Search PubMed⌕ Search

Biomedical subjects

L W Fung

Publications and source records attributed to L W Fung.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1989

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.

Erythrocyte Membrane↗
PubMed1987

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.

Electron Spin Resonance Spectroscopy↗
PubMed1986

Selective detection of rapid motions in spectrin by NMR.

Human erythrocyte spectrin molecules exhibit relatively sharp (30-50 Hz) proton NMR signals in the aliphatic region. A standard solvent presaturation pulse sequence that also partially suppresses the broad envelope from protons with rigid structures in spectrin and selectively enhances the sharp resonances has been used to characterize the behavior of these resonances. The overall resonance pattern strongly resembles that of the denatured spectrin. The observed spectra are also quite similar to the line-broadened spectrum from a mixture of amino acids that corresponds to the composition of the spectrin molecule. These data indicate the existence of regions exhibiting rapid internal motions within the intact spectrin molecule, and suggest that the amino acid composition of the residues giving rise to the sharp resonances is quite similar to that of the full spectrin molecule.

Actins↗
PubMed1986

Molecular properties of cetiedil and its interactions with erythrocyte membranes.

Cetiedil, an antisickling agent and a vascular smooth muscle relaxant, is an amphiphilic molecule. The critical micelle concentration in 5 mM phosphate buffer with 150 mM NaCl is approximately 8.8 mM. The molecule, as the citrate salt, is highly acidic at millimolar concentrations. The UV absorption extinction coefficient at 233 nm, E233, is 2796 M-1 cm-1. The studies of free cetiedil concentrations in the presence of membrane ghosts show that large amounts of cetiedil associate with membrane samples. Spin label electron paramagnetic resonance experiments showed that the lipids and the proteins of erythrocyte membrane samples were both affected by the addition of cetiedil. However, the cetiedil effects on membrane components are reversible. The protein spin label results demonstrate the binding of cetiedil to the membrane with an apparent equilibrium dissociation constant of approximately 2 mM. The binding is saturable. The apparent half-saturation concentrations for the binding at physiological ionic strength and temperature are in the range of 1-3 mmoles of cetiedil per gram of membrane proteins. Our studies also indicate that binding is not affected by the removal of the spectrin and actin network from the membranes. Interaction of cetiedil with Band 3 molecules in the erythrocyte membrane is suggested. The regions near the lipid head groups in the membrane samples are also affected by cetiedil.

Antisickling Agents↗
PubMed1984

Saturation transfer EPR studies of membrane alteration in hereditary spherocytosis.

Our earlier spin label electron paramagnetic resonance (EPR) studies of hereditary spherocytosis (HS) erythrocyte revealed the existence of structural alteration(s) when the membrane is subjected to heat stress. We have now used saturation transfer EPR to show restricted motion in membrane proteins even without subjecting membrane to stress. The abnormal motional behavior is amplified when membranes are incubated at 47 degrees C and is readily detectable by conventional EPR. Gel electrophoresis and lipid assays show that proteins but not lipids are released upon heating. Thus, the more restricted motions in HS membranes may be due to a different membrane protein organization, ultimately resulting in the abnormal morphology of HS cells.

Electron Spin Resonance Spectroscopy↗
PubMed1983

Spin-label detection of sickle hemoglobin--membrane interaction at physiological pH.

The spin-label electron paramagnetic resonance technique has been used to compare the interactions of normal and sickle hemoglobin molecules with human erythrocyte membranes. The sickle hemoglobin molecules show an enhanced binding to membranes when compared to normal hemoglobin (HbA) molecules. Using a simple equilibrium model for hemoglobin--membrane interactions, we obtain an equilibrium dissociation constant for sickle hemoglobin of about half that of HbA at pH 7.4 in 5 mM phosphate at 20 degrees C. The interactions are very low affinity in nature and are stronger at lower pH than at pH 7.4 (Fung, 1981a). The difference between normal and sickle hemoglobin persists at both high (pH 7.4) and low (pH 6.7) pH values. The concentrations of hemoglobin at the saturation level are close to physiological concentrations. Removal of spectrin--actin protein molecules from the membranes causes little change in the interactions, indicating that the remaining membrane proteins play the primary role in hemoglobin--membrane interactions. This observation is further supported by data of spectrin--actin-depleted inside-out vesicle samples. The stronger interaction of sickle hemoglobin than normal hemoglobin with membranes is discussed in relation to the formation of irreversibly sickled cells.

Actins↗
PubMed1982

Models for slow anisotropic rotational diffusion in saturation transfer electron paramagnetic resonance at 9 and 35 GHz.

Model systems of cholestane and 5-doxylstearic acid analogue spin probes in lipid bilayer dispersions of dipalmitoylphosphatidylcholine and cholesterol (9:1 w/w) are used to analyze saturation transfer electron paramagnetic resonance spectral behavior for slow rotational diffusion in an anisotropic medium. Measurements are made at both 9 and 35 GHz to provide enhanced spectral resolution for different types of motion. Parameter correlation plots of spectral parameters from different regions of the saturation transfer spectra appear to be potentially useful in characterizing different types of motion. Anisotropic rotational diffusion about a symmetry axis coincident with the nitroxide y principal axis is clearly distinguishable from isotropic rotational diffusion and may be distinguishable from rotational diffusion about the nitroxide z principal axis. Approximate anisotropic rotational diffusion about a symmetry axis coincident with the nitroxide z principal axis is distinguishable from isotropic rotational diffusion under some, but not all, conditions.

Cholestanes↗
PubMed1982

Oxidation of phenidone and BW755C by prostaglandin endoperoxide synthetase.

1-Phenyl-3-pyrazolidone (phenidone) and 3-amino-1-(m-(trifluoromethyl)-phenyl)-2-pyrazoline (BW755C) are oxidized by the hydroperoxidase component of prostaglandin endoperoxide synthetase and by horseradish peroxidase. The initial oxidation products are radical cations which exhibit visible absorption maxima at 514, 490, and 472 nm (phenidone) and 535, 500, and 488 nm (BW755C). The radical cation of phenidone can be detected by electron paramagnetic resonance spectroscopy as a complex multiline signal centered at g = 2.0039. In addition to being oxidized by peroxidases both compounds are cofactors for the reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid to 15-hydroxy-5,8,11,13-eicosatetraenoic acid by the hydroperoxidase activity of purified and hematin-reconstituted prostaglandin endoperoxide synthetase. As a consequence of their oxidation by prostaglandin endoperoxide synthetase, phenidone and BW755C inhibit the hydroperoxide-dependent oxidation of phenylbutazone, luminol, diphenylisobenzofuran, epinephrine, and guaiacol by ram seminal vesicle microsomes. The inhibition of phenylbutazone oxidation is competitive and exhibits Ki values of 16 microM (BW755C) and 45 microM (phenidone). BW755C and phenidone stimulate prostaglandin biosynthesis by purified and reconstituted prostaglandin endoperoxide synthetase at concentrations up to 100 microM but inhibit at higher concentrations (I50 values approximately 210 microM and 1180 microM, respectively). The ability of phenidone and BW755C to act as peroxidase reducing cofactors or radical scavengers may contribute to their observed biochemical and pharmacological effects.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗