Distribution of a fatty acid spin probe in sarcoplasmic reticulum. Evidence of membrane asymmetry.
The distribution of a lipophilic spin probe, 5-doxyl stearate, between the inner and outer halves of the sarcoplasmic reticulum (SR) bilayer was determined by titration with Ni X EDTA, a spin broadening agent. Titrations were also performed with Fe(CN)3-6 and with the solvated Ni2+ cation. Ni X EDTA titrations reached a clearly defined asymptote at 35% signal reduction. Fe(CN)3-6 and Ni2+ titrations gave biphasic curves but showed 35% of the signal to be readily eliminated at low concentrations. When the Ni2+ cation was used with ionophore, titrations indicated that 96% of the probe is aligned in the bilayer with the spin moiety at either the inner or outer interface. It was concluded that the spin probe distribution between the outer and inner halves of the SR bilayer is 35:65, respectively. Titrations performed on vesicles of purified SR lipids gave a ratio of 60 exposed:40 protected, consistent with the vesicular geometry. In addition the spin probe distribution in SR vesicles did not vary as a function of temperature, salt concentration, or spin probe concentration. On this basis it was concluded that the spin probe distribution gives a reasonable estimation of the volume of fluid lipids available to readily solubilize the probe in each half of the bilayer and that the observed asymmetry in distribution is due to the presence of SR proteins which were eliminated in the pure lipid vesicles. Furthermore, as EDTA is unique in its ability to chelate transition metals, Ca2+ and EGTA can be used in Ni X EDTA titrations without altering the chelation of Ni2+. Known changes in ATPase conformation accompanying Ca2+ and adenyl-5'-yl imidodiphosphate X Mg binding did not affect the spin probe distribution. However, phosphorylation of the enzyme by Pi gave a small, but clearly discernible, protection of spin probe signal. Chemical reduction with ascorbate indicated that this was due to occlusion of a small fraction of spin probes and thus possibly SR lipids.
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000378:metabolism*; D004492:Edetic Acid; D004578:Electron Spin Resonance Spectroscopy; D005295:Ferrocyanides / Q000494:pharmacology; D007832:Lasalocid / Q000494:pharmacology; D008051:Lipid Bilayers / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D009532:Nickel / Q000494:pharmacology; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013113:Spin Labels / Q000378:metabolism*; D014018:Tissue Distribution
D003497:Cyclic N-Oxides; D005295:Ferrocyanides; D008051:Lipid Bilayers; D013113:Spin Labels; D000266:Adenylyl Imidodiphosphate; C005072:5-doxylstearic acid; D009532:Nickel; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; D008274:Magnesium; D001205:Ascorbic Acid; D002118:Calcium; C000170:ferric ferrocyanide; D007832:Lasalocid