Capping of saccharides on the plasma membrane of lymphocytes as studied by fluorescein-labelled lectins

The capping of saccharides on the plasma membrane of rat splenic lymphocytes was studied by means of fluorescein-labelled lectins. Treatment of unfixed splenic lymphocytes with any one of the three lectins, concanavalin A (Con A), Ricinus communis agglutinin (RCA) and wheat germ agglutinin (WGA) led to the formation of caps of each saccharide receptor on the plasma membrane. Treatment of unfixed lymphocytes with Con A was found to result in the formation of caps of saccharide receptors for RCA, whereas cap formations were never noted in such double treatment of the cells with all other combined uses of two lectins. These results are taken to indicate that the saccharide receptors for Con A are associated with those for RCA in the plasma membrane of rat splenic lymphocytes.     

Effect of hexavalent chromium on eukaryotic plasma membrane studied by EPR spectroscopy

The effect of Cr(VI) anion on an ergosterol-producing strain of eukaryotic yeast Candida albicans and its mutant with ergosterol-less membrane was studied with EPR spectroscopy. 5- and 14-doxyl stearic acid spin probes were used to label the protoplast membrane after removal of the cell wall. In control experiments, the mutant strain exhibited larger rigidity in the membrane than its parental strain. Addition of Cr(VI), at a minimum inhibitory concentration of 0.6 mM, increased the rotational mobility of the spin labels significantly and decreased the temperature of the structural changes in both strains, in the temperature range between 0 and 30 degrees C. The ergosterol-less mutant, having a membrane composition with increased polyunsaturated fatty acid content, exhibited higher Cr(VI) sensitivity. Treatment of the membrane with Cr(VI) for 10 min already resulted in an increase in membrane fluidity. An EPR signal of Cr(V) was detected which reached maximum amplitude after 120 min of treatment with Cr(VI). Further chemical reduction of Cr(V) in the absence of extracellular Cr(VI) led to a lack of detectable paramagnetic chromium intermediates within 200 min.     

The gelation of deoxyhemoglobin S in erythrocytes as detected by transverse water proton relazation measurements

At 37 °C, when samples of blood, washed erythrocytes, or isolated hemoglobin from individuals with sickle cell disease are deoxygenated, the transverse water proton relaxation time is sharply decreased. In similar samples from normal adults homozygous for hemoglobin A, only a slight decrease in t₂ is observed upon deoxygenation at 37 °C. In samples containing deoxyhemoglobin S the value of t₂ increases as the temperature is decreased from 37 °C to 4 °C, in contrast to samples containing oxyhemoglobin S, oxyhemoglobin A, or deoxyhemoglobin A where t₂ decreases as the temperature decreases. It is suggested that this decrease in t₂ observed in samples of deoxyhemoglobin S at 37 °C is the result of an increase in the amount of preferentially oriented water at macromolecular interfaces which occurs under conditions known to produce deoxyhemoglobin S gelation. Conditions which reverse deoxyhemoglobin S gelation such as lowering the temperature to 4 °C decrease the amount of preferentially oriented water which results in an increase in the value of t₂. Thus, measurement of the transverse water proton relaxation time can be used to monitor the gelation of deoxyhemoglobin S inside the erythrocyte.     

Lipid-protein interaction in frog nerve membrane as studied by spin-labelling

Molecular dynamics of membrane proteins in frog nerve was studied by the spin labelling technique. By varying the reaction conditions three protein domains were identified which differ in polarity of the micro-environment of the labelled sites (polarity index, h = 0, 0.6 and 1.2) and in mobility (apparent rotational correlation times, tau 2 = 1 ns, 35 ns and 62 ns, respectively). The protein domains reflect different structural stabilities towards specific and non-specific membrane perturbants. One part of the labels has an anisotropic distribution. Modification of the membrane components with different chemicals leads to a change in conformation and/or segmental motion of proteins, as well as to an alteration in the degree of ordering of the attached labels. The experiments support the fact, that the state of the lipid matrix strongly influences the overall conformation of the embedded proteins and their functions in biological processes.     

The ATP depletion process of human erythrocytes studied by freeze fracturing

By the freeze-fracture method it is shown that metabolic depletion of erythrocytes affects three levels of cell organization: the microstructural (erythrocyte form), microstructural (micro-relief of erythrocyte surface) and ultrastructural (ultrastructural state of erythrocyte plasma membranes) ones. As it is established, the size of spikes on the echinocyte surface and that of membrane vesicles budding from a cell coincide with each other. The structural modification of the membrane precedes the stage of erythrocyte crenation. The following model of vesicle budding process is suggested: reduction of ATP level and dephosphorylation of actin-spectrin network--structural modification of the protein and lipid membrane phases with the formation of regions disconnected from the spectrin framework--protrusion of these anomalous regions in the form of spikes--budding of spikes as spherical vesicles.     

Solution and membrane structure of enkephalins as studied by infrared spectroscopy

The backbone conformation of the two opioid pentapeptides Leu5-enkephalin and Met5-enkephalin was studied by the technique of resolution-enhanced infrared spectroscopy. In aqueous solution, the conformation-sensitive amide I bands of the two peptides are identical. The positions of these bands are consistent with the view that in aqueous solution both enkephalins exist as an ensemble of largely unfolded conformers. Interaction of Leu5- and Met5-enkephalins with bilayer membranes of ditetradecylphosphatidylcholine results in a substantial refolding of the peptide backbones. The conformation stabilized by the membrane environment is a hydrogen-bonded turn structure. Conformational transitions in enkephalins induced by a lipid environment may play a role in the specific interactions between these hormones and their receptor sites.     

Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H-ATPase reconstituted into vesicles

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Δψ) generated by the Schizosaccharomyces pombe plasma membrane H⁺-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Δψ because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Δψ at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H⁺-ATPase generates Δψ of about 160 mV on the vesicle membrane. The generated Δψ was stable at least over tens of minutes. An influence of the H⁺ membrane permeability on the Δψ buildup was demonstrated by manipulating the H⁺ permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H⁺-ATPase-mediated Δψ buildup.     

The conformations of nitroxide-labelled fatty acid probes of membrane structure as studied by 2H-NMR

The electron spin resonance (ESR) spectrum of a nitroxide spin probe intercalated in a membrane is influenced by the amplitude of anisotropic motion of the nitroxide group and by the geometry of the oxazolidine ring of the nitroxide. In the analysis of the ESR spectra of nitroxide-labelled fatty acid probes, it is generally assumed that the five-membered oxazolidine ring system is oriented rigidly perpendicular to the long molecular axis of the probe. This assumption is tested in the present study, using 2H-NMR of specifically deuterium-labelled nitroxide spin probes. Evidence is presented that the nitroxide does not display the assumed geometry in membranes. The departure from this geometry depends on the position of the nitroxide label on the acyl chain, with a more pronounced departure for position 5 relative to position 12. These and previous data provide an explanation for the discrepancies between spin-probe ESR and 2H-NMR order parameters in membranes.     

Constitutive plasma membrane targeting and microdomain localization of Dok5 studied by single-molecule microscopy

In the present study, single-molecule fluorescence microscopy was used to examine the characteristics of plasma membrane targeting and microdomain localization of enhanced yellow fluorescent protein (eYFP)-tagged wild-type Dok5 and its variants in living Chinese hamster ovary (CHO) cells. We found that Dok5 can target constitutively to the plasma membrane, and the PH domain is essential for this process. Furthermore, single-molecule trajectories analysis revealed that Dok5 can constitutively partition into microdomain on the plasma membrane. Finally, the potential mechanism of microdomain localization of Dok5 was discussed. This study provided insights into the characteristics of plasma membrane targeting and microdomain localization of Dok5 in living CHO cells.     

Antibody-induced capping of measles virus antigens on plasma membrane studied by electron microscopy.

Antibodies specific for measles virus could redistribute ("cap") virus antigens on infected HeLa cells as shown by transmission and scanning electron microscopy. Using an indirect immunoperoxidase technique, infected cells showed diffuse, circumferential distribution of virus antigens over the cell surface when mixed with antibody at 4 C. At 37 C, virus-coated microvilli concentrated on one pole of the cell, leaving the remainder of the plasma membrane devoid of both viral antigens and microvillus projections. Whereas extreme polar displacement of virus-antibody complexes frequently occurred, endocytosis was rarely seen. The findings indicate that antiviral antibodies can move and cluster virus on plasma membranes and suggest that virus-antibody complexes are stripped and shed from the cell surface.     

Effects of cholesterol on membrane surfaces as studied by high-pressure fluorescence spectroscopy

We have studied the effects of cholesterol on membrane surfaces using fluorescence spectroscopy at high pressure. At atmospheric pressure, the dissociation state of a pH-sensitive fluorophore (6-decanylnaphthol or DECNA) incorporated into several types of membranes showed an apparent increase in dissociation with cholesterol content coming somewhat closer to its dissociation state in solution. Previous studies have shown that when DECNA is free in solution, pressure induces proton dissociation due to the volume decrease that occurs when water electrostricts around the ions. But in phosphatidylcholine (PC) bilayers, proton dissociation is inhibited, either due to the inability of the surface to expand and allow for increased hydration, or other changes in lipid structure. The pressure behavior of DECNA in dioleoyl-PC, dioleoylphosphatidic acid and dioleylphosphatidylglycerol bilayers shows that incorporation of 5-10% cholesterol causes DECNA to behave like it is in a more unrestricted environment. This trend is reversed at higher cholesterol concentrations. These data, together with compressibility measurements, support the model of Sankaram and Thompson [M. Sankaram, T.E. Thompson, Biochemistry 29 (1990) 10676.] whereby in the disordered phase, cholesterol can span the two leaflets causing an increase in the area between the head groups; whereas in the ordered phase, no expansion occurs. Thus, the effect of cholesterol on membrane surfaces depends on its phase diagram.