Donald F.H. Wallach

Multiple thermotropic state transitions in erythrocyte membranes. A laser-Raman study of the CH-stretching and acoustical regions.

1. We have examined the Raman scattering from erythrocyte ghosts at 2700 cm-1 -3000 cm-1 (CH-stretching region), that at low frequencies due to acoustical vibrations, as well as that due to v (-C=C-) and v (=C-C=) of beta-carotene, in the temperature range of -30 degrees C to +30 degrees C. For comparison we have also evaluated the Rama spectra of liposomes composed of lecithins+/-cholesterol. 2. Plots vs. temperature of the intensities (I) of the approx. 2880 cm-1 and 2932 cm-1 bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the (I2880/I2850) and (I2932/I2850) ratios, revealed discontinuities in both erythrocyte membranes and liposomes. 3. In erythrocyte membranes, plots of (I approximately 2880/I2850) and (I approximately 2932/I 2850) reveal several discontinuities. Those reported by (I approximately 2880/I 2850) occur at +17 degrees C and approx. -4 degrees C. The ratio (I approximately 2932/I 2850) reveals a transition between -20 degrees C and +10 degrees C, 1/2 maximal at -8 degrees C. The ratio (I 1527/I 1158), representing the relative intensity of v (-C=C-) vs. v (=C-C=) of membrane-bound beta-carotene, changes discontinuously with the 17 degrees C transition, but is stable over the rest of the temperature change studied. 4. The data indicate that a major membrane-state transition takes place below 0 degrees C but that an additional transition occurs at approx. 17 degrees C.

Separation of EDTA-extractable erythrocyte membrane proteins by isoelectric focussing linked to electrophoresis in sodium dodecyl sulfate

Abstract 1. 1. EDTA-extractable proteins of human erythrocyte membranes can be separated in 8 M urea by isoelectric focussing in polyacrylamide gels 2. 2. Subsequent electrophoresis at right angles in sodium dodecyl sulfate-laden polyacrylamide shows that most of the bands hitherto defined by simple sodium dodecyl sulfate polyacrylamide gel electrophoresis do not represent single proteins/peptides, but comprise mixtures of diverse molecular species with different isoelectric points. 3. 3. The high molecular weight sodium dodecyl sulfate bands 1,2,2.1 and 2.2 possess identical isoelectric points. The isoelectric points and Band 5 components also overlap with those of the Bands 1, 2, 2.1 and 2.2. This suggests a structural relationship between these entities. 4. 4. Membranes from different donors show slight variations in the two-dimensional electrophoretic pattern. 5. 5. The separation techniques presented here provide greater resolution than hitherto attained and should prove useful for the detailed analysis and characterisation of membrane proteins.

A new sensitive, rapid fluorescence technique for the determination of proteins in gel electrophoresis and in solution.

Abstract We describe the use of o -phthalaldehyde (OPT) in a simple, rapid, fluorimetric technique for the proteins in the concentration range of 0.1–50 μg/ml. Fluorescence excitation is near 338 nm and emission is measured near 446 nm. The technique is eminently suitable for simple rapid, high-resolution evaluation of protein/peptide separations in polyacrylamide gel electrophoresis. For this purpose the mixture to be fractionated is treated first with β-mercaptoethanol, followed by OPT, prior to electrophoresis. The separation is evaluated after electrophoresis by exciting the fluorescence of protein/peptide-bound OPT with a conventional (365 nm) “black light”. The technique provides exceptional resolution and far greater sensitivity than conventional detection techniques; its detection limits are ca. 10 ng protein. When used without β-mercaptoethanol, the technique could also allow definition of the distribution of SH-containing proteins/peptides. Quantitative evaluation of protein/peptide distribution is best achieved by photometric scanning of photographic negatives obtained under defined exposure and developing times.

Differential effects of temperature on the nuclear and plasma membranes of lymphoid cells. A study by freeze-etch electron microscopy.

Abstract 1. 1. We have used freeze-fracture electron microscopy to examine the effects of cooling on the core ultrastructure of the plasma and nuclear membranes of normal thymocytes and lymph node cells, as well as concanavalin A-treated thymocytes and mouse lymphoma cells. 2. 2. Chilling below 22 °C produces smooth areas, free of intramembranous particles on both faces of both inner and outer nuclear membranes. This effect is reversible and can be prevented by glutaraldehyde fixation. 3. 3. Plasma membranes, in contrast to the nuclear membranes, exhibit no change in freeze-fracture morphology upon cooling. 4. 4. We hypothesize that the changes observed in the nuclear membranes represent thermotropic lipid phase transitions and that such transitions either do not occur in plasma membranes or are there constrained to very small regions.

Erythrocyte membrane alterations due to infection with Plasmodium berghei.

Abstract 1. 1. The membrane peptide components of erythrocytes from normal mice and animals infected with Plasmodium berghei have been analyzed by sodium dodecyl-sulfate-polyacrilamide electrophoresis. 2. 2. Parasitemia produces significant degration of the “spectrin” bands of the membranes, as well as of another component with an apparent molecular weight of 112 000. This is associated with the appearance of certain new components which most likely represent split-products rather than parasite constituents. 3. 3. The biochemical changes correlate with major surface deformations demonstrated by scanning electron microscopy. 4. 4. We suggest that normal erythrocyte topology requires the integrity of certain protens at the inner membrane surface and that the prelytic topologic alterations of the erythrocyte surfaces arise from the degradation of these proteins.

Diffusion in tissue cultures on gas-permeable and impermeable supports.

We analyze the diffusion of O 2 , CO 2 and non-volatile products in tissue cultures on various gas-permeable and impermeable supports. From the equations presented, it is possible to predict the steady state pericellular P o 2 and P co 2 as well as the accumulation of non-volatile products by cells in culture. In contrast to non-permeable supports, cultures with high respiration rates on highly permeable supports will not deplete their pericellular P o 2 or accumulate high pericellular P co 2 which lowers pericellular pH. Due to a higher steady state P o 2 , dependence on glycolysis and the resulting drop in pericellular pH will not occur on highly permeable supports. We consider various problems in cell culturing in the light of our calculations and conclude that the pericellular environment of cultures on highly permeable supports can be accurately controlled by control of the incubator atmosphere.

A reevaluation of the status of cholesterol in erythrocytes infected by Plasmodium knowlesi and P. falciparum

The cholesterol synthesis of rhesus monkey erythrocytes parasitized by Plasmodium knowlesi and human erythrocytes infected by P. falciparum, as measured by incorporation of [1-14C]acetate and 3H2O, was almost undetectable, concordant with very low levels of measurable 3-hydroxy-3-methyl glutaryl-CoA reductase activity. In addition, both types of infected cells exchanged cholesterol with the plasma at the same rate as uninfected cells. The data do not exclude the possibility of cholesterol transfer from uninfected to infected cells.

Effect of melittin on thermotropic lipid state transitions in phosphatidylcholine liposomes

We have examined the Raman scattering due to CH stretching vibrations, as well as to v(-C=C-) and v(=C-C=) of beta-carotene, of liposomes composed of phosphatidylcholine (egg, dimyristoyl, dipalmitoyl) +/- cholesterol, beta-carotene or melittin in the temperature range of -10 degrees C to 45 degrees C. (2) Plots vs. temperature of the intensities of the 2885 cm-1 and 2930 cm-1 CH stretching bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the I2885/I2850 and I2930/I2850 ratios, reveal a sharp discontinuity in cholesterol-free phosphatidylcholine liposomes; this coincides with the gel leads to liquid-crystal transition temperature of the fatty acyl chains. In cholesterol/phosphatudylcholine liposomes the change in I2885/I2850 occurs over a very broad temperature range and I2930/I2850 remains stable. (3) I1527/I1158, i.e. the intensity of v(-C=C-) relative to that of v(=C-C-) in beta-carotene/phosphatidylcholine liposomes, changes discontinuously at the gel leads to liquid-crystal transition temperature. The values above the transition temperature approach those of the carotenoid in organic solution. (4) The transitions reported in I2885/I2850 for phosphatidylcholine/melittin liposomes (25-56; 1, M/M) are shifted to much higher temperatures than observed in phosphatidylcholine liposomes. In the case of dimyristoyl phosphatidylcholine/melittin the changes in I2930/I2850 also occurs at a higher temperature (28 degrees C) than without melittin (21 degrees C), but the temperature shift is less than the +13 degrees C observed for I2885/I2850. It appears that the apolar moiety of melittin organizes phospholipids adjacent to and more remote from the peptide moiety, to form complexes with an elevated lipid transition temperature. The effect of the peptide moiety is greater on the methylene segments (I2885/I2850) than on the methyl termini (I2930/I2850).

The action of melittin on phosphatide multibilayers as studied by infrared dichroism and spin labeling. A model approach to lipid-protein interactions

Abstract 1. 1. We have studied interaction of melittin with phosphatide multibilayers by infrared absorption spectroscopy, infrared dichroism and spin label methods. 2. 2. The ratio of the infrared absorption peaks at 1460 and 1370 cm −1 depends upon the hydrocarbon chain mobility, in that it increases as chain mobility decreases. The ratio increases upon melittin binding. Concomitantly, the CH 2 -rocking band at 720 cm −1 sharpens, also indicating a decrease in chain mobility. 3. 3. The infrared spectra of phosphatidyl ethanolamine in the region of 1250–800 cm −1 depend upon head group orientation. 4. 4. Melittin changes the transition vector for the bands at 1730 cm −1 (C0 ester stretch), 1240–1220 cm −1 (PO stretch), 1020 cm −1 (POH stretch in phosphatidylethanolamine) and 960 cm −1 (POC deformation in lecithin). This points to reorganization of the polar head groups. 5. 5. The amide I band of melittin incorporated into phosphatide multibilayers lies at 1650 cm −1 , but exhibits no dichroism; this suggests that the peptide chain is in an unordered array. The ESR spectra of a cholestane spin probe, intercalated in phosphatides multibilayers indicate that melittin disrupts the multibilayers but con-currently decreases the mobility of the spin probe. 6. 6. The ESR spectra of a stearate spin probe labelled at C-5 also indicate that melittin disorders the multibilayers and restricts probe mobility. 7. 7. The fact that the amphipathic polypeptide melittin influences both polar and apolar regions of phosphatide multibilayers, suggests a specific type of lipid-protein interaction.

Raman and resonance-Raman scattering by erythrocyte ghosts

1. We present the laser-Raman spectra of human erythrocyte ghosts, isolated by standard conditions and compare these with the spectra of lecithin liposomes and fat-free serum albumin. 2. The hydrocarbon stretching modes of membrane lipids are temperature sensitive and may serve as a index of hydrocarbon chain motion. 3. The Amide I and Amide III bands of ghosts in H-2O and 2-H-2O, indicate a mixture of alpha-helical and unordered conformation, but do not allow a quantitative estimate of secondary structure. 4. Strong, scattering bands at 1530 and 1165 cm-1 are attributable to conjugated double bond systems, probably of membrane-associated carotenoids. Their high intensity is due to resonance enhancement.