L. W M Fung

Ascorbate levels in red blood cells and urine in patients with sickle cell anemia

Ascorbic acid can be important in sickle cell anemia (SCA) because significant oxidative stress occurs in the disease. Ascorbate could contribute to reduction of the increased oxygen free radicals generated in sickle red blood cells (SRBC) and to the recycling of vitamin E in the cells, while renal loss could contribute to the low plasma levels. Evaluation of red blood cell (RBC) and urine ascorbate in SCA has not been reported. Results showed (1) ascorbate levels in SRBC were similar to those in normals; (2) urine ascorbate excretion was increased in 36% of patients; (3) plasma levels of ascorbate were decreased. Conclusions: (1) Ascorbate is present in SRBC, most likely due to ascorbate recycling, despite increased free‐radical generation. (2) The increase in renal excretion may contribute to the low plasma levels of ascorbate. (3) The presence of ample ascorbate in SRBC and decreased plasma ascorbate suggests that ascorbate movement across the SRBC membrane may differ from normal RBC. Am. J. Hematol. 65:174–175, 2000.

Peptides with More than One 106-amino Acid Sequence Motif Are Needed to Mimic the Structural Stability of Spectrin

The primary sequence of human erythrocyte spectrin contains repetitive homologous sequence motifs of approximately 106 amino acids with 22 such motifs in the α-subunit and 17 in the β-subunit. These homologous sequence motifs have been proposed to form domains with a triple-helical bundle type structure (Speicher, D. W., and Marchesi, V. T. (1984) Nature 311, 177-180; Parry, D. A. D., Dixon, T. W., and Cohen, C. (1992) Biophys. J. 61, 858-867). In this study, we show that these sequence motifs, while they do form compact proteolytically resistant units, are not completely independent. Peptides composed of two or three such motifs in tandem are substantially more stable than peptides composed of a single motif, as measured by proteolysis or by fluorescence or circular dichroism studies of urea or thermal denaturation. Circular dichroism and infrared spectroscopy measurements also indicate that these larger, more stable peptides exhibit greater secondary structure. In these respects, the peptides with tandem sequence motifs are more similar to intact spectrin than the peptide with a single sequence motif. Thus, we conclude that peptides with more than one sequence motif model spectrin more adequately than the peptides with one sequence motif, and that these sequence motifs are not completely independent domains.

ANALYSIS OF SPIN‐LABELED ERYTHROCYTE MEMBRANES*

Some of the earlier electron paramagnetic resonance (EPR) studies of proteins labeled with N-( 1 -oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (Mal-6) in intact erythrocyte membranes were mainly qualitative in nature and showed similar, but not identical re~ul t s .~~ .” However, they demonstrated the potential of the EPR method in providing dynamic information for proteirls in red cell membranes. Our biochemical understanding of the erythrocyte membrane components was also limited at that time. Following the electrophoretic identification of several distinct proteins in the membrane^,^ the basic knowledge of the chemical composition, molecular arrangement, and functional properties of erythrocyte membranes has flourished in recent year^.^^.*^ Meanwhile, new EPR techniques have been developed for studying molecules with slow We have compared two of the saturation transfer (ST) EPR techniques (V; and U;) with the conventional EPR method ( Vl) in monitoring molecules in erythrocyte membranes and have shown that V; detection is useful for these systems.* Thus, with the recent enhancements in both red cell membrane biochemistry and EPR techniques, the spin-labeled membrane protein system in erythrocytes can now be studied more quantitatively.

NMR analysis of secondary structure and dynamics of a recombinant peptide from the N-terminal region of human erythroid α-spectrin

We have studied the nuclear magnetic resonance solution secondary structure of the N‐terminal region in human erythroid α‐spectrin using a recombinant model peptide of α‐spectrin consisting of residues 1–156. Pulsed field gradient diffusion coefficient measurements show that the model peptide exists as a monomer under the solution conditions used. The first 20 residues are in a random coil conformation, followed by a helix of 25 residues and then a random coil segment before the next helix. The random coil nature of this linker was confirmed by the presence of fast internal motion from 15N relaxation measurements. The second, third and fourth helices are thought to form the triple helical bundle structural domain, consistent with previous studies. Our study shows that the N‐terminal region of α‐spectrin prior to the first structural domain forms a well behaved helix without its β‐spectrin partner.

Reduced water exchange in sickle cell anemia red cells: a membrane abnormality

We have measured the diffusional water permeability of sickle cell anemia red blood cells under isotonic conditions using pulsed nuclear magnetic resonance (NMR) techniques. We have found that the equilibrium diffusional permeability for sickle cells is about 1.61.10(-3) cm/s, or about 60% of the value measured for normal cells. This abnormality is not related to the heterogeneity generally found in cell populations in sickle red cells with different mean corpuscular hemoglobin concentrations. We speculate that the abnormality of water exchange under isotonic conditions in sickle cells reflects an alteration of membrane proteins responsible for water exchange, possibly caused by oxidation of Band 3 proteins.

Quantitative detection of rapid motions in spectrin by NMR.

Previous high resolution proton NMR data on human erythrocyte spectrin molecules has indicated the existence of regions exhibiting rapid internal motions within the intact molecules [L. W.-M. Fung, H.-Z. Lu, R. P. Hjelm, jr, M. E. Johnson, FEBS Lett., 197, 234 (1986)]. We have extended the studies by developing quantitative NMR methods to determine the fraction of spectrin protons exhibiting rapid internal motions, in both the isolated molecule and within the spectrin-actin network. Using both one-pulse and spin echo pulse sequences, we find that the fraction of the protons in rapid motion is about 15% of the total protons in the spectrin molecule at 37 degrees C in phosphate buffer with 150 mM NaCl at pH 7.4. Quantitative information on these rapid motions will be important in understanding the structural, mechanical and functional properties of spectrin molecules, as well as in understanding filamentous protein structures in general.

Letter to the Editor: 1H, 15N, and 13C NMR backbone assignments of the N-terminal region of human erythrocyte alpha spectrin including one structural domain

Human erythrocyte spectrin is a major constituent of the red blood cell skeletal protein network. Spectrin is thought to be responsible for the remarkable elasticity and stability of the erythrocyte membrane, and is composed of α and β subunits. Each subunit is mainly composed of multiple homologous sequence motifs, each consisting of about 106 amino acid residues in a triple helical bundle conformation. α and β subunits interact laterally in an antiparallel manner with high affinity (KD of ∼10 nM) to form an αβ heterodimer. The heterodimer associates with the N-terminal region of the α subunit (Nα region) and the C-terminal region of the β subunit (Cβ region), producing the physiologically relevant heterotetramer (αβ)2. Sequence homology has shown that the Nα region consists of a partial domain of about 30 residues prior to the first complete domain, with a structure similar to helix C (the third helix) in the triple helical bundle domain, and that the Cβ region also has a partial domain that resembles helices A and B (the first and second helices). It has been suggested that, in the tetramerization process, these regions interact together to form a final structure resembling the triple helical bundle domain. Mutations in these regions lead to altered spectrin tetramerization and eventual weakening of the erythrocyte membrane (Lux and Palek, 1995; Hassoun and Palek, 1996; Gallagher, 1998). Although the 3D structure of a single domain for Drosophila spectrin was reported using X-ray crystallography (Yan et al., 1993)

Recent Developments in Spin Label EPR Methodology for Biomembrane Studies

Publisher Summary This chapter discusses the basic considerations appropriate to the experimental use of spin-label electron paramagnetic resonance (EPR) and discusses several applications in membrane systems. It describes some new developments in spin-label EPR techniques. Moreover, it focuses primarily on saturation transfer EPR studies and on protein systems in membranes. The types of measurements are explained that is done using only conventional EPR techniques. For nonmembrane systems, vanadyl ions, which are oxidized by mitochondria and purified cytochrome c oxidase, have also been used as EPR probes. The chapter also discusses that in addition to studying spin-labeled molecules in solution or in powder form, single-crystal measurements provide information on unpaired spin density, ligand-binding stereochemistry, and molecular dynamics. Spin-labeled molecules are also used for fluorescence quenching to provide information on membrane structure

Hemoglobin - membrane interaction at physiological ionic strength and temperature

The spin-label electron paramagnetic resonance (EPR) technique has been used to study the interaction between human hemoglobin and erythrocyte membranes as a function of temperature and ionic strength. We show, for the first time, experimental evidence for the existence of the interaction at physiological pH, ionic strength and temperature. In addition to the pH dependence that we have previously reported, the interactions are also temperature and ionic strength dependent. Using a simple two-state equilibrium model to analyze the EPR data, we obtain an equilibrium dissociation constant of about 8.1 +/- 5.6 X 10(-5) M for hemoglobin-membrane systems in 5 mM phosphate with 150 mM NaCl at pH 7.4 and 37 degrees C.

The L49F mutation in alpha erythroid spectrin induces local disorder in the tetramer association region: Fluorescence and molecular dynamics studies of free and bound alpha spectrin

The bundling of the N‐terminal, partial domain helix (Helix C′) of human erythroid α‐spectrin (αI) with the C‐terminal, partial domain helices (Helices A′ and B′) of erythroid β‐spectrin (βI) to give a spectrin pseudo structural domain (triple helical bundle A′B′C′) has long been recognized as a crucial step in forming functional spectrin tetramers in erythrocytes. We have used apparent polarity and Stern–Volmer quenching constants of Helix C′ of αI bound to Helices A′ and B′ of βI, along with previous NMR and EPR results, to propose a model for the triple helical bundle. This model was used as the input structure for molecular dynamics simulations for both wild type (WT) and αI mutant L49F. The simulation output structures show a stable helical bundle for WT, but not for L49F. In WT, four critical interactions were identified: two hydrophobic clusters and two salt bridges. However, in L49F, the region downstream of Helix C′ was unable to assume a helical conformation and one critical hydrophobic cluster was disrupted. Other molecular interactions critical to the WT helical bundle were also weakened in L49F, possibly leading to the lower tetramer levels observed in patients with this mutation‐induced blood disorder.