Spin-label studies on rat liver and heart plasma membranes: Do probe-probe interactions interfere with the measurement of membrane properties?

Sauerheber, Richard; Gordon, Larry M.; Crosland, Richard D.; Kuwahara, Melvin D.

doi:10.1007/bf01869402

Cited by 82 publications

(52 citation statements)

References 72 publications

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“…DISCUSSION Spin labels report on the molecular order, motion, and thermal responses of membrane lipids, but the spectral parameters used to determine these properties are influenced by the type of label and its location in the bilayer (9,12). Furthermore, the label should be spectrally dilute such that spin-spin interactions do not occur (19). The spectral parameters used to determine both motion and order in the wheat polar lipids were not altered by a 5-fold increase in the ratio of label to lipid, indicating that preferential partitioning of the label into discrete domains did not occur.…”

Section: Resultsmentioning

confidence: 99%

Maintenance of Membrane Fluidity during Development of Freezing Tolerance of Winter Wheat Seedlings

Pomeroy¹,

Raison²

1981

Plant Physiol.

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Fluidity of membrane lipids of shoot and root tissue and of chloroplasts from young wheat seedlings of contrasting freezing tolerance was investigated by measuring the motion and order parameters after spin labeling. A striking similarity was observed in membrane lipid fluidity of the five cultivars grown at 22 C. After cold hardening by growth at 2 C, a small change in membrane lipid fluidity was observed, but this was not correlated with the development of freezing tolerance, and there was no alteration in the transition temperature of membrane lipids. The results show that neither changes in membrane lipid fluidity nor transition temperature are a necessary feature of cold acclimation in wheat.Changes in both membrane lipid composition and fluidity have been observed in some plants during acclimation to variations in growth temperature (14,17). These changes are thought to be a response ofthe cells to maintain optimal conditions for membraneassociated enzyme function (18). With respect to fatty acid content, growth at low temperature generally favors an increase in the proportion of unsaturated fatty acids (4,14). Under (Triticum aestivum L.) and Falcon spring wheat were used in this investigation. Seedlings were grown in sand, either in dark for 3 days at 22 C or for 2 days at 22 C followed by 4 weeks at 2 C, or in light (12-h day at 400 uE m-2 s-') for 1 week at 22 C or for 5 days at 22 C followed by 6 weeks at 2 C (12-h day at 145 ,uE m-2 s-'). These growth conditions yielded cold-acclimated and nonacclimated seedlings with shoots and leaves approximately 1.5 and 10 cm for dark and light growth conditions, respectively. Cold hardiness of seedlings was determined as the temperature (LD5o) at which 501% of seedlings did not survive exposure for 1 h (1).A total polar lipid fraction was obtained from the plant tissue by a modification of the method of Fishwick and Wright (5). Shoot, leaf, or root tissue was harvested, cut into 1-cm pieces, and added directly to boiling isopropanol (100 ml/10 g fresh weight) containing 1 mg BHT/100 ml isopropanol. Boiling was continued for 10 min and, after cooling, the tissue was homogenized. The homogenate was fitered and the residue homogenized in chloroform:methanol, 2:1 (v/v) (80 ml/10 g fresh weight), filtered, and the residue washed twice with the same solvent. The filtrates were combined, evaporated to dryness, and the dried, bulk lipid dissolved in chloroform:methanol, 2:1 (v/v) (200 ml/10 g fresh weight), washed with water and then with 0.73% NaCl. The polar lipids were then separated by chromatography on silica gel columns (15

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Section: Resultsmentioning

confidence: 99%

Maintenance of Membrane Fluidity during Development of Freezing Tolerance of Winter Wheat Seedlings

Pomeroy¹,

Raison²

1981

Plant Physiol.

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“…The 7 R (= 28.6 x 10"os) in Table 3 confirms that SP-BT-,, is only "weakly immobilized" when bound to SDS. At the peptidellipid ratio used here, Figure 7A indicates minimal aggregation for the spin-labeled peptide in SDS micelles (Sauerheber et al, 1977;Curtain et al, 1987), particularly when compared with the aggregated spectrum of SP-B;-,, in PBS (Fig. 6B).…”

Section: Lm Gordon Et Almentioning

confidence: 86%

“…The first component is spectral broadened, indicating that SP-BT-,, here resides in an environment that is motionally restricted. Besides restricted flexibility, this broadened spectral component may be partially a consequence of nitroxide radical interactions (Le., dipolar broadening and spin-spin exchange) between aggregated SP-BY-,, molecules (Sauerheber et al, 1977;Curtain et al, 1987). The second component shows sharp lines (see arrows in Fig.…”

Section: Lm Gordon Et Almentioning

confidence: 91%

“…Increases in uh indicate a more polar environment for the probe. If experimentally determined low probe concentrations are used (Sauerheber et al, 1977), S is sensitive to the flexibility of the incorporated probe. These order parameters may assume values between 0 and 1, with the extremes indicating that the probe samples fluid and motionally restricted environments, respectively.…”

Section: Evaluation Of the Flexibility And Polarity Of Lipid-incorpormentioning

confidence: 99%

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Conformation and molecular topography of the N‐terminal segment of surfactant protein B in structure‐promoting environments

et al. 1996

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Although the effects of surfactant protein B (SP-B) on lipid surface activity in vitro and in vivo are well known, the relationship between molecular structure and function is still not fully understood. To further characterize protein structure-activity correlations, we have used physical techniques to study conformation, orientation, and molecular topography of N-terminal SP-B peptides in lipids and structure-promoting environments. Fourier transform infrared (FTIR) and CD measurements of SP-B1_25 (residues 1-25) in methanol, SDS micelles, egg yolk lecithin (EYL) liposomes, and surfactant lipids indicate the peptide has a dominant helical content, with minor turn and disordered components. Polarized FTIR studies of SP-B,-25 indicate the long molecular axis lies at an oblique angle to the surface of lipid films. Truncated peptides were similarly examined to assign more accurately the discrete conformations within the SP-B,_,, sequence. Residues Cys-8-Gly-25 are largely a-helix in methanol, whereas the N-terminal segment Phe-1-Cys-8 had turn and helical propensities. Addition of SP-Bl-25 spin-labeled at the N-terminal Phe (i.e., SP-BT_25) to SDS, EYL, or surfactant lipids yielded electron spin resonance spectra that reflect peptide bound to lipids, but retaining considerable mobility. The absence of characteristic radical broadening indicates that SP-B1-25 is minimally aggregated when it interacts with these lipids. Further, the high polarity of SP-BI-25 argues that the reporter on Phe-1 resides in the headgroup of the lipid dispersions. The blue-shift in the endogenous fluorescence of Trp-9 near the N-terminus of SP-B1-25 suggests that this residue also lies near the lipid headgroup. A summary model based on the above physical experiments is presented for SP-BI-2S interacting with lipids.

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“…Unless otherwise stated, spectra were run at 21 + 1 'C. Lineshape was analyzed in terms of the maximum hyperfine splitting parameter, 2T1 (23), or the rotational correlation time, T, (12). For studies on membrane phase transformation temperatures, spectra were run at 1 to 2°C intervals, and the data were analyzed by linear regression.…”

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confidence: 99%

Changes in Tobacco Cell Membrane Composition and Structure Caused by Cercosporin

Daub

Briggs²

1983

Plant Physiol.

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Cercosporin, a toxin produced by Cercospora species, rapidly kills plant cells in the Light. Previous work has shown that cercosporin treatment causes products of lipid peroxidation to be released. We have found that the unsaturated acyl chains of lipids in tobacco (Niotiana tabacum) cell membranes are destroyed when cells are treated with cercosporin. Concomitant with this change in composition is a change in structure of the membranes as detected by two different fatty acid spin labels, 2-(3-carboxypropyl)4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl (denoted 1112,31) and 2-(1'-carboxytetradecyl)-2-ethyl-4,4-dmethyl-3-oxazolidinyloxyl (denoted 111,141). Cercosporin causes the membranes to become more rigid at all temperatures tested and increases the membrane phase transformation temperature from 12.70C to 20.80C.Cercosporin, a toxic compound from Cercospora species, affects both hosts and non-hosts of the producing fungi. Cercosporin was first isolated in 1957 by Kuyama and Tamura (14) from C. kikuchii, a soybean pathogen, and has since been isolated from a large number of Cercospora species (1,8,16) and from Cercosporainfected plants (8,14). The structure of cercosporin was determined independently by Lousberg et al. (15) and Yamazaki and Ogawa (25).Cercosporin is a photosensitizing agent capable of generating both singlet oxygen and superoxide during irradiation (M. Daub and R. Hangarter, unpublished). Plant cells, bacteria, and mice are killed rapidly by cercosporin in the presence of light (5,26). Cercosporin causes electrolytes to leak from plant tissues (6, 17), destroys plant protoplasts (6), and causes peroxidation of plant membrane lipids both in vitro (3) and in vivo (6). These data suggest that cercosporin acts directly on membranes in situ.We report here that cercosporin causes a large increase in the ratio of saturated to unsaturated fatty acids extracted from membranes of toxin-treated cells. Concomitant with this change is a decreased membrane fluidity and the possible appearance of a phase separation in the membranes at the growth temperature of the cells. Electrolyte leakage and cell death may be accounted for by these perturbations of membrane composition and structure. MATERIALS AND METHODS Cercosporin. Cercosporin was isolated and purified from cultures of Cercospora nicotianae as previously described (5). Stock solutions were prepared in acetone and stored at -20°C in the dark. Control cultures were either treated with the same concentration of acetone or treated with cercosporin but incubated in the dark. Final acetone concentrations in both treated and control cultures did not exceed 1% (v/v).Host Material. All experiments were conducted with Nicotiana tabacum cv. 'Wisconsin 38' cell culture line NT575. Liquid suspension cultures were maintained as previously described (5).Protoplast Isolation. Protoplasts were isolated from NT575 suspension cultures 2 d after subculture (early log phase). The cells were preplasmolyzed in 0.6 M mannitol for 2 h followed by centrifugation. Packed ...

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Spin-label studies on rat liver and heart plasma membranes: Do probe-probe interactions interfere with the measurement of membrane properties?

Cited by 82 publications

References 72 publications

Maintenance of Membrane Fluidity during Development of Freezing Tolerance of Winter Wheat Seedlings

Maintenance of Membrane Fluidity during Development of Freezing Tolerance of Winter Wheat Seedlings

Conformation and molecular topography of the N‐terminal segment of surfactant protein B in structure‐promoting environments

Changes in Tobacco Cell Membrane Composition and Structure Caused by Cercosporin

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