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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?

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Summary

The structures of purified rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe,I(12, 3). ESR spectra were recorded with a 50 gauss field sweep, and also with a new technique which “expands” the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The hyperfine splittings measured from the “expanded” spectra were significantly more precise than those obtained from the “unexpanded” spectra. Both procedures were used to study the effects of variousI(12,3) prove concentrations on the spectra of liver and heart membranes, as well as the effects of temperature and CaCl2 additions on the spectra of liver membranes, and revealed the following:

The polarity-corrected order parameters of liver (31°) and heart (22°) membranes were found to be independent of the probe concentration, if experimentally-determined lowI(12,3)/lipid ratios were employed. The absence of obvious radical-interaction broadening in the unexpanded spectra indicated that “intrinsic” membrane properties may be measured at these low probe/lipid ratios. Here, “intrinsic” properties are defined as those which are measured when probe-probe interactions are negligible, and do not refer to membrane behavior in the absence of a perturbing spin label.

At higherI(12,3)/lipid ratios, the order parameters of liver and heart membranes were found to substantially decrease with increasing probe concentration. The increase in the “apparent” fluidity of both membrane systems is attributed to enhanced radical interactions; however, an examination of these spectra (without reference to “low” probe concentration spectra) might incorrectly suggest that radical interactions were absent. For the membrane concentrations employed in these studies, the presence of “liquid-lines” (or “fluid components”) in the unexpanded ESR spectra was a convenient marker of high probe concentrations.

A thermotropic phase separation was observed in liver membranes between 19° and 28°.

Addition of CaCl2 to liver plasma membrane [labelled with “low”I(12,3) concentrations] increased the rigidity of the membrane at 31° and 37°, without inducing a segregation of the probe in the bilayer.

Previously reported data are discussed in relation to these results, and suggested minimal criteria for performing membrane spin label studies are included.

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References

  1. Aoki, K., Foster, J. 1975. Limited hydrolysis of bovine plasma albumin at neutral and alkaline pH catalyzed by associated proteinases. Biochemistry14:3566

    Article  PubMed  CAS  Google Scholar 

  2. Barratt, M.D., Laggner, P. 1974. The pH-dependence of ESR spectra from nitroxide probes in lecithin dispersions.Biochim. Biophys. Acta 363:127

    Article  PubMed  CAS  Google Scholar 

  3. Bartlett, G.R. 1959. Phosphorus assay in column chromatography.J. Biol. Chem. 234:466

    PubMed  CAS  Google Scholar 

  4. Bieri, V., Wallach, D., Lin, P. 1974. Focal erythrocyte membrane perturbations caused by nitroxide lipid analogues.Proc. Nat. Acad. Sci. USA 71:4797

    Article  PubMed  CAS  Google Scholar 

  5. Boggs, J., Hsia, J. 1973. Orientation and motion of amphiphilic spin labels in hexagonal lipid phases.Proc. Nat. Acad. Sci. USA 70:1406

    Article  PubMed  CAS  Google Scholar 

  6. Butler, K., Tattrie, N., Smith, I.C.P. 1974. The location of spin probes in two phase mixed lipid systems.Biochim. Biophys. Acta 363:351

    Article  PubMed  CAS  Google Scholar 

  7. Butterfield, D.A., Chesnut, D.B., Roses, A.D., Appel, S.H. 1974. ESR studies of erythrocytes from patients with myotonic muscular dystrophy.Proc. Nat. Acad. Sci. USA 71:909

    Article  PubMed  CAS  Google Scholar 

  8. Butterfield, D., Roses, A., Cooper, M., Appel, S.H., Chesnut, D.B. 1974. A comparative ESR study of the erythrocyte membrane in myotonic muscular dystrophy.Biochemistry 13:5078

    Article  PubMed  CAS  Google Scholar 

  9. Cannon, B., Polnaszek, C., Butler, K., Eriksson, L., Smith, I.C.P. 1975. The fluidity and organization of mitochondrial membrane lipids of the brown adipose tissue of coldadapted rats and hamsters as determined by nitroxide spin probes.Arch. Biochem. Biophys. 167:505

    Article  PubMed  CAS  Google Scholar 

  10. Chapman, D. 1975. Phase transitions and fluidity characteristics of lipids and cell membranes.Q. Rev. Biophys. 8:185

    Article  PubMed  CAS  Google Scholar 

  11. Chignell, C., Chignell, D. 1975. A spin label study of purple membranes fromHalobacterium halobium, Biochem. Biophys. Res. Commun. 62:136

    Article  PubMed  CAS  Google Scholar 

  12. Cuatrecasas, P. 1974. Membrane receptors.Annu. Rev. Biochem. 43:169

    Article  PubMed  CAS  Google Scholar 

  13. Devaux, P., McConnell, H.M. 1972. Lateral diffusion in spin-labeled phosphatidylcholine multilayers.J. Am. Chem. Soc. 94:4475

    Article  PubMed  CAS  Google Scholar 

  14. Devaux, P., Scandella, C., McConnell, H.M. 1973. Spin-spin interactions between spin-labeled phospholipids incorporated into membranes.J. Magn. Reson. 9:474

    CAS  Google Scholar 

  15. Dodd, N. 1975. PHA and lymphocyte membrane fluidity.Nature (London) 257:827

    Article  CAS  Google Scholar 

  16. Edidin, M. 1974. Rotational and translational diffusion in membranes.Annu. Rev. Biophys. Bioeng. 3:179

    Article  PubMed  CAS  Google Scholar 

  17. Ehrström, M., Eriksson, L., Israelachvili, J., Ehrenberg, A. 1973. Effects of cations and anions on spin labeled cytoplasmic membranes ofBacillus subtilis.Biochem. Biophys. Res. Commun. 55:396

    Article  PubMed  Google Scholar 

  18. Eletr, S., Inesi, S. 1972. Phase changes in lipid moieties of sarcoplasmic reticulum membranes induced by temperature and protein conformational changes.Biochim. Biophys. Acta. 290:178

    Article  PubMed  CAS  Google Scholar 

  19. Eletr, S., Zakim, D., Vessey, D. 1973. A spin-label study of the role of phospholipids in the regulation of membrane-bound microsomal enzymes.J. Molec. Biol. 78:351

    Article  PubMed  CAS  Google Scholar 

  20. Esser, A., Lanyi, J. 1973. Structure of the lipid phase in cell envelope vesicles fromHalobacterium cutirubrum.Biochemistry 12:1933

    Article  PubMed  CAS  Google Scholar 

  21. Esser, A., Souza, K. 1974. Correlation between thermal death and membrane fluidity inBacillus stearothermophilus.Proc. Nat. Acad. Sci. USA 71:4111

    Article  PubMed  CAS  Google Scholar 

  22. Evans, W.H. 1970. Fractionation of liver plasma membranes prepared by zonal centrifugation.Biochem. J. 166:833

    Google Scholar 

  23. Gaffney, B.J. 1974. Spin label measurements in membranes.Methods Enzymol. 32(B):161

    Article  Google Scholar 

  24. Gaffney, B.J. 1975. Fatty acid chain flexibility in the membranes of normal and transformed fibroblasts.Proc. Nat. Acad. Sci. USA 72:664

    Article  PubMed  CAS  Google Scholar 

  25. Hegner, D., Schummer, U., Schnepel, G. 1973. The effect of calcium on temperature-induced phase changes in liquid-crystalline cardiolipin structure.Biochim. Biophys. Acta 307:452

    Article  PubMed  CAS  Google Scholar 

  26. Hegner, D., Schummer, U., Schnepel, G. 1973. The interaction of a lytic peptide, melittin, with spin-labeled membranes.Biochim. Biophys. Acta 291:15

    Article  PubMed  CAS  Google Scholar 

  27. Hubbell, W., McConnell, H.M. 1968. Spin label studies of the excitable membrane of nerve and mucle.Proc. Nat. Acad. Sci. USA 61:12

    Article  PubMed  CAS  Google Scholar 

  28. Hubbell, W., McConnell, H.M. 1969. Orientation and motion of amphiphilic spin labels in membranes.Proc. Nat. Acad. Sci. USA 64:20

    Article  PubMed  CAS  Google Scholar 

  29. Hubbell, W., McConnell, H.M. 1971. Molecular motion in spin-labeled phospholipids and membranes.J. Am. Chem. Soc. 93:314

    Article  PubMed  CAS  Google Scholar 

  30. Hubbell, W., Metcalfe, J., Metcalfe, S., McConnell, H.M. 1970. The interaction of small molecules with spin-labelled erythrocyte membranes.Biochim. Biophys. Acta 219:415

    Article  PubMed  CAS  Google Scholar 

  31. Huestis, W.H., McConnell, H.M. 1974. A functional acetylcholine receptor in the human erythrocyte.Biochem. Biophys. Res. Commun. 57:726

    Article  PubMed  CAS  Google Scholar 

  32. Hui, S.W., Parsons, D.F. 1975. Direct observation of domains in wet lipid bilayers.Science 190:383

    Article  PubMed  CAS  Google Scholar 

  33. Israelachvili, J., Sjösten, J., Eriksson, L., Ehrström, M., Gräslund, A., Ehrenberg, A. 1974. Theoretical analysis of the molecular motion of spin labels in membranes.Biochim. Biophys. Acta 339:164

    Article  PubMed  CAS  Google Scholar 

  34. Jost, P., Griffith, O.H., Capaldi, R., Vanderkooi, G. 1973. Identification and extent of fluid bilayer regions in membranous cytochrome oxidase.Biochim. Biophys. Acta 311:141

    Article  PubMed  CAS  Google Scholar 

  35. Jost, P., Waggoner, A.S., Griffith, O.H. 1971. Spin labeling and membrane structure. In: The Structure and Function of Biological Membranes. L.I. Rothfield, editor Ch. 3, p. 84. Academic Press, New York

    Google Scholar 

  36. Kaplan, J., Canonico, P., Caspary, W. 1973. ESR studies of spin-labeled mammalian cells by detection of surface-membrane signals.Proc. Nat. Acad. Sci. USA 70:66

    Article  PubMed  CAS  Google Scholar 

  37. Keirns, J., Kreiner, P., Bitensky, M. 1973. An abrupt temperature-dependent change in the energy of activation of hormone-stimulated hepatic adenylyl cyclase.J. Supramol. Struct. 1:368

    Article  PubMed  CAS  Google Scholar 

  38. Keith, A.D., Sharnoff, M., Cohn, G. 1973. A summary and evaluation of spin labels used as probes for biological membrane structure.Biochim. Biophys. Acta 300:379

    Article  PubMed  CAS  Google Scholar 

  39. Kidwai, A.M. 1974. Isolation of plasma membrane from smooth, skeletal, and heart muscle.Methods Enzymol. 32 (A):134

    Article  Google Scholar 

  40. Kidwai, A.M., Radcliffe, M., Duchon, G., Daniel, E. 1971. Isolation of plasma membrane from cardiac muscle.Biochem. Biophys. Res. Commun. 45:901

    Article  PubMed  CAS  Google Scholar 

  41. Kreiner, P., Keirns, J., Bitensky, M. 1973. A temperature-sensitive change in the energy of activation of hormone-stimulated hepatic adenylyl cyclase.Proc. Nat. Acad. Sci. USA 70:1785

    Article  PubMed  CAS  Google Scholar 

  42. Kury, P.G., McConnell, H.M. 1975. Regulation of membrane flexibility in human erythrocytes.Biochemistry 14:2798

    Article  PubMed  CAS  Google Scholar 

  43. Landsberger, F., Lenard, J., Compans, R. 1971. Spin-label electron spin resonance study of the lipid-containing membrane of Influenza virus.Proc. Nat. Acad. Sci. USA 68:2579

    Article  PubMed  CAS  Google Scholar 

  44. Landsberger, F., Paxton, J., Lenard, J. 1971. A study of intact human erythrocytes and their ghosts using stearic acid spin labels.Biochim. Biophys. Acta 266:1

    Google Scholar 

  45. Lenard, J., Compans, R.W. 1974. The membrane structure of lipid-containing viruses.Biochim. Biophys. Acta 344:519

    Google Scholar 

  46. Linden, C.D., Keith, A.D., Fox, C.F. 1973. Correlations between fatty acid distribution in phospholipids and the temperature dependence of membrane physical state.J. Supramol. Struct. 1:523

    Article  PubMed  CAS  Google Scholar 

  47. Linden, C.D., Wright, K.L., McConnell, H.M., Fox, C.F. 1973. Lateral phase separations in membrane lipids and the mechanism of sugar transport inEscherichia coli.Proc. Nat. Acad. Sci. USA 70:2271

    Article  PubMed  CAS  Google Scholar 

  48. McCalley, R., Shimshick, E., McConnell, H.M. 1972. The effect of slow rotational motion of paramagnetic resonance spectra.Chem. Phys. Lett. 13:115

    Article  CAS  Google Scholar 

  49. Mehlhorn, R.J., Keith, A.D. 1972. Spin labeling of biological membranes.In: Membrane Molecular Biology. C.F. Fox and A.D. Keith, editors. p. 192. Sinauer Associates, Stamford

    Google Scholar 

  50. Mehlhorn, R., Snipes, W., Keith, A.D. 1973. Spin label motion in fatty acids.Biophys. J. 13:1223

    Article  PubMed  CAS  Google Scholar 

  51. Morse, P.D., Ruhlig, M., Snipes, W., Keith, A.D. 1975. A spin-label study of the viscosity prifile of sarcoplasmic reticular vesicles.Arch. Biochem. Biophys. 168:40

    Article  PubMed  CAS  Google Scholar 

  52. Nozawa, Y., Iida, H., Fukushima, H., Ohki, K., Ohnishi, S. 1974. Studies onTetrahymena membranes: Temperature-induced alterations in fatty acid composition of various membrane fractions inTetrahymena pyriformis and its effect on membrane fluidity as inferred by spin-label study.Biochim. Biophys. Acta 367:134

    Article  PubMed  CAS  Google Scholar 

  53. Ohnishi, S., Ito, T. 1973. Clustering of lecithin molecules in phosphatidylserine membranes induced by calcium ion binding to phosphatidylserine.Biochem. Biophys. Res. Commun. 51:132

    Article  PubMed  CAS  Google Scholar 

  54. Ohnishi, S., Ito, T. 1974. Calcium-induced phase separations in phosphatidylserine-phosphatidylcholine membranes.Biochemistry 13:881

    Article  CAS  Google Scholar 

  55. Oldfield, E., Chapman, D. 1972. Dynamics of lipids in membranes: Heterogeneity and the role of cholesterol.FEBS Lett. 23:285

    Article  PubMed  CAS  Google Scholar 

  56. Oldfield, E., Keough, K., Chapman, D. 1971. The study of hydrocarbon chain mobility in membrane systems using spin-label probes.FEBS Lett. 20:344

    Article  Google Scholar 

  57. Rottem, S. 1975. Heterogeneity in the physical state of the exterior and interior regions ofMycoplasma membrane lipids.Biochem. Biophys. Res. Commun. 64:7

    Article  PubMed  CAS  Google Scholar 

  58. Rottem, S., Hubbell, W.L., Hayflick, L., McConnell, H.M. 1970. Motion of fatty acid spin labels in the plasma membrane ofMycoplasma.Biochim. Biophys. Acta 219:104

    Article  PubMed  CAS  Google Scholar 

  59. Rottem, S., Samuni, A. 1973. Effect of proteins on the motion of spin-labeled fatty acids inMycoplasma membranes.Biochim. Biophys. Acta 298:32

    Article  PubMed  CAS  Google Scholar 

  60. Sackmann, E., Träuble, H. 1972. Studies of the crystalline-liquid crystalline phase transition of lipid model membranes.J. Am. Chem. Soc. 94:4482

    Article  PubMed  CAS  Google Scholar 

  61. Sackmann, E., Träuble, H. 1972. Studies of the crystalline-liquid crystalline phase transition of lipid model membranes.J. Am. Chem. Soc. 94:4492

    Article  PubMed  CAS  Google Scholar 

  62. Sackmann, E., Träuble, H., Galla, H., Overath, P. 1973. Lateral diffusion, protein mobility, and phase transitions inEscherichia coli membranes. A spin label study.Biochemistry 12:5360

    Article  PubMed  CAS  Google Scholar 

  63. Sauerheber, R.D., Gordon, L.M. 1975. Spin label studies on rat liver plasma membrane: Calcium effects on membrane fluidity.Proc. Soc. Exp. Biol. Med. 150:28

    Article  PubMed  CAS  Google Scholar 

  64. Scandella, C.J., Devaux, P., McConnell, H.M. 1972. Rapid lateral diffusion of phospholipids in rabbit sarcoplasmic reticulum.Proc. Nat. Acad. Sci. USA 69:2056

    Article  PubMed  CAS  Google Scholar 

  65. Schreier-Muccillo, S., Niculitcheff, G., Oliveira, M., Shimuta, S., Paiva, A. 1974. Conformational changes at membranes of target cells induced by the peptide hormone angiotensin. A spin label study.FEBS Lett. 47:193

    Article  PubMed  CAS  Google Scholar 

  66. Schummer, U., Hegner, D., Schnepel, G., Wellhöner, H. 1975. Investigations of thermotropic phase changes in peripheral nerve of frog and rat.Biochim. Biophys. Acta 394:93

    Article  PubMed  CAS  Google Scholar 

  67. Seelig, J. 1970. Spin label studies of oriented smectic liquid crystals (a model system for bilayer membranes).J. Am. Chem. Soc. 92:3881

    Article  CAS  Google Scholar 

  68. Seelig, J., Hasselbach, W., 1971. A spin label study of sarcoplasmic vesicles.Eur. J. Biochem. 21:17

    Article  PubMed  CAS  Google Scholar 

  69. Seeman, P. 1972. The membrane actions of anesthetics and tranquilizers.Pharm. Rev. 24:583

    PubMed  CAS  Google Scholar 

  70. Sefton, B., Gaffney, B.J., 1974. Effect of the viral proteins on the fluidity of the membrane lipids in Sindbis virus.J. Mol. Biol. 90:343

    Article  PubMed  CAS  Google Scholar 

  71. Smith, I.C.P. 1972. The spin label method. In: Biological Applications of Electron Spin Resonance. H.M. Swartz et al., editors. p. 483. Wiley-Interscience, New York

    Google Scholar 

  72. Snipes, W., Keith, A.D. 1970. Spin labels extend applications of ESR.Res. Dev. 21(2): 22

    CAS  Google Scholar 

  73. Verma, S., Wallach, D. 1975. Evidence for constrained lipid mobility in the erthrocyte ghost. A spin label study.Biochim. Biophys. Acta 382:73

    Article  PubMed  CAS  Google Scholar 

  74. Walaas, O., Walaas, E., Grønnerød, O. 1974. Molecular events in the action of insulin on cell metabolism. The significance of cyclic AMP-dependent protein kinases.Acta Endocrinol. Copenhagen 77:93 (suppl. 191)

    CAS  Google Scholar 

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  1. Richard D. Crosland

    Present address: Dept. of Biochemistry, School of Medicine, University of Southern California, Los Angeles, California

  2. Melvin D. Kuwahara

    Present address: Dept. of Pharmacology, University of Minnesota, Minneapolis, Minnesota

Authors and Affiliations

  1. Department of Chemistry, San Diego State University, 92182, San Diego, California

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

  2. California Metabolic Research Foundation, 92038, La Jolla, California

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

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  1. Richard D. Sauerheber
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Sauerheber, R.D., Gordon, L.M., Crosland, R.D. et al. Spin-label studies on rat liver and heart plasma membranes: Do probe-probe interactions interfere with the measurement of membrane properties?. J. Membrain Biol. 31, 131–169 (1977). https://doi.org/10.1007/BF01869402

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