Structural changes in erythrocyte membranes during cooling studied by a spin probe method

Peculiarities of structural changes in erythrocyte membranes during freezing (from -20 degrees to -50 degrees) were studied by electron paramagnetic resonance method using spin-labelled derivative of stearic acid-5-doxylstearate. It was established that membranes underwent a number of structural reconstructions due to the temperature decrease and water freezing-out. Differences were found in temperature dependences that characterize lipid ordering during probe insertion into membranes of native erythrocytes, white ghosts, and liposomes from total lipids of erythrocyte membranes. The data obtained indicate the impairment in the structure of lipid components and lipid-protein interactions in erythrocyte membranes during cooling.     

Presynaptic plasma membranes and synaptic vesicles of cholinergic nerve endings demonstrated by means of specific antisera

Summary Antisera were raised to cholinergic presynaptic plasma membranes and synaptic vesicles isolated from the electric organ of Torpedo marmorata and tested by immunochemical and immunohistochemical methods. The antisera responded to many antigens not specific to nerve endings, but it was possible to eliminate these antibodies by means of simple absorption procedures with fractions containing the unwanted antigens. After absorption, staining of thin sections of electric organ by immunofluorescence was limited to the region of nerve endings in the tissue.The remaining antibodies responded in the case of the plasma membrane antisera predominantly to a 33,000 molecular-weight polypeptide and a chloroform/methanol-soluble antigen. In cross reactivity studies it was found that this antiserum not only stains cholinergic nerve endings in Torpedo but also those in mammalian tissue. The antigen responsible for the cross reactivity is restricted to the chloroform/methanol-soluble material.The vesicle antiserum labels cholinergic nerve endings in mammalian tissue as well; the relevant antigen in this case is different from the one described above and is likely to be a glycosaminoglycan. The antisera provide valuable markers for cholinergic nerve terminals. In addition, the vesicle antiserum may now be used to study axonal transport and the life cycle of this organelle in the cholinergic neurone.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - EGTA ethylenebis (oxoethylenenitrilo) tetra-acetic acid - MW apparent molecular weight Enzymes. Na⁺, K⁺-activated ATPase (EC 3.6.1.3); acetylcholine esterase (EC 3.1.1.7); choline acetyl-transferase (EC 2.3.1.6)     

Antigenic structure of phytohemagglutinin-stimulated mouse lymphocytes using specific antisera]

The use of antiserum to the intact and the PHA-transformed lymphocytes showed that a new antigenic determinant (or determinants) appeared on the surface of lymphocytes after 68-hour PHA-stimulation. Intact lymphocytes or cells stimulated by the PHA for 2 hours only didn't carry this antigenic marker. At the same time the quantity (or density) of the antigenic markers present on the surface of the intact lymphocytes was lowered in the lymphocytes stimulated with the PHA for a long time.     

Conformational changes in soluble mitochondrial ATPase by the spin probe method]

Modification of soluble mitochondrial ATPase (factor F1) by spin-labelled iodoacetamide and spin-labelled methyleneketone does not cause and change in the catalytic properties of the enzyme. The temperature dependence of tau corr. of labels bound to factor F1 testifies to conformational changes in the enzyme at temperatures of 18--20 degrees C and 34--37 degrees C. At these temperature intervals, breaks are observed in the temperature dependence of the ATPase reaction rate in the Arrenius plot. The results obtained indicate that the thermally induced conformational changes in factor F1 affect large areas of the protein molecule. The interaction of factor F1 with the hydrophobic spin probes, namely fatty acid derivatives, was studied. It was shown that the interaction of foctor F1 with Mg2+, Mg-ATP, Mg-ADP and ADP, results in an increase in the ability of the enzyme to adsorb spin probes.     

Study of the conformational changes of serum albumin molecules within a pre-denaturation temperature range by a spin probe technique]

To study conformational changes of protein molecules in a pre-denaturation temperature range, nitroxyl radicals adsorbed by a protein are suggested to be used. A spin probe technique is specially developed to be implied for this aim by using a probe specifically bound to the bovine serum albumin molecule, it was possible to reveal a dependence of the rotation correlation time of the adsorbed radical and the environment polarity on the temperature. The data obtained testify in favour of a change occurring in the intramolecular structure of the protein under study due to a temperature change within the pre-denaturation temperature range.     

Mechanisms of the structural changes in erythrocyte membranes under the action of a shift in pH]

The effect of pH shifts (5--10) on erythrocyte "ghosts" has been studied using the IR spectroscopy. The process of regulation has been shown to take place within pH 7.15--8 range involving both protein (transition testified by absorption band changes 1520, 1540, 1635, 1645, 1585, 1696, 3305 CM-1) and lipid membrane components testified by absorption band splits 1745, 1470, 1380, 720 AND 1242 cm-1. The transition from a lesser to a higher regulating state of membrane structure happens unevenly, since the above changes take place within the narrow pH 7.15--8 interval, near the homeostase limit, being never observed at pH 5--7.15 or 8--10. It is supposed that the increase in regulation of both protein, phospholipid and lipid membrane fields is of phasic nature which presumably constitutes the basis for pH action mechanism as a regulation factor.     

Estimation of spin probe clustering in biological membranes

An iterative spectral subtraction technique has been developed which accurately estimates the proportion of 'dilute' and 'clustered' I(12, 3) (i.e., 5-nitroxide stearate) in human erythrocyte ghosts at 37 degrees C, even if subtractant spectra free from probe-probe interactions cannot be measured due to technical limitations. Gordon et al. ((1985) J. Membrane Biol. 84, 81-95) earlier showed that I(12, 3) occupies a class of high-affinity sites in ghosts at probe/total lipid ratios (P/L) less than 1/2250. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membrane-bound clusters of variable size. Although this model allows determination of the dilute/clustered probe ratio, it requires subtraction of experimental spectra with a 'magnetically dilute' spectrum obtained using P/L less than 1/4600. The new methodology accurately profiles the % probe clustering in human erythrocyte ghosts over the entire P/L range, even if the lowest P/L for the subtractant spectrum contains substantial probe-probe interactions (i.e., P/L of 1/604 or 1/303). Application of either the subtraction technique in Gordon et al. (1985) or the iterative subtraction protocol described here should allow determination of probe clustering in a wide range of I(12, 3)-labeled biological membranes.     

Structural reorganization of the brain synaptic membranes and aging

Results of the authors' studies and data from literature underlie the development of a notion on structural rearrangement of the brain synaptic membranes in aging. Reorganization results in conformational changes of the key membrane-bound enzymes and receptors underlying the age alterations of neuronal functions.     

Heterogeneity of thylakoid membranes studied by EPR spin probe

A lipophilic nitroxyl radical, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 1-adamantylacetate, has been applied to EPR spin probe study of chloroplasts and subchloroplast fragments of different types. The latter originate from grana and the grana core regions. The binding of the spin probe to the membranes was revealed by specific changes in a shape of the EPR spectra. A share of membrane-bound spin probe was different for chloroplasts and subchloroplast fragments, as well as its rotational correlation time and apparent enthalpy and entropy activation of nitroxide rotational motion. The binding of the spin probe induced a significant decrease in the amount of the oxidized P700 and changes in the kinetics of its light oxidation and dark recovery. This suggests that one of the sites of nitroxyl radical binding is the nearest surrounding of the pigment-protein complexes of Photosystem I (PSI). Distinctions in mobility of spin probe immobilized by chloroplasts and their fragments can be caused by the different environment of the PSI complexes located in various regions of thylakoid membranes. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 5, pp. 690–698.     

The interaction of fructose-1,6-biphosphate aldolase with liposome membranes: a spin probe technique study

Thermotropic properties of liposome membranes prepared of bulk bovine erythrocyte membrane lipids, native, or aldolase-modified, were investigated by the ESR method. Breaks were observed in the log 2T parallel vs 1/T plots for two spin labels: tempopalmitate and 5-doxyl-palmitate methyl ester. These phenomena have been interpreted as reflecting structural changes near the lipid bilayer polar heads region. Upon modification with aldolase, the temperature at which the breaks occurred was decreased for both spin probes.     

Structural lability of membranes]

The present-day state of the problem of membrane structural lability is reviewed. The ways of initiation and the nature of structural rearrangements, the mechanisms of generalization of local structural perturbations in membranes and the effects of rearrangements on the functional activity of organoids and cells are discussed.     

Structural changes in spores under high temperature exposure]

The electron microscopy of ultrathin sections of B. cereus spores showed that no lysis and destructive changes occurred in the main structural components of the spores when heated to 99 degrees C (in distilled water). By the time 99% of the population were destroyed, the spores seemed to preserve the exosporium of the sporoderm, the cortex and the sporoblast intact. Even autoclaving at 120 degrees C for 15 min brought about no visible changes in the ultrastructure of the spores, though it killed the whole spore population.     

Biological activity of human alpha-interferons studied using specific antisera

Preparation of highly active rabbit antisera (AS) to human recombinant alpha 2-interferon and their use for studying biological properties of natural and plasmid alpha-interferons are described. By exhaustion of AS by alpha 3-interferon there were prepared practically monospecific AS not reacting with antigenic determinants of alpha 3-interferon. It was found that alpha 3-interferon represented a significant portion of human lymphoblastoid interferons and was included in PH-labile alpha-interferon from serum of patients with Kaposi carcinoma. AS to alpha 2-interferon completely neutralized antiviral and antiproliferative activity of the homologous subtype alpha-interferon and stimulation of cytotoxicity of human natural killer cells induced by it. It neutralized also the same effects of the heterologous subtypes (alpha 3 and alpha F/D) and leukocytic interferon, but the neutralization level was lower. The results of the study confirmed the polyfunctional nature of the interferon molecule.     

Structural reorganizations of the lipids and proteins of erythrocyte membranes under the action of low temperatures

The reversible structural rearrangement of lipids and protein oligomerization has been shown to occur during cooling in membranes of model systems (liposome, erythrocyte shadows) and native erythrocytes. Analysing the dependence of Azz in membrane probes (5- or 15-doxylstearic acids) in the Arrhenius plots a conclusion on the structural changes at 13-19 degrees C and within the range of interior water freezing from -17 up to -19 degrees C has been drawn, the last transition is smoothed out in the presence of glycerin. Using diamide joining spectrin and electrophoresis in polyacrylamide gel it has been determined that the low temperatures cause the spatial approach of proteins of spectrin-actinic complex and formation connections between the erythrocyte membrane proteins which aren't destroyed by dodecylsulfate.     

Effect of amphotericin B on membranes: a spin probe study

The effect of the polyene antibiotic amphotericin B on the electron paramagnetic resonance spectra of lipid probes intercalated in model membranes was examined. When the antibiotic was added to the aqueous phase, no spectral effects occurred. However, when the antibiotic was incorporated during membrane preparation, changes in spectral parameters suggested the appearance of a new phase. The spectral changes do not necessarily corroborate the pore models proposed previously for amphotericin B in membranes. With a spin probe that partitions between water and membrane, an interaction between the amphotericin B and probe is observed. This interaction does not occur in the membrane, but in the aqueous phase, between the probe and the aggregated antibiotic. Some of the equilibria involving the antibiotic appear to be achieved slowly.     

Separation of enriched synaptosomes, synaptic vesicles and synaptic plasma membranes]

A rapid and simple method is described for separation of intact synaptosomes, synaptic plasma membranes and vesicles. Two synaptosome fractions were obtained by modified differential centrifugation. The rate zonal zentrifugation in a linear sucrose gradient (very low density) is suitable to obtain fractions highly enriched in synaptic plasma membranes and vesicles. Examination of the prepared fractions was done by enzyme marker activities and electron microscopy