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D A Butterfield

Publications and source records attributed to D A Butterfield.

At least 127 records · Page 7Linked to original sources
PubMed1992

Membrane-altering effects of velnacrine and N-methylacridinium: relevance to tacrine and Alzheimer's disease.

The interaction of pharmacological agents potentially useful in Alzheimer's disease, 9-amino-1,2,3,4-tetrahydroacridine (THA or tacrine) and its major metabolite velnacrine (or HP-029), along with related compounds with cytoskeletal proteins in human erythrocyte membrane was investigated using electron paramagnetic resonance spin labeling techniques. The results suggest that: (1) the position of the positive charge of tacrine may be important in the mechanism of its interaction with the membrane cytoskeleton; (2) like tacrine, velnacrine also strengthens cytoskeletal protein-protein interactions in erythrocyte membranes, but appears to be only about half as potent as tacrine. These results are discussed with relevance to therapeutic use of these agents in Alzheimer's disease.

Acridines↗
PubMed1992

Spin-labeling studies of the conformation of the Ca(2+)-regulatory protein calmodulin in solution and bound to the membrane skeleton in erythrocyte ghosts: implications to transmembrane signaling.

Electron paramagnetic resonance (EPR) studies of the Ca(2+)-regulatory protein calmodulin (CaM) have been performed. The conformation of CaM in solution changes upon binding of Ca2+ allowing the protein to bind to target proteins existing in the red blood cell membrane. In this study a maleimide spin label, covalently attached to the single cysteine residue of CaM located in the first Ca(2+)-binding domain, was used to monitor allosteric conformational changes induced by interaction of CaM with Ca2+ and subsequently with the red blood cell membrane. The results show, relative to apo-CaM, a significant increase in the apparent rotational correlation time, tau, of the spin label when Ca2+ was present in solution (P less than 0.001). When apo-CaM exposed to red blood cell membrane ghosts in the absence of Ca2+, no significant difference in spin label motion was seen relative to solution, consistent with the idea that Ca2+ is required for CaM to bind to skeletal proteins. When Ca2+ was added to CaM which was then exposed to ghosts, a highly significant increase in tau (decrease in motion) (P less than 0.000001) relative to apo-CaM exposed to ghosts was found. This latter increase in tau is significantly greater than that produced by the addition of Ca2+ to CaM in solution (P less than 0.001). The major interaction sites of CaM were found by photoaffinity labeling and autoradiography on SDS-PAGE to be on the principal skeletal protein, spectrin. EPR was also used to investigate the biophysical correlates of transmembrane signaling. Spin-labeled CaM was bound to the membrane skeleton in the presence of Ca2+. On the opposite side of the erythrocyte membrane a lectin was bound to the external glycoconjugate of Band 3, the major transmembrane protein of the erythrocyte. A highly significant increase in T of the maleimide spin probe was found relative to the control system in which the lectin was absent. (P < 0.00001). These results suggest that electron paramagnetic resonance spectra of spin-labeled CaM can provide useful information about protein structure and function when in solution and when bound to membranes.

Autoradiography↗
PubMed1991

Spin labeling studies of wheat germ calmodulin in solution.

Electron paramagnetic resonance was used to investigate the physical state of plant calmodulin in solution. Wheat germ calmodulin contains a single cysteine residue (Cys-27) on the first of four calcium binding loops. In this study the nitroxide spin label 2,2,6,6-tetramethyl-4-maleimidopiperidine-1-oxyl (MAL-6) was covalently attached to Cys-27 to produce a Ca(2+)-sensitive, biologically-active, labeled protein. The rotational correlation time of the spin label, a measure of its rotational mobility and reflective of the physical state of this region of the protein, was calculated under various conditions. Relative to control, changes in the physical state of the protein reflected by increased motion of the spin label were observed at high pH, low ionic strength and upon addition of Ca2+. These results extend knowledge of the structure of the protein, previously known from solid state and biochemical studies, to calmodulin in solution.

Calmodulin↗
PubMed1990

The effects of the extracellular manganese concentration and variation of the interpulse delay time in the CPMG sequence on water exchange time across erythrocyte membranes.

There has been broad disagreement in the literature regarding the dependence of water exchange times (Te) across erythrocyte membranes studied by the 1H-NMR Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence on extracellular Mn2+ concentration. While some workers saw no change in Te with Mn2+, others reported a 35-50% decrease in Te with this extracellular paramagnetic relaxation agent. We present 1H-NMR evidence that a 30-50% change in Te can be produced by interdependence of the interpulse delay time of the CPMG pulse sequence and the external Mn2+ concentration. Such a large dependency is interpreted in terms of the diffusional effect as a major source. However, it is shown experimentally that if a large number of refocusing pi pulses are used, the observed transverse relaxation times are unaffected by Mn2+. Under these conditions excellent agreement of Te obtained in our study (13.0 +/- 0.64 ms (N = 36) at 21 degrees C) and that of 12.8 +/- 3.6 ms at 20-23 degrees C reported by the radiotracer method was found. Our findings suggest new and important implications for evaluating the previous reports of the 1H-NMR CPMG method concerning the [Mn2+] effect in the decrease of Te, and provide conditions where studies of water transport across erythrocyte membranes using this magnetic resonance method can be used with confidence.

Biological Transport↗
PubMed1990

Structure-activity investigation of the alteration of the physical state of the skeletal network of proteins in human erythrocyte membranes induced by 9-amino-1,2,3,4-tetrahydroacridine.

The oral administration of 9-amino-1,2,3,4-tetrahydroacridine (THA) is purported to increase the mental function of Alzheimer's disease patients (Summers et al. (1986) N. Engl. J. Med. 315, 1241-1245). Numerous erythrocyte membrane proteins are known to be identical or highly similar to neuronal proteins. In a previous study (Butterfield and Palmieri [1990) Free Radical Res. Commun., in press), we showed that THA greatly increased skeletal protein-protein interactions in erythrocyte membranes as monitored by a spin label specifically bound to membrane proteins. In this report, a structure-activity study has been performed to determine which THA structural components are involved in its effect on the physical state of human erythrocyte membrane skeletal proteins. The results imply that both the planarity of the molecule and the amino group at the 9-position of the parent acridine molecule are important in the mechanism of interaction with membrane proteins.

Alzheimer Disease↗
PubMed1990

Membrane processes associated with the osmotic-pulse incorporation of inositol hexaphosphate.

In previous studies (Biochem. Biophys. Res. Commun. 144, 779-786 (1987); Prog. Clin. Biol. Res. 292, 65-75 (1989)), we showed that inositol hexaphosphate (IHP), when added to erythrocyte membrane ghosts in the range 0.6-2.5 mM, caused a large disruption of skeletal protein-protein interactions as monitored by electron paramagnetic resonance techniques. IHP incorporated into intact cells by an osmotic-pulse method (J. Cell. Physiol. 129, 221-229 (1986)) leads to cells with markedly decreased oxygen affinity. Exposure of the red cells to higher levels of IHP during the osmotic pulse leads to less lysis and more normal cellular indices after healing of the transiently-disrupted membrane (J. Lab. Clin. Med. 113, 58-66 (1989)). In order to determine what effect higher levels of IHP had on skeletal proteins and bilayer lipids of membrane ghosts, spin labeling studies were performed. The main findings were: (a) There was a concentration-dependent alteration in skeletal protein interactions. At concentrations greater than 25 mM IHP, the effectiveness of IHP to disrupt skeletal protein interactions was diminished. (b) No apparent alteration of the motion or order of phospholipids or the lipid water interface of intact cells into which IHP was incorporated occurred, suggesting that higher levels of IHP do not alter the physical state of the lipid bilayer.

Electron Spin Resonance Spectroscopy↗
PubMed1990

Effects of domain-specific erythrocyte membrane modulators on acetylcholinesterase and NADH:cytochrome b5 reductase activities.

Previously, we showed using electron paramagnetic resonance that the physical state of one side of erythrocyte membranes could be modulated by agents which interact with the opposite side (reviewed in Butterfield, 1989, Biological and Synthetic Membranes, A. R. Liss, Inc., New York). The present study was undertaken to determine whether membrane-bound enzymes would exhibit a similar transmembrane modulation effect. The effects of known, domain-specific modulators of the physical state of erythrocyte membranes on the activity of two membrane-bound enzymes were investigated. Acetylcholinesterase, an enzyme having its active site situated on the extracellular side of the membrane, seemed to be unaffected by most of the modulators employed in this study, with the exception of reversible inhibition by benzyl alcohol. Conversely, the activity of NADH:cytochrome b5 reductase, an enzyme whose active site is located on the cytoplasmic side of the erythrocyte membrane, was increased by those agents that interact primarily with skeletal proteins to increase skeletal protein-protein interactions; however, those agents which interact primarily with the skeleton to decrease protein-protein interactions decreased the activity of NADH:cytochrome b5 reductase. This enzyme's activity was also significantly altered by lectins which bind specifically to the external face of glycophorin A on the opposite side of the membrane, but it's activity was unaffected by concanavalin A, a lectin which binds to the external face of band 3. The results of these biochemical studies suggested that NADH:cytochrome b5 reductase can interact with and its activity can be modulated by skeletal or transmembrane proteins. In addition, these results support the hypothesis that in transmembrane signaling processes, biophysical and biochemical changes are correlated.

Acetylcholinesterase↗
PubMed1990

Spin-labelling studies of the interaction of 9-amino-1,2,3,4-tetrahydroacridine (THA), a proposed drug for the treatment of Alzheimer's disease, with erythrocyte membranes.

ESR spin labels specific for skeletal proteins or cell-surface sialic acid have been used to monitor the interaction of 9-amino-1,2,3,4-tetrahydroacridine (THA) and its structural analogs with human erythrocyte membranes. The results suggest that THA significantly increases skeletal protein-protein interactions and may secondarily alter the physical state of the opposite side of the membrane. The fully aromatic analog of THA, 9-aminoacridine, showed even more pronounced effects on skeletal proteins than did THA. These results are discussed in relation to possible interaction sites of THA in erythrocyte ghosts and to potential mechanisms by which THA reportedly increases mental function of victims of Alzheimer's disease.

Alzheimer Disease↗
PubMed1989

Morphological alterations in erythrocyte membranes induced by 9-amino-1,2,3,4-tetrahydroacridine and 9-aminoacridine.

The effects of 9-amino-1,2,3,4-tetrahydroacridine (THA) and its fully aromatic analogue 9-aminoacridine (9-AA) on erythrocyte membrane morphology were investigated via scanning electron microscopy. The ghost population was categorized into four distinct classes and alterations in the relative amounts of these populations with drug addition were noted. The samples incubated in 9-AA had a significantly higher (p less than 0.001) flat, two-dimensional cell population. This shift in morphology may be attributable to the unwinding of spectrin and the subsequent collapse of the membrane.

Aminacrine↗
PubMed1989

Interaction of hemin with erythrocyte membranes: alterations in the physical state of the major sialoglycoprotein.

Hemin has been shown to disrupt erythrocyte membrane skeletal protein-protein interactions, initially those involving band 4.1 (Shaklai et. al. (1986) Biochem. Int. 13, 467-477). We have used electron spin resonance (ESR) spin labels specific for cell-surface carbohydrates, skeletal membrane proteins, or bilayer lipids to find: (1) simultaneous reaction of the protein-specific spin label, MAL-6, which binds to skeletal protein SH residues, and 10 microM hemin suggested that hemin decreased skeletal protein-protein interactions; (2) 10 microM hemin markedly decreased (greater than 60%, P less than 0.001) the rotational motion of spin-labeled erythrocyte membrane cell-surface sialic acid residues, 70% of which are located on the major transmembrane sialoglycoprotein, glycophorin A; and (3) 10 microM hemin caused a small, but significant (P less than 0.02), decrease in the motion of a lipid bilayer specific spin label (5-NS) in the erythrocyte membrane. Since glycophorin A is reportedly linked to the erythrocyte membrane skeletal protein network by band 4.1, it is conceivable that hemin-induced disruption of skeletal protein interactions, particularly those of band 4.1, could subsequently lead to the alterations in the motion of cell-surface sialic acid presented in this report.

Cyclic N-Oxides↗
PubMed1989

Electron spin resonance investigation of the interaction of the anion and glucose transport inhibitor, p-azidobenzylphlorizin, with the human red cell membrane.

The membrane perturbations caused by the interaction of p-azidobenzylphlorizin (p-AzBPhz), a potential photoaffinity labeling agent of the anion and D-glucose transporters in the human erythrocyte, have been studied using electron spin resonance (ESR) spectrometry. Two lipid-specific spin labels have been employed; one of these agents, a hexadecyl-quarternary amine with the nitroxide reporter group covalently attached to the cationic nitrogen, (CAT-16), has been used to monitor changes in the physical state of the membrane's extracellular phospholipid/water interface. The other spin label, 5-doxylstearic acid (5-NS), is designed to examine the order and motion of the lipid bilayer near the cell surface. In separate experiments, intact human red cells labeled with these lipid-specific spin labels were exposed to small amounts of the phlorizin azide. A dose-dependent alteration in CAT-16 motion was observed, but the p-AzBPhz interaction with the membrane had no effect on the spectrum of 5-NS. The half-maximal effect of the phlorizin derivative on the CAT-16 spectrum occurred when about 2 million molecules were bound to each cell. This is also the combined amount of band 3 and band 4.5 present in the red cell membrane and represents the concentration necessary to inhibit both anion and glucose transport. Our results suggest that the first p-AzBPhz molecules binding to the red cell membrane interact with the anion and sugar transporters, and not with the bulk lipid bilayer.

Anion Transport Proteins↗
PubMed1988

Electron spin resonance and biochemical studies of the interaction of the polyamine, spermine, with the skeletal network of proteins in human erythrocyte membranes.

Spermine (N, N'-bis(aminopropyl)-1,4-butanediamine) is a polyamine thought to be important in several cell regulatory processes. Previous studies had shown that spermine prevented the lateral diffusion of transmembrane proteins in human erythrocyte ghosts (Schindler et al. (1980) Proc. Natl. Acad. Sci. USA 77, 1457-1461). In this paper, we present results of studies on the effect of spermine on erythrocyte membranes by employing electron spin resonance spin-labeling techniques in conjunction with spin labels specific for skeletal proteins, bilayer lipids or cell-surface sialic acid of the membrane and by employing SDS-polyacrylamide gel electrophoresis analysis of extracted spectrin and Triton shells. The major findings are: (1) spermine significantly decreases the segmental motion of protein spin-label binding sites (P less than 0.0001), which are predominantly on cytoskeletal proteins; (2) addition of spermine leads to a significant increase in the rotational motion of spin-labeled terminal sialic acid residues (P less than 0.001), most of which are located on glycophorin A, a result which may be secondarily caused by spermine-induced aggregation of cytoskeletal proteins and the cytoplasmic pole of this transmembrane sialoglycoprotein; (3) spermine completely inhibits the low-ionic strength extraction of spectrin, the major protein of the skeletal network which is attached to the bilayer proteins by two or more connecting proteins; (4) pretreatment of ghosts with spermine followed by Triton extraction resulted in the retention of significantly increased amounts of Band 3 and other skeletal and bilayer proteins including Bands 4.2, 6 and 7 in Triton X-100 shells relative to that of control-treated ghosts. These results suggest that spermine acts both to increase protein-protein interactions in the cytoskeletal protein network and to bridge skeletal and bilayer proteins and are discussed with reference to possible molecular mechanisms by which spermine may influence cell functions.

Binding Sites↗
PubMed1987

Electron spin resonance (ESR) studies of skeletal protein interactions in human erythrocyte membranes exposed to polyanions and in membranes prepared from inositol hexaphosphate (IHP)-incorporated low-affinity erythrocytes.

Previous biophysical investigations, including those from our laboratories, have reported that polyphosphates weaken RBC membrane skeletal protein-protein interactions and decrease hemoglobin affinity for oxygen. We have additionally demonstrated that low-affinity intact RBC's may be produced by inositol hexaphosphate (IHP) incorporation via an osmotic pulse method. In the present electron spin resonance (ESR) study, IHP was shown to cause a concentration-dependent increase in the segmental motion of ghost membrane skeletal proteins, but no alterations in spin-labeled terminal sialic acid. Pyrophosphate and inositol hexasulfate were significantly less effective in altering the physical state of skeletal proteins than was IHP. Additional ESR studies of both the interaction of IHP with membrane skeletal proteins in the presence of hemoglobin and of membranes obtained from osmotic pulse-treated intact cells were performed. The results of all these studies are discussed in terms of previous biophysical investigations of the effects of polyphosphates on membranes and of possible molecular events that occur during the osmotic pulse procedure.

2,3-Diphosphoglycerate↗
PubMed1987

Alternations in lipid membrane fluidity and the physical state of cell-surface sialic acid in zinc-deficient rat erythrocyte ghosts.

Erythrocyte ghosts, prepared from the blood of rats fed zinc-deficient diets, were evaluated for membrane fluidity and surface sialic acid properties using spin-labeled probes and electron spin resonance (ESR) spectroscopy. These physical parameters of the erythrocyte ghosts from the zinc-deficient group were compared to those for erythrocyte ghosts obtained from ad libitum and pair fed controls consuming zinc-adequate diets. As the animals became progressively zinc deficient, the erythrocyte ghost membranes became more fluid than those from the control groups. In addition, the apparent rotational correlation time of Tempamine spin probes on surface sialic acid residues was smaller for the zinc deficient group, indicative of an increased rotational mobility of the spin label. These results suggest that zinc deficiency can have pronounced effects on the physical state of membrane bilayer lipids and cell surface carbohydrates and supports the view that many of the pathological signs of zinc deficiency are due to a general membrane defect.

Animals↗