Site-directed spin labeling of proteins. Applications to diphtheria toxin.
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ESR spectra were recorded from rat epididymal adipocyte ghosts labeled with the 5-nitroxide stearic acid spin probe, I(12,3). Polarity-corrected and approximate order parameters, that are sensitive to the flexibility of the incorporated label, were used to evaluate the membrane lipid fluidity. Addition of CaCl2 a 37 degrees C decreased the fluidity, as indicated by positive increases in the order parameters. The ordering effect of Ca2+ was concentration-dependent, reached saturation at approx. 3--4 mM, and was completely reversed by excess EGTA. Previous studies indicated that low- and high-affinity sites on adipocyte plasma membranes are able to bind 45Ca2+, and our results suggest that Ca2+-induced alterations in the lipid fluidity involve cation binding to low-affinity sites. The cellular movements of Ca2+ and, in particular, the binding of Ca2+ to the plasma membrane may play important roles in insulin's action on fat cell function. The possibility that insulin directly alters the membrane fluidity was tested by adding hormone to freshly-prepared I(12,3)-labeled adipocyte ghosts. Insulin, at concentrations (10(-6) M) that enhance glucose uptake into intact adipocytes, did not affect the fluidity of ghosts suspended in buffers with or without Ca2+. The fluidities of I(12,3)-labeled rat adipocyte ghosts or human erythrocyte ghosts were also unaffected by various forms of human growth hormone.
Using a recently described technique of electron spin resonance spectroscopy, we determined the rate of transbilayer mobility (flip) of the four major simian-erythrocyte phospholipids in the erythrocyte membrane after infection by Plasmodium knowlesi. The development of the malarial parasite induces a very large increase in the flip rate of these phospholipids (mainly during the ring stage and at the beginning of trophozoite maturation). The half flip time fell from 2 and 3 hr in the case of choline phospholipids in healthy erythrocytes to less than 15 min in erythrocytes infected with the last stage of the parasite.
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