition of atractyloside or of other specific ligands, spin-labeled long-chain acyl-ATRs bound to the ADP carrier are displaced from their binding site toward the lipid phase of the mitochondrial membrane and the short chain (0,2)acyl-ATR is released into the aqueous phase. Spin-labeled long-chain acyl-ATRs do not show any evidence of binding to a protein when incubated with "inside out" submitochondrial particles, in spite of the fact that these particles are able to transport ADP. These results are discussed with respect to the size and the asymmetry of the ADP carrier in the mitochondrial membrane and the mechanism of ADP transport.

Adenosine Diphosphate↗
PubMed1984

Selective spin labeling of terminal galactose and N-acetylgalactosamine residues on the membrane surface of erythrocytes.

A method to selectively spin label galactose and N-acetylgalactosamine residues of erythrocyte membrane glycoconjugates is described. The method is based on the activation of the C-6 CH2OH group of these two sugars by galactose oxidase followed by reductive amination with 2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl in the presence of a mild reducing agent, NaBH3CN. The extent and distribution of the spin labeling suggest that the major sialoglycoprotein, PAS-1, incorporates the greatest amount of spin label while the glycolipids incorporate less than 10% of the spin label.

Acetylgalactosamine↗
PubMedDate not supplied

Nonenzymatic bioreduction in rat liver and kidney of nitroxyl spin labels, potential contrast agents in magnetic resonance imaging.

Paramagnetic nitroxyl spin labels have potential clinical utility as contrast agents in proton magnetic resonance imaging. Reduction of the nitroxyl moiety in vivo results in the formation of the diamagnetic hydroxylamine, which lacks contrast-enhancing activity. Bioreduction is therefore an important determinant of the imaging behavior of these agents. Both enzymatic and nonenzymatic reduction mechanisms have been suggested for nitroxyl spin labels. This study examines the nonenzymatic mechanisms in rat liver and kidney, mammalian tissues that demonstrate high reducing activity. Protein-free preparations, obtained by heat precipitation or ultrafiltration of rat liver and kidney homogenates, were used to test piperidine and pyrrolidine nitroxyl spin-label derivatives, for which imaging properties and bioreduction had previously been examined. For the piperidine derivative, the initial reduction rates in ultrafiltrates and supernatant fluids were 25-60% of those in whole liver and kidney homogenates. However, the pyrrolidine derivative was reduced at rates much slower than those in whole tissue homogenates. The reduction in whole tissue homogenates was NADPH-dependent, while reduction in ultrafiltrates was unaffected by the addition of NADPH. Preincubation of the ultrafiltrates and supernatant fluids with ascorbic acid oxidase caused almost complete inhibition of the reduction. The reduction rates of these nitroxyl derivatives were determined in ascorbic acid solution; second order rate constants were 0.45 +/- 0.04 and 0.0042 +/- 0.001 mM-1 min-1 for the piperidine and pyrrolidine derivatives, respectively. The concentrations of ascorbic acid in the supernatant fluids and ultrafiltrates of rat liver and kidney were then predicted from the observed reduction rates and found to be virtually identical with those from spectrophotometric determinations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Quantitative basal CBF and CBF fMRI of rhesus monkeys using three-coil continuous arterial spin labeling.

A three-coil continuous arterial-spin-labeling technique with a separate neck labeling coil was implemented on a Siemens 3T Trio for quantitative cerebral blood flow (CBF) and CBF fMRI measurements in non-human primates (rhesus monkeys). The optimal labeling power was 2 W, labeling efficiency was 92+/-2%, and optimal post-labeling delay was 0.8 s. Gray matter (GM) and white matter (WM) were segmented based on T1 maps. Quantitative CBF were obtained in 3 min with 1.5-mm isotropic resolution. Whole-brain average DeltaS/S was 1.0-1.5%. GM CBF was 104+/-3 ml/100 g/min (n = 6, SD) and WM CBF was 45+/-6 ml/100 g/min in isoflurane-anesthetized rhesus monkeys, with the CBF GM/WM ratio of 2.3+/-0.2. Combined CBF and BOLD (blood-oxygenation-level-dependent) fMRI associated with hypercapnia and hyperoxia were made with 8-s temporal resolution. CBF fMRI responses to 5% CO2 were 59+/-10% (GM) and 37+/-4% (WM); BOLD fMRI responses were 2.0+/-0.4% (GM) and 1.2+/-0.4% (WM). CBF fMRI responses to 100% O2 were -9.4+/-2% (GM) and -3.9+/-2.6% (WM); BOLD responses were 2.4+/-0.7% (GM) and 0.8+/-0.2% (WM). The use of a separate neck coil for spin labeling significantly increased CBF signal-to-noise ratio and the use of small receive-only surface coil significantly increased signal-to-noise ratio and spatial resolution. This study sets the stage for quantitative perfusion imaging and CBF fMRI for neurological diseases in anesthetized and awake monkeys.

Animals↗
PubMed1996

Site-directed spin labeling and chemical crosslinking demonstrate that helix V is close to helices VII and VIII in the lactose permease of Escherichia coli.

Site-directed chemical cleavage of lactose permease indicates that helix V is in close proximity to helices VII and VIII. To test this conclusion further, permease containing a biotin-acceptor domain and paired Cys residues at positions 148 (helix V) and 228 (helix VII), 148 and 226 (helix VII), or 148 and 275 (helix VIII) was affinity purified and labeled with a sulfhydryl-specific nitroxide spin label. Spin-spin interactions are observed with the 148/228 and 148/275 pairs, indicating close proximity between appropriate faces of helix V and helices VII and VIII. Little or no interaction is evident with the 148/226 pair, in all likelihood because position 226 is on the opposite face of helix VII from position 228. Broadening of the electron paramagnetic resonance spectra in the frozen state was used to estimate distance between the 148/228 and the 148/275 pairs. The nitroxides at positions 148 and 228 or 148 and 275 are within approximately 13-15 A. Finally, Cys residues at positions 148 and 228 are crosslinked by dibromobimane, a bifunctional crosslinker that is approximately 5 A. long, while no crosslinking is detected between Cys residues at positions 148 and 275 or 148 and 226. The results provide strong support for a structure in which helix V is in close proximity to both helices VII and VIII and is oriented in such a fashion that Cys-148 is closer to helix VII.

Amino Acid Sequence↗
PubMed1990

Residual motion of hemoglobin-bound spin labels and protein dynamics: viscosity dependence of the rotational correlation times.

The residual motion of spin labels bound to cysteine beta 93 and to lysines of methemoglobin has been studied by electron paramagnetic resonance spectroscopy. To separate the influences of the solvent and the protein environment of the label fluctuations, the correlation times, tau, were analyzed as a function of temperature for fixed solvent viscosities, eta. Results show that over a wide range of viscosity the dependence of tau on eta may be empirically described by a power law tau approximately eta kappa. The exponent kappa depends strongly on the location of the label on the protein surface. If one regards the spin labels as artificial amino acid side chains, characteristic values of correlation times and amplitudes of the rotational motion at the surface can be given. For eta = 1 cP and T = 297 K the correlation time of the labels bound to lysines is found to be tau = 9.10(-10) S and the rotational diffusion is nearly isotropic. The spin label bound to cysteine beta 93 occupies a protein pocket, its rotational motion is therefore restricted. The correlation time of the label motion within a limited motion cone of semi angle theta = 30 degrees +/- 3 degrees is found to be tau = 1.3.10(-9) S for eta = 1 cP and T = 297 K.

Animals↗
PubMed1995

Spin-labeled psoralen probes for the study of DNA dynamics.

Six nitroxide spin-labeled psoralen derivative have been synthesized and evaluated as probes for structural and dynamic studies. Sequence specific photoaddition of these derivatives to DNA oligonucleotides resulted in site-specifically cross-linked and spin-labeled oligomers. Comparison of the general line shape features of the observed electron paramagnetic resonance (EPR) spectra of several duplexes ranging in size from 8 to 46 base pairs with simulated EPR spectra indicate that the nitroxide spin-label probe reports the global tumbling motion of the oligomers. While there is no apparent large amplitude motion of the psoralen other than the overall tumbling of the DNA on the time scales investigated, there are some indications of bending and other residual motions. The (A)BC excinuclease DNA repair system detects structural or dynamic features of the DNA that distinguish between damaged and undamaged DNA and are independent of the intrinsic structure of the lesion. NMR studies have shown that psoralen-cross-linked DNA has altered backbone dynamics and conformational populations in the immediate vicinity of the adduct [Emsley et al. (1993) J. Am. Chem. Soc. 115, 7765-7771; Spielmann et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 2345-2349]. We suggested that the signal for recognition of a lesion to be repaired is in the sugar--phosphate backbone and not in the damaged base(s).

Base Sequence↗
PubMed2005

Multiquantum EPR spectroscopy of spin-labeled arrestin K267C at 35 GHz.

Three- and five-quantum absorption and dispersion multiquantum electron paramagnetic resonance spectra of a spin-labeled protein have been obtained for the first time at Q-band (35 GHz). Spectra of arrestin spin-labeled at site 267 were recorded at room temperature as a function of microwave power. The separation of irradiating microwave frequencies, Deltaf, was 10 kHz, and a newly-designed multiquantum Q-band electron paramagnetic resonance bridge was utilized, operating in a superheterodyne detection mode. The sample volume was 30 nL using a 3-loop-2-gap resonator. Most spectra were obtained at a 300 microM concentration in single, 2-min scans, but spectra were also successfully obtained at 30 microM, corresponding to one picomole of protein. Enhanced sensitivity to T(1) and T(2) was evident in the spectra, and linewidths varied considerably across the spectra. The pure absorption displays are beneficial relative to field modulation methods for spectral characterization. The presence of two states of the nitroxide spin-label with different relaxation times is evident, particularly in the dispersion spectra, which are expected to exhibit enhanced sensitivity to lineshape variation relative to absorption. Feasibility has been established for the use of this technique for site-directed spin-labeling studies of biologically relevant samples, particularly the study of protein structure and dynamics.

Absorption↗
PubMed1987

Positioning of a spin-labeled substrate analogue into the structure of delta 5-3-ketosteroid isomerase by combined kinetic, magnetic resonance, and X-ray diffraction methods.

We have shown by kinetic and magnetic resonance measurements that a spin-labeled substrate analogue, spiro[doxyl-2,3'-5' alpha-androstan]-17'beta-ol, binds at the substrate site of crystalline delta 5-3-ketosteroid isomerase (steroid delta-isomerase; EC 5.3.3.1) of Pseudomonas testosteroni. The spin-labeled steroid is a linear competitive inhibitor with a Ki value (25 +/- 5 microM) that is consistent with dissociation constants obtained by direct binding measurements based on changes in the electron paramagnetic resonance spectrum of the nitroxide, longitudinal relaxation rates of water protons, and longitudinal and transverse relaxation rates of carbon-bound protons of the isomerase. These binding studies yield a stoichiometry for the nitroxide of 1 per subunit of the enzyme. Measurements of the longitudinal relaxation rates of water protons indicate that the 3-doxyl portion of the spin-label is highly immobilized yet is exposed to solvent. Paramagnetic effects of the nitroxide on T1 defined distances to several previously assigned [Benisek, W. F., & Ogez, J. R. (1982) Biochemistry 21, 5816-5825] and newly assigned protons of the enzyme. These distances were then used to locate (with an accuracy of +/- 2 A) the nitroxide moiety at a unique position in a partially refined 2.5-A resolution X-ray structure of native isomerase. Three of five additional proton resonance peaks, attributed to ring-shielded methyl groups, could be assigned to specific residues on the basis of distances from the spin-label in the X-ray structure. The remaining portion of the spin-labeled steroid was then docked into the X-ray structure in a hydrophobic cavity of the enzyme. This position of the steroid is consistent with the steroid binding site previously proposed [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103]. However, the rotational orientation of this steroid about its long axis could not be unambiguously established. If we assume that steroid substrates and the spin-labeled inhibitor bind to the same site, but with reversal of the 3- and 17-positions, then the phenolic hydroxyl of Tyr-55 is optimally positioned to function as the general acid that protonates the 3-keto group of the substrate, facilitated by the negative end of the dipole of a 10-residue alpha-helix, the only helix in the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed2002

Structure of the inhibitory region of troponin by site directed spin labeling electron paramagnetic resonance.

Site-directed spin labeling EPR (SDSL-EPR) was used to determine the structure of the inhibitory region of TnI in the intact cardiac troponin ternary complex. Maeda and collaborators have modeled the inhibitory region of TnI (skeletal 96-112: the structural motif that communicates the Ca(2+) signal to actin) as a kinked alpha-helix [Vassylyev, D., Takeda, S., Wakatsuki, S., Maeda, K. & Maeda, Y. (1998) Proc. Natl. Acad. Sci. USA 95, 4847-4852), whereas Trewhella and collaborators have proposed the same region to be a flexible beta-hairpin [Tung, C. S., Wall, M. E., Gallagher, S. C. & Trewhella, J. (2000) Protein Sci. 9, 1312-1326]. To distinguish between the two models, residues 129-145 of cardiac TnI were mutated sequentially to cysteines and labeled with the extrinsic spin probe, MTSSL. Sequence-dependent solvent accessibility was measured as a change in power saturation of the spin probe in the presence of the relaxation agent. In the ternary complex, the 129-137 region followed a pattern characteristic of a regular 3.6 residues/turn alpha-helix. The following region, residues 138-145, showed no regular pattern in solvent accessibility. Measurements of 4 intradomain distances within the inhibitory sequence, using dipolar EPR, were consistent with an alpha-helical structure. The difference in side-chain mobility between the ternary (C.I.T) and binary (C.I) complexes revealed a region of interaction of TnT located at the N-terminal end of the inhibitory sequence, residues 130-135. The above findings for the troponin complex in solution do not support either of the computational models of the binary complex; however, they are in very good agreement with a preliminary report of the x-ray structure of the cardiac ternary complex [Takeda, S. Yamashita, A., Maeda, K. & Maeda, Y. (2002) Biophys. J. 82, 832].

Animals↗
PubMed1984

Electron spin resonance spin label studies of plasma fibronectin: effect of temperature.

Plasma fibronectin was chemically modified by 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl (maleimide spin label). Only the free sulfhydryl groups of plasma fibronectin were modified by the label under the experimental conditions. The ESR spectrum of spin-labeled fibronectin showed that the sites of labeling were highly immobilized, suggesting that the sulfhydryl groups of the protein are in small, confined environments. The conversion of the strongly immobilized ESR spectrum into a weakly immobilized one was observed when the spin-labeled protein was heated from 30 to 60 degrees C, indicating the thermal unfolding of the protein molecules. The midpoint temperature for the thermal unfolding of plasma fibronectin is about 50 degrees C. The results suggest that plasma fibronectin is stable to about 40 degrees C and starts unfolding above this temperature. The rotational correlation time estimated from the ESR spectrum of spin-labeled fibronectin at 21 degrees C was about 2.0 X 10(-8) s. The rotational correlation time calculated from the Stokes-Einstein equation, assuming a rigid globular configuration for fibronectin with a Stokes radius of 10 nm, was about 7.8 X 10(-7) s. The differences in rotational correlation time by a factor of 39 between experimental and calculated values do not support a globular configuration for plasma fibronectin.

Binding Sites↗
PubMed1981

Effect of phospholipid substitution on the mobility of spin labels bound to the ATPase of sarcoplasmic reticulum.

The mobility of spin labels covalently bound to the Ca2+-transport ATPase (ATP phosphohydrolase [EC 3.y.1.3]) was studied by electron spin-resonance spectroscopy in purified ATPase and reconstituted vesicles. The purified ATPase of sarcoplasmic reticulum of rabbit skeletal muscle was covalently labeled with maleimide spin-labels of different chain length and the phospholipids were exchanged for dipalmitoylphosphatidylcholine. The spectrum of the short-chain maleimide spin-label, bound to purified ATPase indicates reduced mobility after substitution of endogenous phospholipids with dipalmitoylphosphatidylcholine. With the long-chain maleimide derivative no difference was detected in the spectra, measured at 20-35 degrees C temperature before and after substitution with dipalmitoylphosphatidylcholine. Below 10 degrees C temperature the substitution with dipalmitoylphosphatidylcholine decreased the mobility of the prove, indicating that the microviscosity of environment in the vicinity of nitroxide groups was influenced by changes in the fatty acid composition. With both short and long chain spin-labels bound to purified ATPase adn sarcoplasmic reticulum vesicles the amplitude of weakly immobilized component sharply decreased in media containing 20-50% glycerol. Therefore, the mobility of covalently bound nitroxide group in short or long chain maleimide derivatives is also sensitive to the viscosity of the water phase.

Animals↗
PubMed1975

Initial membrane reaction in the biosynthesis of peptidoglycan. Spin-labeled intermediates as receptors for vancomycin and ristocetin.

Phospho-N-acetylmuramyl-pentapeptide translocase (UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla:undecaprenyl phosphate, phospho-MurNAc-pentapeptide transferase) catalyzes the initial membrane reaction in the biosynthesis of peptidoglycan. The spin-labeled nucleotide, UDP-MurNAc-Ala-DGlu-Lys (Nepsilon-2,2,5,5-tetramethyl-N-oxyl-pyrroline-3-carbonyl)-DAla-DAla, was used as a substrate by this enzyme for the synthesis of membrane-associated undecaprenyl-diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Tempyo)-DAla-DAla. The spin-labeled substrate and product complex with the antibiotics vancomycin and ristocetin. The association constants for the spin-labeled nucleotide are 6.2 times 10(5) and 6.2 times 10(4) M-1 for vancomycin and ristocetin, respectively. The association constants for the spin-labeled lipid intermediate are 3.0 times 10(4) and 2.1 times 10(4) M-1 for vancomycin and ristocetin, respectively. These results indicate that the acyl-DAla termini of membranes-associated spin-labeled undecaprenyl-diphosphate-MurNAc-pentapeptide are accessible to vancomycin and ristocetin and that the association constants are smaller than those determined for the corresponding antibiotic spin-labeled UDP-MurNAc-pentapeptide complexes.

Binding Sites↗
PubMed1991

Binding of spin-labeled fatty acids and lysophospholipids to hydrophobic region of calmodulin.

In the presence of bovine brain calmodulin activated by calcium, the sharp triplet electron spin resonance (ESR) lines of free doxyl stearic acids decreased, and the broad resonance lines increased concomitantly, suggesting that the doxyl stearic acids bound to calmodulin calcium-dependently. The bound molecules were displaced by a calmodulin inhibitor, W-7, whereas their nitroxide radicals were hardly reduced by ascorbic acid, suggesting that the spin-labeled fatty acids bind to hydrophobic regions of calmodulin, and consequently inhibit calmodulin-dependent phosphodiesterase activity. These binding characteristics to calmodulin were different from those to bovine serum albumin. Moreover, the ESR spectra of two spin-labeled derivatives of lysophospholipid having a spin-labeled acyl group or a spin-labeled polar head group showed that it is the acyl chain of lysophospholipid that interacts with the hydrophobic region of calmodulin. The interactions of fatty acids and lysophospholipids with calmodulin seem to be quite different from those of acidic phospholipids, described previously [Suzuki, T., Katoh, H., & Uchida, M.K. (1986) Biochim. Biophys. Acta, 873, 379-386]. Thus, from the results of ESR study, we can obtain information on the function of fatty acids and lysophospholipids on calmodulin. Instead of enzyme assay, ESR spectroscopy is a useful means to examine lipid-protein interaction.

Animals↗