On the use of the spin labeling technique in the study of erythrocyte membranes

Butterfield, D. Allan; Whisnant, Carol C.; Chesnut, D. B.

doi:10.1016/0005-2736(76)90134-6

Cited by 35 publications

(22 citation statements)

References 6 publications

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“…Probe titration experiments conducted on intact human platelets indicated that S and S(TD decreased with increasing probe concentration at sufficiently high probe/lipid ratios, while S(~I) remained constant. Similar results were obtained in previous I(12,3) probe studies performed on rat liver and heart plasma membrane (Gordon et al, 1978;Sauerheber et al, 1977) and human erythrocytes 1 (Butterfield, Whisnant & Chesnut, 1976). We earlier proposed two distinct physical models to account for the spectral alterations and order parameter effects seen at high probe concentrations (Gordon et al, 1978) : (i) The uniform distribution model in which the probe is viewed as being homogeneously dispersed throughout the accessible lipid domains for the entire probe concentration range; and (ii) thepatch model in which the membrane contains only a limited number of binding sites in the lipid matrix that can be occupied by the I(12,3) probe.…”

Section: The Interaction Of Ca 2 + and La 3 + With I(123)-labeled Husupporting

confidence: 91%

Effects of calcium, lanthanum, and temperature on the fluidity of spin-labeled human platelets

et al. 1980

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Previous platelet studies have shown that calcium plays important roles in stimulus-secretion coupling, aggregation, and other membrane-associated functions. In addition, lanthanum induces platelet aggregation and the platelet release reaction and also influences platelet responsiveness to various stimuli. The spin-label results presented here suggest that one mechanism through which calcium and lanthanum mediate their effects on platelet functions may be by decreasing the lipid fluidity of the surface membrane. The structure of platelet membrane lipids was examined with the spin-label method. Washed human platelets were labeled with the 5-, 12- and 16-nitroxide stearic acid spin probes. Order parameters which measure the fluidity of the lipid environment of the incorporated probe may be calculated from the electron spin resonance (ESR) spectra of 5-nitroxide stearate [I(12,3)]-labeled cells. Evidence is presented which indicates that these spectra principally reflect properties of the platelet surface membrane lipids. The membrane fluidity increased with temperature for the range 17 to 37 degrees C. Either calcium or lanthanum additions to intact cells increased the rigidity of the platelet membranes at 37 degrees C, although the La3+ effect was larger and occurred at lower concentrations than that of Ca2+. For example, addition of 1 mM La3+ or 4 mM Ca2+ increased the order parameter of I(12,3)-labeled platelets by 4.3 +/- 1.7% or 2.1 +/- 0.5%. Preliminary studies conducted on purified platelet plasma membranes labeled with I(12,3) indicated that 1 mM LaCl3 or 4 mM CaCl2 additions similarly decreased the lipid fluidity at 37 degrees C. The above cation-induced effects on the fluidity of whole platelets were reversed by the use of the divalent cation-chelating agent ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetra-acetic acid (EGTA). Lastly, lanthanum (0.2-1 mM) caused rapid aggregation of platelets which were suspended in a 50-mM Tris buffer pH 7.4 that did not contain adenosine.

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Section: The Interaction Of Ca 2 + and La 3 + With I(123)-labeled Husupporting

confidence: 91%

Effects of calcium, lanthanum, and temperature on the fluidity of spin-labeled human platelets

et al. 1980

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“…At these high membrane concentrations, no 'liquid-lines' due to 1(12,3) tumbling rapidly in aqueous solution were present. Similar spectral alterations have been reported with l(12,3)-Iabeled whole erythrocytes [5] or erythrocyte ghosts [16,39].…”

Section: Effects Of I(123) Concentration On Spectra Of Human Erythrosupporting

confidence: 86%

“…An important finding centers on our observation that significant I(12,3) clustering occurs at much lower P/L (e.g., 1/2250 to 1/359) than has been previously reported [5,16]. This is a radical departure from the conventional picture of 'magnetically dilute' I(12,3) located in a homogeneous fluid phase for P/L <1/100.…”

Section: Discussionsupporting

confidence: 50%

“…This is a radical departure from the conventional picture of 'magnetically dilute' I(12,3) located in a homogeneous fluid phase for P/L <1/100. One explanation for the discrepancy is the incorrect assumption of Butterfield et al [5] and Gordon and Mobley [16] that erythrocyte membranes quantitatively incorporated I(12,3) over the entire P/L range. Figure 2 indicates that such an assumption leads to a serious overestimation of the probe concentration in the membrane.…”

Section: Discussionmentioning

confidence: 99%

“…[7]). At sufficiently high loading, probe concentration-dependent effects characteristically perturb the order parameters and polarity of the 5-nitroxide stearate spin probe, I (12,3), in rat heart plasma membranes [20,33], rat liver plasma membranes [16,18,28,33], human lymphocytes [8], rat mast cells It 1], rat fat-cell ghosts and plasma membranes [1,34], human platelet plasma membranes [18,35], erythrocytes [4,5,16], microsomal membranes ~Abbreviations used: ESR, electron spin resonance; I (12,3), the N-oxyl-4',4'-dimethyloxazolidine derivative of 5'-ketostearic acid; P/L, ratio of probe molecules to total lipid molecules; dil, 3,3-dioctadecylindocarbocyanine; NBD-PE, N-(7-nitro-2, 1,3-benzoxadiazol-4-yl )phosphatidylet hanolamine; NBD-Chol, N ~-cholesterylcarbamoyI-N~-(7-nitro-2,1,3-benzoxadiazol-4-yl)-3,6-dioxaoctane-l,8-diamine; Lm lateral diffusion coefficient (cm-'/sec).…”

Section: Introductionmentioning

confidence: 99%

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Spin probe clustering in human erythrocyte ghosts

1985

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A model has been developed for 5-nitroxide stearate, I(12,3), distribution in human erythrocyte ghosts which accurately predicts ESR spectral alterations observed with increased probe/total lipid (P/L) at 37 degrees C. This spin probe occupies a class of high-affinity, noninteracting sites at low loading. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membrane-bound clusters of variable size. No 'low' probe remains at high P/L where all I(12,3) clusters in a 'concentrated' phase. This model allows determination of the dilute/clustered probe ratio, and shows that I(12,3) segregates in erythrocytes at what might otherwise be considered low P/L (e.g., 1/359). These findings validate the earlier use of empirical parameters to estimate probe sequestration in biological membranes.

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Electron spin resonance spin label studies of the erythrocyte membrane in Duchenne muscular dystrophy

Nash

Patel

et al. 1983

Am. J. Med. Genet.

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The membrane organisation of erythrocytes from patients with Duchenne muscular dystrophy (DMD) and from asymptomatic carriers was studied by the electron spin resonance (ESR) spin label technique. Following the work of Sato et al [1978], we used 2-(14-carboxytetradecyl)-2-ethyl-4, 4-dimethyl-3-oxazolidinyloxyl as probe. We found no significant difference in lipid fluidity at 30 degrees C, measured by the ratio of the line height at central field to that at high field ho/h-1, in DMD patients or carriers compared to appropriate control persons. Our conclusions for the dystrophic boys differ from those of Sato et al [1978], although our data are consistent with theirs within experimental error. We also studied the variation of the ratio ho/h-1 over the temperature range 5-35 degrees C in these individuals. For normal erythrocytes there is a discontinuity in this plot around 15 degrees C that is absent in the erythrocytes of DMD patients [in agreement with the findings of Sato et al, 1978] and also absent in DMD carriers. We suggest the slope of this logarithmic temperature plot over the range 15-35 degrees C is a more useful empirical parameter as the presence of these discontinuities is sometimes uncertain, and using this parameter, we find a clear separation between carriers and controls.

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On the use of the spin labeling technique in the study of erythrocyte membranes

Cited by 35 publications

References 6 publications

Effects of calcium, lanthanum, and temperature on the fluidity of spin-labeled human platelets

Effects of calcium, lanthanum, and temperature on the fluidity of spin-labeled human platelets

Spin probe clustering in human erythrocyte ghosts

Electron spin resonance spin label studies of the erythrocyte membrane in Duchenne muscular dystrophy

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