Alec D. Keith

A spin-labeled lipid for probing biological membranes

Abstract A spin-labeled lipid has been synthesized in which the nitroxide is rigidly bound to a stearic acid chain at the 12-position. The spin-labeled lipid is sufficiently stable for general use in studying biological membranes and model membrane systems. Interactions of this new spin label with sodium dodecyl sulfate and lecithin micellar solutions have been investigated by electron spin resonance. The electron spin resonance spectra of the solubilized spin labeled lipid are sensitive to changes in micellar structure which affect the mobility of the label.

Membrane properties of saturated fatty acid mutants of yeast revealed by spin labels

Abstract (I) Several yeast mutants deficient in the synthesis of saturated fatty acids were isolated. (II) These mutants cannot grow when supplemented with a highly purified unsaturated fatty acid. They demonstrate a rigid requirement for a dietary saturated fatty acid. (III) They can be enriched with different fatty acids including odd chain length fatty acids. (IV) The enriched mutants demonstrate a characteristic lyotropic mesomorphic phase transition dependent on fatty acid composition but independent of pre-enzyme or physical treatment. (V) It is inferred that the ionic interaction between membrane lipids and membrane proteins is detectable by spin labels. (VI) The apparent “fluidity” of membranes detectable by spin labels requires re-evaluation.

Erythrocyte membranes - compression of lipid phases by increased cholesterol content

Abstract Lipid and protein interactions were studied in guinea pig erythrocytes containing a normal or a two-fold increased amount of cholesterol. The electron spin resonance (ESR) spectra of cholesterol-loaded cells labeled with fatty-acid probes showed an increase in the local viscosity of the membrane as compared with control cells. This increase reflects changes in the interior of the lipid matrix of the membrane because the probes resisted destruction by ascorbate, were unaffected by the action of pronase, and gave spectra similar to those of liposomes. No differences were observed between control and cholesterol-loaded cells in the conformation of the membrane proteins by either the infrared spectra or the ESR spectra of cells labeled with maleimide probes.

Relation of unsaturated fatty acid composition of rat liver mitochondria to oscillation period, spin label motion, permeability and oxidative phosphorylation

Abstract An investigation was made of the influence of mitochondrial unsaturated fatty acid composition on the following mitochondrial parameters: oscillation period, spin label motion (ESR), permeability, and oxidative phosphorylation. Liver mitochondria from rats fed diets deficient in or supplemented with essential fatty acids showed approximately the same total number of unsaturated fatty acids but changed unsaturation levels. Electron microscopy showed that the morphology of the inner membrane compartment was unchanged. Two differences were correlated with unsaturated fatty acid composition: (1) a slower frequency (or time period) of the oscillatory state of energy-dependent ion transport and (2) a reduction in the motional freedom of each of three spin labels (12NS, 5N10 and 7N14). The increase in oscillation period could arise from a number of rate-limiting processes, including permeability of mitochondria to various anions, cations, and substrate metabolites. However, when the permeability of mitochondria to such substances was tested, no changes were observed in passive or active uptake of these substances or in the efficiency of oxidative phosphorylation under steady-state conditions. Thus, the two parameters, oscillation period and freedom of spin label motion, which are dependent upon large domains of the mitochondrial membranes, are significantly influenced by the change in unsaturated fatty acid composition in essential fatty acid-depleted mitochondria, even though processes such as permeability of ionic materials and oxidative phosphorylation were not measurably affected by these changes in unsaturated fatty acid composition.

Syntheses and novel uses of nitroxide motion probes

Abstract A number of nitroxide spin lables of different molecular geometry were synthesized. These were used to infer the effect of molecular geometry and matrix ordering upon the extent of rotational anisotropy. Simulations were used in conjunction with selected spin labels to demonstrate that enhanced tumbling about the x- and y-principal axes of the nitroxide gives rise to unique spectra and can be recognized qualitatively.

Role of lipids in mitochondrial energy coupling: Evidence from spin labeling and freeze-fracture electron microscopy

Abstract The structure of lipid in mitochondria under oscillatory state conditions was investigated using spin labeling and freeze-fracture electron microscopy. An examination of the temperature dependence of the time course of mitochondrial oscillations disclosed a break in the Arrhenius plot at approximately 24°C. A similar break was observed for the partitioning of the hydrocarbon spin label, 6N11, between hydrophobic and polar domains of the membrane. Spin labeling was also used to study mitochondria fixed by glutaraldehyde in contracted and expanded states. These studies revealed that: (a) glutaraldehyde fixation appeared to restrict the motion of membrane lipids; (b) 6N11 displayed an enhanced polar partitioning in expanded samples relative to contracted preparations; and (c) the partitioning phenomenon in contracted mitochondria showed a break in the Arrhenius plot at about 24°C while no discontinuity was observed for expanded samples. Mitochondrial expansion in the oscillatory state resulted in a change in particle density and aggregation in the outer membrane, but not in the inner membrane as shown by freeze-fracture electron microscopy. Our results, therefore, suggest an active involvement of lipid in the mitochondrial oscillatory state.

Spin labeled lipid probes in serum lipoproteins

Abstract Observations are made relating to the local environment and motional behavior of spin labeled lipids incorporated into human serum lipoproteins. Use has been made of ascorbic acid as a spin subtracting agent to determine the accessibility of the labeled site to the aqueous region.

Interactions of spin-labeled lipid molecules with natural lipids in monolayers at the air-water interface.

The pressure-area curves of two widely-used spin-labeled lipids, 3-nitroxide cholestane and the acid form of 12-nitroxide stearate, were measured at the air-water interface. The 3-nitroxide cholestane molecule produced a curve similar to that of cholesterol or dihydrocholesterol (cholestanol), but the area per molecule was somewhat greater than those of the natural sterols. 12-Nitroxide stearate produced a very expanded pressure-area curve. n nMixed monolayers containing 3-nitroxide cholestane and either cholesterol or 1-palmitoyl-2-oleoyllecithin were measured to determine possible interactions, and these were compared with mixed monolayers containing cholesterol plus 1-palmitoyl-2-oleoyllecithin, or dihydrocholesterol plus this lecithin. The 3-nitroxide cholestane molecule interacts with cholesterol in such a manner as to expand the mixed films; however, this spin label exhibits little interaction with lecithin. In contrast, both cholesterol and dihydrocholesterol condensed lecithin films. n nThe application of this information to the interpretation of spin-label studies on lipid systems of membranes or membrane models is discussed.

Effect of sucrose and uncouplers on lipid spin labeling of mitochondria

Abstract The functional activity of isolated mitochondria is known to be inhibited by sucrose. An investigation of the effect of sucrose on membrane structure as judged by lipid spin-label localization and mitochondrial function as judged by energizedproton translocation was undertaken. High sucrose concentrations inhibit oxygen-dependent hydrogen transport in mitochondrial and submitochondrial preparations and concomitantly change the partitioning of spin-label probes between polar and nonpolar membrane phases. It was concluded that sucrose probably interacts directly with the phospholipids of the membrane to cause disorder of the lipid structure. This conclusion is supported by using several types of nitroxide spin labels. Uncoupling agents such as fluorocarbonyl cyanide phenylhydrazone and 2,4-dinitrophenol influence lipid spin-label behavior in the same way as sucrose, only to a lesser degree. Changes in membrane structure are not brought about by sucrose in glutaraldehyde-fixed SMP which implies that the lipid domain is protein bounded.