Electron spin resonance investigations of membrane proteins in erythrocytes in muscle diseases. Duchenne and myotonic muscular dystrophy and congenital myotonia.

Comparison of electron spin resonance spectra of spin labeled erythrocyte membranes from patients with the dystrophic conditions Duchenne and myotonic muscular dystrophy with those of normal controls suggests that alterations in membrane protein conformation and/or organization are present in these disease states. These protein alterations are not apparent in the non-dystrophic disease congenital myotonia. The results suggest a correlation between changes in the physical state of proteins in membranes with the presence of dystrophy. In addition, the present results from erythrocytes lend support for the concept of a generalized membrane defect in these diseases.     

Progressive muscular dystrophy type Duchenne. I. Spin label studies on the physico-chemical state of erythrocyte membranes.

Electron spin resonance studies of erythrocyte membranes from patients with Duchenne muscular dystrophy exhibit changes in the physical state of lipids and proteins in membranes when compared to membranes from normal subjects. The results suggest that the alterations in membrane lipid-protein organization are present in this disease.     

Effects of intramembrane particle aggregation on erythrocyte membrane fluidity: an electron spin resonance study in normal and in dystrophic subjects

Mobilization and aggregation of intramembrane particles (IMPs) are physiological events observed in various cells. In erythrocyte membranes, aggregation of IMPs can be induced by the exposure of partially desprectrinized erythrocyte membranes to acidic pH. We investigated the association between IMPs aggregation, protein mobility, and membrane fluidity in erythrocyte membranes of healthy controls and Duchenne muscular dystrophy (DMD) patients by using electron spin resonance and specific spin labels for membrane proteins and lipids. In erythrocyte membranes of control subjects, the partial spectrin removal induced a decreased segmental motion of protein spin label indicating an increase of protein-protein interactions. Stearic acid spin labels 5- and 16-(N-oxyl-4,4'-dimethyloxazolidine) showed that the treatment induces an increase of membrane fluidity. In DMD patients, both treated and untreated erythrocyte membranes showed changes of membrane fluidity when compared to those of the controls. Our results suggest that defects in the interactions between skeletal proteins and/or between membrane and skeleton components may contribute to the alterations of erythrocyte membranes in DMD.     

Phosphorylation of component a of the human erythrocyte membrane in myotonic muscular dystrophy.

Endogenous membrane protein kinase activity in fresh erythrocyte ghosts is altered in myotonic muscular dystrophy. Phosphorylation of erythrocyte Component a, which migrates with an apparent molecular weight of 90,000 to 100,000, is significantly reduced compared to age- and sex-matched controls. The difference in endogenous membrane protein kinase activity in fresh RBC membranes lends confirmation to the suggestion that myotonic dystrophy is a disease of widespread membrane alterations.     

Fluidity of erythrocyte membranes from patients with Duchenne muscular dystrophy: studies in intact erythrocytes and in ghosts

We have studied the alterations of fluidity in intact erythrocytes and in erythrocyte membranes from patients with Duchenne muscular dystrophy. The interest of this study was to comparison directly two types of results; these demonstrate an increase of fluidity in the erythrocytic membranes, no changes are present when the label is incorporated in intact erythrocytes. It might be inferred that hypotonic haemolysis removes components that are more weakly bound in Duchenne membranes, and that exert an immobilizing effect on the membrane lipids.     

Interaction of antitumor drugs with human erythrocyte ghost membranes and mastocytoma P815: a spin label study.

The cytotoxic and mutagenic properties of antitumor drugs such as adriamycin, acridines, diacridine, actinomycin D and Pt compounds are related to their interaction with nucleic acids and inhibition of protein synthesis. We have examined their interaction with human erythrocyte ghost membranes and murine mastocytoma cells using spin labeling techniques. These drugs induce changes in electron spin resonance of the spin labeled ghost membranes and in the mastocytoma cells. These alterations suggest that these drugs induce changes in protein conformation of the membranes. The membrane binding properties of these drugs may be important in their mechanism of action.     

Interaction of tacrine and velnacrine with neocortical synaptosomal membranes: Relevance to Alzheimer's disease

The acridine-based, potential Alzheimer's disease therapeutic agents, tacrine and velnacrine, were incubated with rat or gerbil neocortical synaptosomal membranes. Electron paramagnetic resonance employing a protein-specific spin label was used to monitor this interaction. Analogous to their effects in erythrocyte membranes [Butterfield and Rangachari (1992) Biochem. Biophys. Res. Commun. 185: 596–603], in the present studies both agents decreased segmental motion of spin labeled synaptosomal membrane proteins, consistent with increased cytoskeletal protein-protein interactions (0.001相似文献   

Transport enzymes in the cell membranes of cultured fibroblasts; alterations in dystrophic cells

In support of the widely held belief that membrane defects are present in the muscular dystrophies, alterations have been found in some transport-related enzymes of cells from affected donors. Cell membranes were isolated from cultured dermal fibroblasts of victims of myotonic muscular dystrophy, and of Duchenne's muscular dystrophy, and from cells of normal age- and sex-matched donors. Myotonic cells had an elevated Na+, K+ ATPase. gamma-Glutamyl transpeptidase was elevated in Duchenne cells. Among all cells' 5'nucleotide phosphatase exhibited a remarkably constant specific activity.     

Temperature-induced protein conformational changes in barley root plasma membrane-enriched microsomes: I. Effect of temperature on membrane protein and lipid mobility

A protein spin label and lipid spin probes were used to study the temperature-dependent motion of protein and lipid, respectively, in barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes. Using membranes from seedlings grown at 20°C, the temperature-dependence of the relative motion of membrane surface spin probes and a spin label covalently attached to membrane proteins suggested abrupt changes in the lipid and protein mobilities at about 12°C. Spin probe spin-spin exchange broadening and fluorescent probe eximer formation indicated apparent temperature-induced alterations in probe lateral diffusion within the membrane at about 12 to 14°C. The results suggest the presence of temperature-induced quasicrystalline lipid clusters which may influence the activity of membrane-bound enzymes.     

Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Aging of the erythrocyte IV. Spin-label studies of membrane lipids,proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Electron spin resonance, hematological, and deformability studies of erythrocytes from patients with Huntington's disease.

Electron spin resonance, hematologic, and deformability studies of erythrocytes from patients with Huntington's disease have been performed A decreased deformability of Huntington's disease erythrocytes compared to normal controls was demonstrated. No difference in erythrocyte hematologic indices, osmotic fragility, reticulocyte counts, or intracellular Na+ concentration was found. Huntington's disease serum had no demonstrable effect on electron spin resonance parameters of a protein-specific spin label attached to membrane proteins in control erythrocytes compared to the effect of control serum. This finding suggests that under the conditions employed no serum component or circulating factor is responsible for the changes in the physical state of membrane proteins in Huntington's disease erythrocytes (Butterfield, D.A., Oeswein, J.Q. and Markesbery, W.R. (1977) Nature 267, 453--455). No alteration in lipid fluidity of Huntington's disease erythrocyte membranes could be discerned suggesting that the underlying molecular defect in Huntington's disease involves a membrane protein. The results of the present studies on erythrocytes strongly support the concept that Huntington's disease is associated with a generalized membrane abnormality.     

Electron spin resonance analysis of irreversible changes induced by calcium perturbation of erythrocyte membranes.

Introduction of calcium during hemolysis of erythrocytes causes irreversible membrane changes, including protein aggregation. These changes have been investigated by incorporation of one protein and three fatty acid spin label probes into washed membranes from erythrocytes hemolyzed with a range of Ca2+ concentrations. Electron spin resonance spectra of the lipid probes were analyzed for changes in the order parameters, isotropic coupling constants and mean angular deviations of the lipid hydrocarbon chains. The results generally indicated an increased freedom of mobility of the probes with increased Ca2+ concentration during hemolysis, but the response of each probe showed a different concentration dependence. The maximal response was obtained with the I(5, 10) probe. Variations in the responses were interpreted to reflect different modes of protein-lipid or protein-probe interactions arising from Ca2+ -induced membrane protein alterations. Spectra from membranes treated with the protein spin label showed an increased ratio of immobilized to mobile label with increased Ca2+ concentrations at hemolysis. This is consistent with the membrane protein aggregation phenomena previously observed. It is suggested that the increased protein-protein interactions formed as a result of calcium treatment permit an increased lipid mobility in the membrane regions monitored by the fatty acid probes.     

Erythrocyte membrane fluidity in chicken muscular dystrophy.

An increased rigidity of erythrocyte membranes in four-week old dystrophic chickens compared to closely related normal controls has been suggested using electron spin resonance. These findings suggest that similar to the case of human Duchenne muscular dystrophy, chicken muscular dystrophy may be associated with a generalized membrane defect.     

Differential molecular dynamics and transmembrane fluidity gradients in canine myocardial sarcolemma and sarcoplasmic reticulum.

The molecular dynamics of highly purified preparations of canine myocardial sarcolemma (SL) and sarcoplasmic reticulum (SR) were quantified by electron spin resonance spectroscopy (ESR). Canine myocardial SL and SR have substantially different motional regimes in their membrane interiors as demonstrated by alterations in the relative peak height ratios, peak widths and peak splittings in ESR spectra of 16-doxylstearate incorporated into SL and SR. Quantification of the apparent order parameters (S) of 16-doxylstearate in SL and SR by analyses of ESR spectra demonstrated that the interior of the SL membrane was substantially more immobilized than the interior of the SR membrane (e.g. S = 0.168 +/- 0.002 for SL and S = 0.128 +/- 0.003 for SR). In contrast, only modest differences in membrane dynamics near the hydrophobic-hydrophilic interface were present in SL and SR as ascertained by ESR spectra of the probe 5-doxylstearate incorporated into these membranes. Myocardial sarcolemma contained heretofore unsuspected amounts of cholesterol (1.4 +/- 0.1 mumol cholesterol/mg protein) while sarcoplasmic reticulum contained only small amounts of cholesterol (0.17 +/- 0.06 mumol cholesterol/mg protein). Model systems employing binary mixtures of plasmenylcholine/cholesterol and phosphatidylcholine/cholesterol demonstrated that the observed alterations in molecular dynamics were due, in large part, to the differential cholesterol content in these two subcellular membrane compartments. Taken together, these results demonstrate that these two functionally distinct myocardial subcellular membranes have markedly disparate molecular dynamics and transmembrane fluidity gradients which may facilitate their performance of specific functional roles during excitation-contraction coupling in myocardium.     

The Lipid Peroxidation Product, 4-Hydroxy-2-trans-Nonenal, Alters the Conformation of Cortical Synaptosomal Membrane Proteins

Abstract: Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid β-peptide (Aβ). Aβ-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2- trans -nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of Aβ and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6(2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1–10 µ M HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and Aβ-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 µ M ). The results obtained are discussed with relevance to the hypothesis of Aβ-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.     

Erythrocyte membrane in Duchenne muscular dystrophy. II. Fluidity of lipids in dystrophic patients and their families

Electron spin resonance techniques was used to study the fluidity of intact and hemoglobin free erythrocyte membranes from patients with Duchenne muscular distrophy and from members of their family. A greater mobility of spin label motion was observed only at the surface of hemoglobin free membranes in the patients. These studies suggest alterations in lipid-protein interaction, indicating in Duchenne muscular distrophy a generalized membrane abnormality.     

Erythrocyte membrane alterations in Huntington disease: Effects of γ-aminobutyric acid

The interaction of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) with erythrocyte membranes from patients with Huntington disease and normal controls has been studied by electron spin resonance. GABA affects the physical state of erythrocyte membrane proteins in control and Huntington disease differently. In addition, after exposure of spin-labeled Huntington disease erythrocyte membranes to 0.1 mM GABA, the relevant electron spin resonance parameters reflecting the physical state of membrane proteins are indistinguishable from those of untreated control membranes. These findings support the concept that this disease is associated with a generalized membrane defect.     

Evidence for a membrane surface defect in erythrocytes in Huntington's disease

Electron spin resonance studies of erythrocyte membranes from patients with Huntington's disease and normal controls have been performed. Intact erythrocytes in each case were either untreated or subjected to proteolysis with the membrane impermeable enzymes, pronase, chymotrypsin, or trypsin. Membrane ghosts were prepared from untreated and protease-treated intact cells and spin labeled with protein- or lipid-specific spin probes. Comparison of the resulting electron spin resonance spectra confirmed our previous findings that in untreated samples the relevant parameter of the protein-specific spin label was increased in Huntington's disease (P < 0.02) suggesting an altered physical state of membrane proteins in this disorder, while no difference in erythrocyte lipid fluidity could be discerned. No significant difference in the physical state of membrane proteins in Huntington's disease and control as judged as spin labeling methods could be detemined in membrane ghosts prepared from protease-treated intact cells. These results, together with the known specificity of the proteases used in this study, suggest that a molecular defect in Huntington's disease erythrocytes is manifested in an exterior part of a membrane protein and supports our hypothesis that Huntington's disease is associated with a generalized cell membrane defect.     

Dystrophin in electric organ of Torpedo californica homologous to that in human muscle

We have found that dystrophin is highly concentrated at neuromuscular junctions and innervated membranes of the electric organ of Torpedo californica. In acetylcholine receptor-rich Torpedo membrane preparations dystrophin represents approximately 0.4% of total protein and can be extracted from these membranes by alkaline treatment in the absence of detergent, indicating that it is a peripheral membrane protein. Polyclonal antibodies raised against electrophoretically isolated Torpedo dystrophin cross-react with dystrophin in human muscle and unequivocally discriminate between normal and Duchenne muscular dystrophy patient's muscle. These results indicate that dystrophin is phylogenetically a highly conserved protein and that the relatively abundant dystrophin in electric organ would facilitate further investigations of its structure and function.