Biomedical subjects
Publications and source records attributed to D A Butterfield.
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Previous studies in our laboratory had demonstrated alterations in the physical state of membrane proteins in erythrocytes in Huntington's disease. In order to assess the specificity of our findings, the results of electron spin resonance studies of protein and lipid components, scanning electron-microscopic studies, enzymatic analyses of membrane-bound sodium plus potassium stimulated, magnesium-dependent adenosine triphosphatase and protein kinase, and cell deformability studies of erythrocyte membranes have been performed in the neurological disorders, Huntington's disease, Friedreich's ataxia, Alzheimer's disease, amyotrophic lateral sclerosis, and myotonic and Duchenne muscular dystrophy. Comparison of the results revealed that alterations in the biophysical and biochemical states of erythrocyte membranes in each disorder are specific to the particular disease state with the exception of those in Friedreich's ataxia and Alzheimer's disease. In the latter instance, the clinical and pathological alterations suggest that these two diseases have different primary defects. Our studies suggest that the molecular basis of each disease is different. In addition, the results suggest that biophysical and biochemical investigations of extraneural tissue in Huntington's disease and other neurological disordes have the potential of clarifying the molecular mechanisms by which these diseases arise.
Electron spin resonance, enzymatic, and SDS-polyacrylamide gel electrophoretic investigations of erythrocyte membranes from patients with Alzheimer's disease were performed. Alterations in the physical state of membrane proteins in Alzheimer's disease erythrocytes were found by spin labeling studies. However, no alterations in membrane lipid fluidity or in the activities of membrane-bound sodium plus potassium-stimulated, magnesium-dependent adenosine triphosphatase or acetylcholinesterase could be demonstrated. Also, no changes in staining profiles of AD erythrocyte membrane proteins subjected to electrophoresis were observed. The altered conformation and/or organization of extraneural membrane proteins in Alzheimer's disease suggests the possibility that this disorder may have more widespread membrane involvement than was originally thought.
The sialic acid present in erythrocyte ghosts was estimated by methane chemical ionization mass fragmentography of the trimethylsilyl methyl glycosides. The internal standard was 3,4,6-tris-trimethylsilyl-alpha-phenyl-2-deoxy-2-acetamide-D-glucosaminide, as proposed by Yu and Ledeen (J. Lipid Res. 1970. 11: 506-516). The [MH-16]+ions (m/e 610 for the TMS-methylglycoside of N-acetylneuraminic acid and m/e 498 for the internal standard) were used for quantifying nanogram levels of sialic acid in the presence of other contaminating substances. This effectively raises the signal-to-noise ratio for the Yu and Ledeen method by at least two orders of magnitude. The sensitivity and linearity of the method, without use of isotopic carriers, were tested using known quantities of N-acetylneuraminic acid. The limit of detection was below 0.4 nanograms (approximately one picomole). The useful range of detection was 10 ng-1 microgram, showing a large dynamic range.
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, scanning electron microscopic, and SDS-polyacrylamide gel electrophoretic studies of erythrocytes in Friedreich's ataxia have been performed. No alteration in the physical state of membrane lipids, in morphology, or in the staining profile of erythrocytes in Friedreich's ataxia could be demonstrated. An altered conformation and/or organization of proteins in erythrocyte membranes in this disorder was suggested by spin labeling studies (P less than 0.025), favoring the possibility of a generalized membrane abnormality in Friedreich's ataxia. These findings are discussed in relation to other inherited neurological diseases where similar studies have been performed.
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Dopa-decarboxylase, acetylcholinesterase, sodium plus potassium stimulated adenosine triphosphatase (Na+ + K+-ATPase), and membrane-bound protein kinase were compared in the erythrocytes of patients with Huntington's disease and normal controls. All these enzymes also exist in the basal ganglia. The Na+ +K+-ATPase level was elevated (p less than 0.05) in Huntington's disease, while no significant changes were observed in the other enzymes. This finding is consistent with the concept that Huntington's disease is associated with a general membrane abnormality.
The interaction of the inhibitory neurotransmitter gamma-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.
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.
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Electron spin resonance experiments have been performed on erythrocyte membranes from rats with myotonia induced by treatment with 20,25-diazacholesterol. The results suggest that erythrocyte membranes in this animal model of human congenital myotonia possess a highly significantly increased surface membrane fluidity compared to that of controls. Alterations in the physical state of membrane proteins were not apparent. These findings, also present in human congenital myotonia [Butterfield, Chesnut, Roses & Appel, 1976, Nature (London) 263:159; Butterfield, 1977 (Submitted for publication)], strengthen the concepts that increased membrane fluidity is associated with the presence of myotonia and that congenital myotonia may be a diffuse membrane disease.
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