Electron Spin Resonance Studies of Spin-Labeled Mammalian Cells by Detection of Surface-Membrane Signals

Abstract

Lipid-soluble spin labels were incorporated into human lymphocytes and mouse L-cells and the resulting electron spin resonance spectra were compared with spectra obtained from similarly labeled human erythrocytes. Spin labels were found in all subcellular fractions of the nucleated cells that contained membranes. Spinlabeled cells remained viable and capable of replicating in vitro. Electron spin resonance signals from spin-labeled nucleated cells underwent a time-and temperaturedependent decay that was reversed by bathing the cells in K3Fe(CN)6. The demonstration of a relative cell impermeability to ferricyanide, as measured by both colorimetric and radioisotopic label methods, indicated that only spin-labeled molecules in the surface membrane were reactivated when ferricyanide was added to spin-labeled cells after the electron spin resonance signal had decayed.Electron spin resonance (ESR) spectroscopy and spin-label techniques have been used recently to probe the structure of artificial membranes (1, 2) and membranes of erythrocytes (3), neurospora (4), viruses (5), lobster walking nerves (6), sarcoplasmic reticulum (7), and mycoplasma (8). However, information is not available regarding application of spinlabel techniques to studies with intact nucleated mammalian cells. A major problem in the application of ESR techniques to studies of surface membrane phenomena in intact cells is differentiating between labels attached to cell surface and those bound to internal membrane structures.This report describes a technique for differential detection of surface-membrane signals from spin-labeled, intact nucleated mammalian cells. The cellular distribution and spectral characteristics of various lipid-soluble spin labels incorporated in membranes of mouse L-cells and human lymphocytes are described. ESR spectra of these spin-labeled nucleated cells are compared with spectra of spin-labeled human erythrocytes. In addition, factors affecting signal stability and the effect of label incorporation on cell viability and replication are reported. MATERIALS AND METHODSSpin Labels. All spin labels were purchased from Syva Assoc., Palo Alto, Calif. The spin labels used were ( Fig. 1 in ME medium to 50% of the original volume of homogenate, the nuclear pellet was given three additional strokes in the Duall homogenizer and again centrifuged. Supernates were combined and centrifuged at 25,000 X g for 10 min to obtain a mitochondrial fraction (M), and at 48,000 X g for 60 min to obtain a microsomal fraction (P). The final supernate was the soluble fraction (S). N, M, and P fractions were concentrated by suspending them to 50% or 25% of the homogenate's original volume. Relative concentrations of label in each of the subcellular fractions were determined. 66

Effect of the viral proteins on the fluidity of the membrane lipids in Sindbis virus

“…It has been observed (Landsberger et al, 1971) that the membrane of influenza virus was at least as rigid as that of human erythrocytes. Human erythrocytes, in turn, have been demonstrated to be distinctly more rigid than either mouse L cells (Kaplan et al, 1973) or secondary chick fibroblasts . Thus, while a direct comparison of the fluidity of the influenza virus membrane and the membrane of its host cell has not been made, it seems fair to conclude that influenza virus, like Sindbis virus, has a membrane that is more rigid than the membrane8 of typical tissue culture fibroblasts.…”

High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C--dependent activation of NAD(P)H oxidase in cultured vascular cells.

“…Measurement of ROS by ESR spectroscopy. The procedures for ESR with spin probes and nitroxide radicals in cultured cells were described previously (37)(38)(39)(40). Briefly, the cells were removed from the dishes by treatment with 0.25% trypsin, centrifuged at 800g for 5 min, and suspended in the medium without serum at a concentration of 1 ϫ 10 7 cells/ml.…”

Drying Increases Intracellular Partitioning of Amphiphilic Substances into the Lipid Phase

“…In fact, the lack of extracellular EPR signal in the vapor-rehydration experiments made the use of ferricyanide as a quencher superfluous. However, some ferricyanide (10 mm) was necessary as an external electron acceptor to prevent reduction of TEMPONE to a non-free-radical form (Kaplan et al, 1973;Miller, 1978). The filtered pollen sample loaded with the spin probe was rapidly dried overnight in a flow of dry air (3% RH) in a dry box, and retained good viability during the procedure.…”