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L W Fung

Publications and source records attributed to L W Fung.

At least 19 recordsLinked to original sources
PubMed1999

Interactions of the alpha-spectrin N-terminal region with beta-spectrin. Implications for the spectrin tetramerization reaction.

Spectrin of the erythrocyte membrane skeleton is composed of alpha- and beta-spectrin, which associate to form heterodimers and tetramers. It has been suggested that a fractional domain (helix C) in the amino-terminal region of alpha-spectrin (Nalpha region) bundles with another fractional domain in the carboxyl-terminal region of beta-spectrin (Cbeta region) to yield a triple alpha-helical bundle and that this helical bundling is largely responsible for tetramer formation. However, there are certain objections to assigning a preeminent role to this helical bundling in the tetramerization reactions. We prepared several recombinant peptides of alpha-spectrin fragments spanning only the Nalpha region (lacking the dimer nucleation site) and quantitatively studied their interaction with beta-spectrin. We found that a majority of the interactions were localized, as expected, in the Nalpha-helix C region but that there was also some contribution from the nonhomologous region. More importantly, the temperature and ionic strength dependence of this interaction in our model peptides was different from that in intact spectrin. We suggest that, although the regions involving the putative helical bundling in alpha- and beta-spectrin undoubtedly play a significant role in tetramerization, regions distal to the Nalpha-helix C region in spectrin are also involved in tetramer formation. Structural flexibility and lateral interactions may play a role in spectrin tetramerization.

Biopolymers↗
PubMed1999

A new model system for lipid interactions in stratum corneum vesicles: effects of lipid composition, calcium, and pH.

We prepared large unilamellar vesicles (LUVs) with three different stratum corneum lipid compositions: constant amounts of ceramides (55 wt %) and fatty acids (15%) with varying amounts of cholesterol sulfate (0-15%) and cholesterol (15-30%). One of the compositions served as a model for normal stratum corneum, while the second one served as a model for recessive X-linked ichthyosis stratum corneum. The third composition consisted of no cholesterol sulfate. Intervesicle lipid interactions in these LUVs were monitored by fluorescence methods for content leakage, and contents mixing at pH 9, in the absence and presence of Ca2+, and at pH 6. Since the content leakage and contents mixing assays were originally developed for phospholipid vesicles, we characterized the probe binding and the probe quenching properties for stratum corneum LUV systems, and modified the assays slightly accordingly. The time-dependent fluorescence intensity changes in the probe-containing LUVs at pH 9 and 6 and in response to the addition of calcium were monitored. Our results demonstrated that all three types of LUVs were relatively stable at pH 9. Addition of Ca2+ or decreasing the pH to 6 activated intervesicle lipid mixing followed by vesicle fusion and lysis. We found that the LUVs with no cholesterol sulfate and 30% cholesterol exhibited a more extensive Ca2+- or low-pH-activated intervesicle lipid interaction than LUVs with either 5% cholesterol sulfate and 25% cholesterol or 15% cholesterol sulfate and 15% cholesterol. These results suggest that fusogenic agents such as Ca2+ and H+ act to neutralize the fatty acids in the lipid bilayer of stratum corneum vesicles. The inclusion of 5-15% cholesterol sulfate helps to prevent the collapse of fused vesicles into other structures.

Animals↗
PubMed1998

Ionic strength effect on the thermal unfolding of alpha-spectrin peptides.

In previous work, we have shown that the ionic strength-mediated differences found for the hydrodynamic dimensions of the human erythrocyte spectrin are not caused by secondary structural changes, but are caused more probably by subtle changes in tertiary interactions (LaBrake, C. C., Wang, L., Keiderling, T. A., and Fung, L. W.-M. (1993) Biochemistry 32, 10296-10302.). The substructure of spectrin has been suggested to be composed largely of triple alpha-helical bundle structural domains in tandem. In the present study, we used fluorescence and circular dichroism methods to study ionic strength effects on intact spectrin dimers and on recombinant peptides of spectrin domains of different lengths. We observed little ionic strength effect on the thermal unfolding temperature, Tm, values in these systems. However, we found that ionic strength-induced cooperativity in the unfolding processes was similar for the spectrin dimer and for peptides with two or three domains, as measured by entropy changes (DeltaSm). Although single-domain peptides exhibited rather variable DeltaSm values, depending on the specific domain, they showed little salt effects on the DeltaSm values themselves. This suggests that spectrin undergoes subtle ionic strength-induced conformational changes, probably near the interdomain regions of the molecule. These conformational changes may be responsible for the observed hydrodynamic and unfolding properties in intact spectrin under different ionic strength conditions. We suggest that recombinant peptides of various lengths may serve as models for studying the structural flexibility in spectrin.

Circular Dichroism↗
PubMed1998

Sickle hemoglobin is more fusogenic than normal hemoglobin at physiological pH and ionic strength conditions.

We used electron microscopy, quasi-elastic light scattering and static light scattering to show that human hemoglobin (Hb) interacts with bovine brain phosphatidylserine lipid vesicles and promotes vesicle fusion in an isotonic buffer at pH 7.4. The fusogenic properties of Hb were observed in both small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs). A simple turbidity measurement method was used to follow increases in vesicle size (scattering diameter) as a function of time. For the first 3 h, upon incubation with oxygenated Hb, the scattering diameters of vesicles increased at a rate of 7.8 nm/h for LUVs. Continuous incubation with Hb led to complicated vesicle fusion, probably due to the oxidation products of Hb and lipid molecules. In the absence of both Hb and lipid oxidation, using Hb liganded with carbon monoxide, we obtained, for the entire 20 h incubation period, a fusion rate of 2.9 nm/h for LUVs. We also studied interactions between sickle Hb and vesicles under the same conditions and found that the vesicle fusion rates for sickle Hb were about 2 times faster than those for normal Hb. These results showed that sickle Hb exhibited more extensive interactions with lipid bilayer than normal Hb at physiological pH and ionic strength conditions, and provide insights toward understanding the molecular mechanisms in sickle cell abnormalities.

Carboxyhemoglobin↗
PubMed1997

Titanium-porcelain system. Part II: Bond strength of fired porcelain on nitrided pure titanium.

Commercially pure titanium (CPT) substrate was subjected to porcelain firing and bond strengths under three-point bending mode (span length: 15 mm; crosshead speed: 0.5 mm/min) were evaluated. Experimental variables included surface treatments of CPT and porcelain firing schedules. Variables for the surface treatments were (1) sandblasting, (2) mono- and triple-layered nitridation, and (3) mono-layered chrome-doped nitridation. Variables for the porcelain firing schedule included (4) bonding agent application, (5) bonding agent plus gold bonding agent application, and (6) Procera porcelain application. All together eleven sample groups were prepared with different combination of aforementioned experimental variables. Statistically all of them exhibited no significant differences. Hence, we employed two further criteria; (I) the minimum bond strength should exceed the maximum porcelain strength per se, and (II) the CPT substrate should not be heated close to the beta-transus temperature. After applying these criteria, it was concluded that mono-layered nitridation and mono-layered application of chrome-doped nitridation on both (with and without) sandblasted and non-sandblasted surfaces were the most promising conditions for a successful Titanium-Porcelain System.

Dental Porcelain↗
PubMed1996

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 alpha-subunit and 17 in the beta-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.

Circular Dichroism↗
PubMed1996

Erythrocyte spectrin maintains its segmental motions on oxidation: a spin-label EPR study.

The segmental motions of cross-linked erythrocyte skeletal protein (spectrin-actin-protein 4.1) samples, labeled with nitroxide spin labels, were monitored by conventional first-harmonic and saturation transfer second-harmonic electron paramagnetic resonance methods. Skeletal proteins were extracted from human red blood cells and treated with three oxidative reagents (diamide, hydrogen peroxide, and phenylhydrazine) to cross-link sulfhydryl groups and with one fixative reagent (glutaraldehyde) to cross-link lysine residues. The treatments provided extensive cross-linking between spectrin-actin-protein 4.1 molecules, as determined by gel electrophoresis, and surface charge modification, as determined by pl measurements. However, segmental motions of the cross-linked skeletal proteins remained generally similar to those in normal skeletal proteins. Both the weakly immobilized and the strongly immobilized motions were similar in cross-linked and control samples. Small differences in some motional components were detected. In some cases, faster mobilities were observed, with approximately 5% of the strongly immobilized motions converted to the weakly immobilized motions in the cross-linked samples. It is often believed that the consequence of membrane protein oxidation is restricted protein dynamics, giving membrane rigidity. However, our studies provide needed experimental evidence to indicate that segmental motions are maintained with very little modification even in the presence of extensive cross-linking. Thus cross-linking does not restrict the internal molecular flexibility that gives rise to segmental motions.

Actins↗
PubMed1995

Molecular properties of a stratum corneum model lipid system: large unilamellar vesicles.

Stratum corneum lipids are relatively complex, and there is little detailed understanding of their chemical and physical properties at the molecular level. Large unilamellar vesicles (LUVs) with lipid compositions similar to those of stratum corneum were prepared at pH 9 with commercially available lipids. This system was used as a model system for molecular studies of stratum corneum lipids. LUVs were chosen as the model system as they are comparatively more stable and can be characterized more quantitatively in terms of lipid concentration, surface area, and volume than model systems such as lipid mixture suspensions, lipid films, and small unilamellar vesicles. Results from freeze-fracture and cryo electron microscopy studies of our LUVs showed spherical vesicles. Quasi-elastic light scattering measurements revealed a narrow size distribution, centering around 119 nm. At room temperature, the LUVs were stable for several weeks at pH 9 and for more than 15 h but less than 24 h at pH 6. Differential scanning calorimetry measurements indicated broad endothermic transitions centered near 60-65 degrees C, closely matching the transition temperature reported for stratum corneum lipid extracts. Spin probes, 5-doxylstearic acid and 12-doxylstearic acid, were used for electron paramagnetic resonance (EPR) studies of the molecular dynamics of the lipids. EPR results indicated more restricted motion near the polar headgroup region than near the center of the alkyl chain region. Motional profiles of the spin labels near the polar headgroup and within the alkyl chain region in the LUVs were obtained as a function of temperature, ranging from 25 to 90 degrees C. We also found that the partitioning between the lipid and aqueous phases for each spin probe was temperature dependent and was generally correlated with phase transitions observed by differential scanning calorimetry and with alkyl chain mobility observed by EPR. Thus, this LUV system is well suited for additional molecular studies under different experimental conditions.

Animals↗
PubMed1994

The first human alpha-spectrin structural domain begins with serine.

The 106-amino acid sequence motifs of spectrin have been suggested to fold into stable structural domains, consisting mostly of coiled coils of triple helices. With the advent of molecular biology and biophysical techniques, structural studies of these spectrin 106-amino acid structural domains became approachable. However, one of the difficulties in such an approach is determination of the correct phasing of the structural domains, which may or may not coincide with the phasing of the sequence motifs. Proper identification of the domain phasing is vital to the construction of stable spectrin domains for molecular studies. A previously published phasing shift for Drosophila alpha-spectrin indicated a downstream phase-shift of 26 amino acids for the structural domain (Winograd, E., Hume, D., and Branton, D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10788-10791). Using this phase-shift, we prepared a recombinant spectrin peptide with the sequence from residue 49 to residue 155 of human erythrocyte alpha-spectrin and found this peptide to be unstable relative to other peptides that we prepared. Using several other recombinant alpha-spectrin peptides and following the protease digestion approach, we digested spectrin peptides with elastase and chymotrypsin and analyzed the amino acid sequence of the digestive products. We provide the first experimental evidence in identifying the first amino acid residue of the first spectrin domain in human erythrocyte alpha-spectrin as residue 52 (Ser).

Amino Acid Sequence↗
PubMed1994

The roles of ascorbic acid and other antioxidants in the erythrocyte in reducing membrane nitroxide radicals.

Fatty acid nitroxide radicals in CO-gassed erythrocytes are reduced by intracellular components with a half-life of about 160 min. In this study, using reduction rate constants of fatty acid spin labels to determine the reduction quantitatively, we found that catalase, glutathione, glutathione peroxidase, superoxide dismutase, and vitamin E--as well as hemoglobin, individually or in concerted manner, contributed little in reducing membrane nitroxides. Ascorbic acid appeared to be the predominant component in the erythrocyte to reduce membrane nitroxides. However, ascorbic acid solutions at 0.1 mM or less, concentrations similar to those found in the erythrocyte, produced no observable reduction in spin labeled membranes during the 2 h monitoring period. Ascorbic acid solutions at about 1 mM were needed to exhibit rate constants similar to those observed in labeled erythrocyte samples. It was also found that beta-nicotinamide adenine dinucleotide, beta-nicotinamide adenine dinucleotide phosphate, and heat-sensitive components in the erythrocyte enhanced the ability of ascorbic acid to scavenge nitroxide radicals in the erythrocyte membrane near the membrane surface.

Adult↗
PubMed1993

Fourier transform infrared spectroscopic studies of the secondary structure of spectrin under different ionic strengths.

Spectrin, a highly dynamic skeletal membrane protein, plays an important role in maintaining the disk biconcave shape of the human erythrocyte. The sequence of spectrin is mostly composed of repeating segments of 106 amino acids which have been proposed to form unique structural domains. Electronic and vibrational circular dichroism and Fourier transform infrared (FTIR) spectroscopy were used as complementary techniques to study the secondary structure of spectrin. The amide I and II regions of the FTIR absorbance spectra were analyzed using partial least-squares analysis. The secondary structure of spectrin under physiological buffer conditions was estimated to be about 70% alpha-helix, 10% beta-sheet, and 20% other. We believe that this is the first detailed experimental evidence of significant beta-sheet content in spectrin secondary structure. The antiparallel beta-sheet SH3 domain in the center of the alpha-subunit in spectrin accounts for only about 1.5% of the total amino acid residues in the dimer. Hydrodynamic studies have shown spectrin to be sensitive to changes in ionic strength and to addition of denaturing agents. Our FTIR results showed that the secondary structure of spectrin treated with detergent or NaOH changed by 10-20%. The Stokes radii of the spectrin samples used for FTIR measurements were found to vary as a function of the ionic strength, but their secondary structures did not change as a function of ionic strength. These results indicate that while the overall hydrodynamic dimension of spectrin depends on the medium ionic strength, the secondary structure remains essentially constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Buffers↗
PubMed1992

Phospholipid vesicles promote human hemoglobin oxidation.

We have studied the heme oxidation kinetics of purified human hemoglobin (Hb) in the presence of lipid vesicles of dipalmitoyl phosphatidylcholine and bovine brain phosphatidylserine that exhibited minimal lipid peroxidation. We showed that the lipid vesicles enhanced Hb oxidation and that small unilamellar vesicles (SUVs) exerted a larger effect than large unilamellar vesicles (LUVs). We have determined pseudo first-order rate constants for the initial disappearance of oxygenated ferrous Hb (k0) and for the initial formation of several ferric Hb species (methemoglobin, hemichrome, and choleglobin) in the presence of SUVs and LUVs. k0 and other rate constants depended linearly on lipid-to-hemoglobin molar ratio (lipid/Hb), with k0SUV (h-1) = k0auto (h-1) + 3.7 x 10(-3) x lipid/Hb, and k0LUV (h-1) = k0auto (h-1) + 0.2 x 10(-3) x lipid/hb, where k0auto is the rate constant for Hb autoxidation in the absence of vesicles. Thus, in the absence of lipid peroxidation products, lipid vesicles themselves promote Hb oxidation by enhancing the rate of Hb oxidation. The enhanced oxidation was inhibited by catalase, but not by butylated hydroxytoluene. The rate constants were independent of Hb concentration, in the range of about 3.1 to 100 microM. We suggest that the lipid surface properties, including surface curvature, surface energy, and hydrophobicity, promote hemoglobin oxidation.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Dynamic light scattering investigations of human erythrocyte spectrin.

Dynamic light scattering measurements were performed on spectrin from human erythrocytes in 25 mM Tris buffer at pH 7.6 with 100 mM NaCl and 5 mM EDTA. Measurements were made on spectrin solutions prepared as dimers and tetramers over the temperature range from 23 to 41 degrees C, as a function of the square of the scattering vector (K2) over the range of 0.7 x 10(10) cm-2 less than or equal to K1 less than or equal to 20 x 10(10) cm-2. Analysis of the autocorrelation functions collected for these solutions revealed the presence of two predominant motional components over the entire range of K2. Plots of the diffusion coefficients (D20) of these components, with viscosity and temperature corrected to water at 20 degrees C, as a function of K2 indicated three rather distinct regions, flat regions at low and high K2 joined by a sloping intermediate region. At small K2 (less than or equal to 4 x 10(10) cm-2) the D20 values were (7.3 +/- 2.0) x 10(-8) cm2/s for the slow component and (20.3 +/- 2.0) x 10(-8) cm2/s for the fast component. At large K2 (greater than or equal to 10 x 10(10) cm-2) the values increased to (13.0 +/- 2.0) x 10(-8) cm2/s for the slow component and (39.4 +/- 2.0) x 10(-8) cm2/s for the fast component. In the intermediate K2 region, D20 is a linear function of K2 and appears as a transition between the low and high K2 regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Diffusion↗
PubMed1992

Antisickling activity of hydroxybenzoic acids in Cajanus cajan.

The amounts of phenylalanine and hydroxybenzoic acid in a Cajanus cajan methanolic extract were estimated. Results showed that the amount of phenylalanine and hydroxybenzoic acid per gram weight of bean was 4.92 mg +/- 0.13 mg and 21.0 mg +/- 3.0 micrograms, respectively. Sickling inhibition was observed to be efficient with the extract which contains a mixture of phenylalanine (0.69 mg/ml) and p-hydroxybenzoic acid (10.5 micrograms/ml), equivalent to those found in bean extract. The additive antisickling effect of both compounds can be therapeutically exploited for the treatment of sickle cell anemia.

Antisickling Agents↗
PubMed1990

A method to evaluate the antioxidant system for radicals in erythrocyte membranes.

The erythrocyte defense system against cellular oxidants is complex and efficient. Free radicals generated in cell membranes, however, are relatively sequestered from the cell's antioxidant mechanisms. When an oxidant challenge exceeds the capacity of the erythrocyte's antioxidant system, membrane damage may occur, causing red cell destruction and hemolytic anemia. In this study, we present a method for monitoring radical reduction in erythrocyte membranes, using fatty acid spin labels with nitroxide radicals on the hydrocarbon chains. About 50 microL of packed (about 5-6 x 10(8)), carbon monoxide (CO)-gassed red blood cells are used. The electron paramagnetic resonance signals of the 5-doxylstearic acid spin labels in the intact cells are obtained as a function of time, at 37 degrees C over a period of 2 h. The pseudo first-order rate constant for reduction of the spin label in normal adult intact cells under our experimental conditions is 4.3 +/- 1.8 x 10(-3)/min. The reproducibility and variability of the measurements are discussed. Since the measurements we describe reflect the extent of radical reductions occurring in cell membranes, we suggest that this method can be used to measure the ability to defend oxidants in membranes of erythrocytes with defective antioxidant systems. This method is particularly useful for measuring the modification of the antioxidant system toward radicals in membranes by drugs, chemicals, or environmental toxins.

Adult↗
PubMed1990

Secondary structure prediction for the spectrin 106-amino acid segment, and a proposed model for tertiary structure.

A collective secondary structure prediction for the human erythrocyte spectrin 106-residue repeat segment is developed, based on the sequences of nine segments that have been reported in the literature, utilizing a consensus of several secondary structure prediction methods for locating turn regions. The analysis predicts a five-fold structure, with three alpha-helices and two beta-strand regions, and differs from previous models on the lengths of the helices and the existence of beta-strand structure. We also demonstrate that this structural motif can be folded into tertiary structures that satisfy the experimental spectrin data and several general principles of protein organization.

Circular Dichroism↗
PubMed1989

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.

Anemia, Sickle Cell↗