Bio


Professor Wray Huestis’ research concerns the molecular mechanisms whereby cells control their shape, motility, deformability and the structural integrity of their membranes. Metabolic control of interprotein and protein-lipid interactions is studied by a variety of biochemical, spectroscopic and radiochemical techniques, including fluorescence and EPR spectrometry, autoradiography and electron microscopy. The role of lipid metabolism and transport in regulating the fluid dynamics of cell suspensions (red blood cells, platelets, lymphocytes) is examined using circulating cells and cells grown in culture. Cell-cell and cell-liposome interactions are studied using model membrane systems with widely differing physical properties. Complexes of liposomes and encapsulated viruses are used as selective vectors to deliver water-soluble compounds across the membranes of intact cells. The particular projects described in the listed publications have as a common goal an understanding of the molecular workings of the cell membrane.

Academic Appointments


Administrative Appointments


  • Vice-Chair, Department of Chemistry, Stanford University (2005 - 2011)
  • Associate Professor, Department of Chemistry, Stanford University (1980 - 1986)
  • Assistant Professor, Department of Chemistry, Stanford University (1974 - 1980)
  • Sloan Fellow, Stanford University (1977 - 1979)

Honors & Awards


  • Distinguished Teaching Award, Stanford University (1988)
  • Sloan Foundation Fellow, Alfred P. Sloan Foundation (1977-79)
  • National Institutes of Health Research Career Development, NIH (1977 - 1981)

Boards, Advisory Committees, Professional Organizations


  • Member, Stanford University Committee on Libraries (2012 - 2013)
  • Member, Stanford Chemistry Department Graduate Studies Committee (2011)
  • Member, Stanford Chemistry Department Curriculum Committee (2011)
  • Member, ad hoc committee on design of Teaching and Learning Center Chemistry and Biology Departments (2011 - 2013)
  • Ombuds, Chemistry Department (2003 - 2011)
  • Member, Phi Beta Kappa (1997)
  • Chair, Stanford Chemistry Department Committee on Teaching Assignments (1990 - 2011)
  • Member, National Science Foundation Graduate Fellowship Panel (1989 - 1992)
  • Member, Red Cell Club (1980)
  • Member, Biophysical Society (1979)
  • Member, National Institutes of Health Study Section on Biophysics and Biophysical Chemistry (1977 - 1980)
  • Member, American Chemical Society (1967)

Professional Education


  • NIH Postdoctoral Fellow, Stanford University, Chemistry (1974)
  • PhD, California Institute of Technology, Biophysics, minor in Chemistry (1972)
  • BA, Macalester College, Chemistry (1967)

All Publications


  • Selective amphipathic nature of chlorpromazine binding to plasma membrane bilayers BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Chen, J. Y., Brunauer, L. S., Chu, F. C., Helsel, C. M., Gedde, M. M., Huestis, W. H. 2003; 1616 (1): 95-105

    Abstract

    Chlorpromazine (CPZ), an antipsychotic agent shown to inhibit the action of various neurophysiological receptors, also exhibits preferential association with the plasma membrane, inducing stomatocytic morphological response in red blood cells (RBC). Given the cationic nature of CPZ, fluorimetry, pH titration, and red cell morphological studies were performed to assess the associative predilection of CPZ for anionic membrane components. CPZ fluorescence intensity increased 320-370% upon addition of phosphatidylcholine (PC) small unilamellar vesicles (SUVs) to aqueous CPZ, indicating an affinity of the drug for lipidic phases. After removal of unbound drug, CPZ fluorescence increased up to 92% with increasing phosphatidylserine (PS) in the lipid phase (up to 30 mol% of total lipid), suggesting a preferential association of the drug with anionic lipids. In studies of pH titration, the pK(a) of CPZ in the presence of Triton X-100 micelles or phospholipid SUVs increased with increasing anionicity of the lipidic phase [7.8 with Triton X-100, 8.0 with PC, 8.3 with phosphatidylglycerol (PG)], lending further support to preferential drug interaction with anionic lipidic components. At 0 degrees C, CPZ-induced red cell shape change was less extensive in cells made echinocytic by adenosine triphosphate (ATP) depletion, compared to cells made echinocytic by PS treatment following vanadate preincubation. This suggests that polyphosphoinositide lipids are CPZ membrane binding sites. Since polyphosphoinositide lipids are implicated as important intermediates in a number of receptor-mediated cell signaling pathways, evidence of association with these specific lipids provides a means by which psychoactive drugs may induce neurophysiological effects through direct interaction with general membrane components.

    View details for DOI 10.1016/S0005-2736(03)00229-3

    View details for Web of Science ID 000185590700003

    View details for PubMedID 14507423

  • Preparation and analysis of small unilamellar phospholipid vesicles of a uniform size BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pitcher, W. H., Huestis, W. H. 2002; 296 (5): 1352-1355

    Abstract

    The interaction of carbonmonoxyhemoglobin and heme with small unilamellar phospholipid vesicles was studied using dynamic light scattering. Addition of carbonmonoxyhemoglobin to dimyristoylphosphatidylcholine:dimyristoylphosphatidylserine small unilamellar vesicles resulted in an increase of average vesicle size from 17.4 to 32.0nm. Addition of heme to vesicles produced a smaller size increase, from 17.4 to 21.0nm. Also reported is a method for preparing small unilamellar lipid vesicles of a uniform size, suitable for use in NMR spectroscopy.

    View details for Web of Science ID 000177987600050

    View details for PubMedID 12207924

  • Interaction of nominally soluble proteins with phospholipid monolayers at the air-water interface BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Pitcher, W. H., Keller, S. L., Huestis, W. H. 2002; 1564 (1): 107-113

    Abstract

    The interactions of carbonmonoxyhemoglobin (HbCO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and polyhistidine with phospholipid monolayers at the air-water interface were studied at physiological pH and ionic strength. HbCO and GAPDH both interact more strongly with monolayers containing negatively charged lipids. The interaction of HbCO and GAPDH with lipid monolayers decreases with increasing pH. Both the HbCO-monolayer and the GAPDH-monolayer interactions can be modeled as diffusion-limited processes, with kinetic data fit to a stretched exponential equation. The significance of these kinetics are discussed. Polyhistidine interacts only with monolayers containing lipids with negatively charged headgroups. In total, the results presented are consistent with an HbCO-lipid interaction with a large electrostatic component, a GAPDH-lipid interaction with comparable electrostatic and hydrophobic components, and a polyhistidine-lipid interaction that is solely electrostatic.

    View details for Web of Science ID 000176896700015

    View details for PubMedID 12101002

  • Selective virus-mediated intracellular delivery of membrane-impermeant compounds by means of plasma membrane vesicles ANTIVIRAL RESEARCH Trigiante, G., Huestis, W. H. 2000; 45 (3): 211-221

    Abstract

    The impermeability of the cell plasma membrane is a major obstacle to intracellular delivery of large hydrophilic molecules, such as many kinds of drugs. This contribution describes a general-purpose delivery system that employs the membrane fusion capacity of enveloped viruses to circumvent cell impermeability. Vesicles were generated from the plasma membrane of HEp-2 cells, a human cell line host for the Newcastle disease virus (NDV). They could be loaded with a fluorescent, high molecular weight dye (FITC/dextran, MW 70 KDa) or with the enzyme ribonuclease A (MW 14 KDa). These vesicles were found to fuse and deliver their lumen contents to cultured HEp-2 cells in the presence of NDV virions. When ribonuclease was employed as the encapsulated solute, viral replication was inhibited and death of the infected cells was accelerated. Implications and possible applications of this technique in antiviral therapy are discussed.

    View details for Web of Science ID 000086736100005

    View details for PubMedID 10771084

  • Resolution of the paradox of red cell shape changes in low and high pH BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Gedde, M. M., Yang, E. Y., Huestis, W. H. 1999; 1417 (2): 246-253

    Abstract

    The molecular basis of cell shape regulation in acidic pH was investigated in human erythrocytes. Intact erythrocytes maintain normal shape in the cell pH range 6.3-7.9, but invaginate at lower pH values. However, consistent with predicted pH-dependent changes in the erythrocyte membrane skeleton, isolated erythrocyte membranes evaginate in acidic pH. Moreover, intact cells evaginate at pH greater than 7.9, but isolated membranes invaginate in this condition. Labeling with the hydrophobic, photoactivatable probe 5-[125I]iodonaphthyl-1-azide demonstrated pH-dependent hydrophobic insertion of an amphitropic protein into membranes of intact cells but not into isolated membranes. Based on molecular weight and on reconstitution experiments using stripped inside-out vesicles, the most likely candidate for the variably labeled protein is glyceraldehyde-3-phosphate dehydrogenase. Resealing of isolated membranes reconstituted both the shape changes and the hydrophobic labeling profile seen in intact cells. This observation appears to resolve the paradox of the contradictory pH dependence of shape changes of intact cells and isolated membranes. In intact erythrocytes, the demonstrated protein-membrane interaction would oppose pH-dependent shape effects of the spectrin membrane skeleton, stabilizing cell shape in moderately abnormal pH. Stabilization of erythrocyte shape in moderately acidic pH may prevent inappropriate red cell destruction in the spleen.

    View details for Web of Science ID 000079122500006

    View details for PubMedID 10082800

  • Interactions of a vesicular stomatitis virus G protein fragment with phosphatidylserine: NMR and fluorescence studies BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Hall, M. P., Burson, K. K., Huestis, W. H. 1998; 1415 (1): 101-113

    Abstract

    The interaction of a 19 amino acid vesicular stomatitis virus G protein fragment (GTWLNPGFPPQSCGYATVT) with phosphatidylserine-containing model membranes was investigated using solution-phase 1d and 2d 1H NMR spectroscopy and intrinsic tryptophan fluorescence. Results of these studies show that this peptide interacts with model membranes containing negatively charged phospholipids. The interaction is modulated by both ionic and hydrophobic factors and appears to be dependent on the fluidity and lipid packing of the target bilayer. The data further suggest the existence of two isomeric forms of this peptide, which react differentially with model membranes. Upon binding, 2d 1H NOESY and tryptophan fluorescence data indicate penetration of the tryptophan residue into the bilayer. A model is proposed for the interaction of the peptide with model membranes, consistent with the experimental findings.

    View details for Web of Science ID 000077781500009

    View details for PubMedID 9858700

  • Red blood cell lipids form immiscible liquids PHYSICAL REVIEW LETTERS Keller, S. L., Pitcher, W. H., Huestis, W. H., McConnell, H. M. 1998; 81 (22): 5019-5022
  • Membrane potential and human erythrocyte shape BIOPHYSICAL JOURNAL Gedde, M. M., Huestis, W. H. 1997; 72 (3): 1220-1233

    Abstract

    Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The process is fast and reversible at room temperature, so it seems to involve shifts in weak inter- or intramolecular bonds. This shape change has been reported to depend on changes in membrane potential, but control experiments excluding roles for other simultaneously varying cell properties (cell pH, cell water, and cell chloride concentration) were not reported. The present study examined the effect of independent variation of membrane potential on red cell shape. Red cells were equilibrated in a set of solutions with graduated chloride concentrations, producing in them a wide range of membrane potentials at normal cell pH and cell water. By using assays that were rapid and accurate, cell pH, cell water, cell chloride, and membrane potential were measured in each sample. Cells remained discoid over the entire range of membrane potentials examined (-45 to +45 mV). It was concluded that membrane potential has no independent effect on red cell shape and does not mediate the membrane curvature changes known to occur in red cells equilibrated at altered pH.

    View details for Web of Science ID A1997WJ82400023

    View details for PubMedID 9138568

  • Cytoplasmic pH and human erythrocyte shape BIOPHYSICAL JOURNAL Gedde, M. M., Davis, D. K., Huestis, W. H. 1997; 72 (3): 1234-1246

    Abstract

    Altered external pH transforms human erythrocytes from discocytes to stomatocytes (low pH) or echinocytes (high pH). The mechanism of this transformation is unknown. The preceding companion study (Gedde and Huestis) demonstrated that these shape changes are not mediated by changes in membrane potential, as has been reported. The aim of this study was to identify the physiological properties that mediate this shape change. Red cells were placed in a wide range of physiological states by manipulation of buffer pH, chloride concentration, and osmolality. Morphology and four potential predictor properties (cell pH, membrane potential, cell water, and cell chloride concentration) were assayed. Analysis of the data set by stratification and nonlinear multivariate modeling showed that change in neither cell water nor cell chloride altered the morphology of normal pH cells. In contrast, change in cell pH caused shape change in normal-range membrane potential and cell water cells. The results show that change in cytoplasmic pH is both necessary and sufficient for the shape changes of human erythrocytes equilibrated in altered pH environments.

    View details for Web of Science ID A1997WJ82400024

    View details for PubMedID 9138569

  • Role of membrane lipid distribution in chlorpromazine-induced shape change of human erythrocytes BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Chen, J. Y., Huestis, W. H. 1997; 1323 (2): 299-309

    Abstract

    This is a study of the morphology and transbilayer lipid distribution of human erythrocytes treated with chlorpromazine (CPZ) over extended time courses. At 0 degree C, treatment of dilauroylphosphatidyl[1-14C]choline-labeled erythrocytes with 120 microM CPZ produced an immediate stomatocytic transformation (t1/2 < 5 min) with no concurrent change in transbilayer distribution of radiolabeled lipid, as determined by bovine serum albumin extractability. At 37 degrees C, CPZ treatment of cells produced two sequential morphological effects: immediate stomatocytosis (t1/2 < 1 min) with no concurrent change in radiolabel transbilayer distribution, followed by gradual increase in stomatocytic extent over several hours, with concurrent redistribution of radiolabeled lipid to the inner monolayer. Cells pretreated with vanadate at 37 degrees C exhibited a triphasic morphological response: CPZ produced immediate stomatocytosis, followed by a transient reversion to echinocytes lasting about 2 h, before returning to stomatocytic morphologies over the next several hours. The echinocytic reversion was accompanied by exposure of phosphatidylserine on the cell surface, as indicated by increased activation of exogenous prothrombinase. These findings suggest that while CPZ induces transbilayer lipid redistribution over extended time periods (which may mediate the complex morphological transformations observed), the early stomatocytic response elicited by addition of CPZ is not due to lipid reorganization.

    View details for Web of Science ID A1997WH04800013

    View details for PubMedID 9042352

  • Hemoglobin oxidation products extract phospholipids from the membrane of human erythrocytes BIOCHEMISTRY MOXNESS, M. S., Brunauer, L. S., Huestis, W. H. 1996; 35 (22): 7181-7187

    Abstract

    Hydrogen peroxide oxidation of human erythrocytes induces a transfer of phospholipid from the membrane into the cytosol [Brunauer, L.S., Moxness, M.S., & Huestis, W.H. (1994) Biochemistry 33, 4527-4532]. The current study examines the mechanism of lipid reorganization in oxidized cells. Exogenous phosphatidylserine was introduced into the inner monolayer of erythrocytes, and its distribution was monitored by microscopy and radioisotopic labeling. Pretreatment of cells with carbon monoxide prevented both hemoglobin oxidation and the transfer of phosphatidyserine into the cytosolic compartment. The roles of the various hemoglobin oxidation products in lipid extraction were investigated using selective oxidants. Nitrite treatment of intact cells produced almost complete conversion to methemoglobin, but no detectable lipid extraction. Treatments designed to produce the green hemoglobin derivatives, sulfhemoglobin and choleglobin, resulted in cytosolic extraction of phosphatidylserine. Ion exchange and size exclusion chromatography of oxidized cytosolic components revealed a lipid-hemoglobin complex. The interaction between lipid and hemoglobin oxidation products was verified in a model system. Purified hemoglobin, enriched in sulfhemoglobin and choleglobin by treatment with H2O2, H2S, or ascorbate, extracted phospholipid from small unilamellar phospholipid vesicles. Electron paramagnetic resonance studies demonstrated that hemoglobin oxidation products also adsorb fatty acids from solution. This newly described activity of hemoglobin may play a role in the clearance of oxidatively damaged and senescent cells from circulation.

    View details for Web of Science ID A1996UP43000027

    View details for PubMedID 8679546

  • Physical determinants of intermembrane protein transfer BIOCHEMISTRY WATERS, S. I., Sen, R., Brunauer, L. S., Huestis, W. H. 1996; 35 (13): 4002-4008

    Abstract

    Intermembrane protein transfer between erythrocytes and phospholipid vesicles was examined under a variety of conditions to investigate physical factors governing this process. Human erythrocytes were incubated with sonicated dimyristoylphosphatidylcholine vesicles containing trace [14C]dipalmitoylphosphatidylcholine. Protein-vesicle complexes were separated from cells and from membrane fragments by density gradient centrifugation. The yield of isolated protein vesicles was determined from the 14C-vesicle marker; protein compositions were analyzed by SDS-polyacrylamide gel electrophoresis. Enzymatic removal of portions of the cytoplasmic or exoplasmic domains of cell membrane proteins had little effect on the extent of protein transfer. Membrane additives such as cholate produced a 2-fold increase in protein-vesicle yield. The selectivity of protein transfer from erythrocytes was influenced by the lipid composition of recipient vesicles: inclusion of cholesterol increased band 3 content while the presence of anionic phospholipids reduced transfer. Proteins transferred from 32P-labeled cells differed in specific radioactivity from bulk cell proteins: glycophorin, highly phosphorylated in the cell membrane, showed no detectable labeling in the corresponding protein-vesicle band. These observations suggest that cell-to-vesicle protein transfer is insensitive to bulk steric and electrostatic properties of cell membranes, but enhanced by membrane defects. Recipient membrane composition influences the selectivity of transferred proteins and may reveal subtle differences in the membrane association of protein subpopulations.

    View details for Web of Science ID A1996UC97700018

    View details for PubMedID 8672433

  • SHAPE RESPONSE OF HUMAN ERYTHROCYTES TO ALTERED CELL PH BLOOD Gedde, M. M., Yang, E. Y., Huestis, W. H. 1995; 86 (4): 1595-1599

    Abstract

    Alteration of red blood cell (RBC) pH produces stomatocytosis (at low pH) and echinocytosis (at high pH). Cell shrinkage potentiates high pH echinocytosis, but shrinkage alone does not cause echinocytosis. Mechanisms for these shape changes have not been described. In this study, measured dependence of RBC shape on cell pH was nonlinear, with a broad pH range in which normal discoid shape was maintained. Transbilayer distribution of phosphatidylcholine and phosphatidylserine, measured by back-extraction of radiolabeled lipid, was the same in control and altered pH cells. Possible roles of pH-titratable inner monolayer phospholipids were examined by assessing pH-dependent shape in cells in which their levels had been perturbed. In metabolically depleted cells and calcium-treated cells, which have altered levels of phosphatidic acid, phosphatidylinositol-4-phosphate, and/or phosphatidylinositol-4,5-bisphosphate, low cell pH was stomatocytogenic and high cell pH was echinocytogenic, as in control cells. Thus, neither change in membrane lipid asymmetry nor normal levels of the pH-titratable inner monolayer lipids is necessary for cell pH-mediated shape change.

    View details for Web of Science ID A1995RN46700048

    View details for PubMedID 7632969

  • WHEAT-GERM-AGGLUTININ STABILIZATION OF ERYTHROCYTE SHAPE - ROLE OF BILAYER BALANCE AND THE MEMBRANE SKELETON BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Lin, S. S., Huestis, W. H. 1995; 1233 (1): 47-56

    Abstract

    The effects of wheat germ agglutinin (WGA), Limulus lectin, and concanavalin A on cell shape changes were examined in human erythrocytes. These agents inhibited echinocytosis in cells having elevated cytosolic Ca2+ or incorporated foreign phosphatidylcholine, but had no effect on cell stomatocytosis in response to incorporated phosphatidylserine. The role of the membrane skeleton in this selective membrane fixation was examined. WGA inhibited echinocytosis in cells previously depleted of polyphosphoinositides to reduce membrane skeleton binding to transmembrane proteins, treated with phorbol ester to enhance protein 4.1 phosphorylation, heat-treated to denature spectrin, alkylated with p-chloromercuribenzoate to dissociate glycophorin from the membrane skeleton, or subjected to elevated cell 2,3-diphosphoglycerate to alter organization of the spectrin-actin-protein 4.1 complex. Limulus lectin and increased concentrations of WGA also stabilized discoid shape in pronase-digested cells containing no detectable intact glycophorin. In contrast, cell digestion with sialidase abolished the shape-stabilizing effect of WGA. The results suggest that the membrane skeleton is not involved in WGA shape stabilization. Rather, they suggest that glycoproteins and glycolipids interact with the lectin to stabilize cell surface molecular associations, forming a superficial calyx that inhibits outward, but not inward, membrane bending.

    View details for Web of Science ID A1995QD64000008

    View details for PubMedID 7833349

  • QUANTIFICATION OF 2-DIMENSIONAL NOE SPECTRA VIA A COMBINED LINEAR AND NONLINEAR LEAST-SQUARES FIT JOURNAL OF BIOMOLECULAR NMR Brown, J. W., Huestis, W. H. 1994; 4 (5): 645-652

    Abstract

    Determining the volumes of peaks in 2D NMR spectra can be prohibitively difficult in cases of overlapping, broad lines. Deconvolution and parameter estimation can be attempted on either the time-domain or the frequency-domain data. We present a method of estimating spectral parameters from frequency-domain data, using a combination of Lorentzian and Gaussian lineshapes for reference lines. This approach combines a previously published method of projecting the data on a linear space spanned by reference lines with a nonlinear least-squares fitting algorithm. Comparison of this method with other published methods of frequency-domain deconvolution shows that it is both more precise and more accurate when estimating 2D volumes.

    View details for Web of Science ID A1994PE35200005

    View details for PubMedID 7919951

  • RELATIONSHIP OF PHOSPHOLIPID DISTRIBUTION TO SHAPE CHANGE IN CA2+-CRENATED AND RECOVERED HUMAN ERYTHROCYTES BIOCHEMISTRY Lin, S. S., Yang, E. Y., Huestis, W. H. 1994; 33 (23): 7337-7344

    Abstract

    Echinocytosis induced by elevation of intracellular Ca2+ in human erythrocytes can be reversed by removal of the cation. Using back-extraction of radiolabeled dilauroyl phospholipid analogs which had been incorporated into the cell membrane, we examined the relationship between this reversible shape transformation and phospholipid distribution. Upon Ca2+ crenation of cells, surface exposure of phosphatidylserine and phosphatidylethanolamine was observed simultaneously with inward diffusion of phosphatidylcholine. Removal of Ca2+ allowed resequestration of exposed phosphatidylserine to the membrane inner monolayer, but randomized phosphatidylethanolamine and phosphatidylcholine were not redistributed to their original states. Both shape reversion and retranslocation of phosphatidylserine were reversibly inhibited vanadate. On the other hand, the cell shape recovery was found to be independent of membrane skeleton and phosphoinositide metabolism and was supported by ATP resynthesis only under conditions where the aminophospholipid translocator is active. Other Ca(2+)-mediated biochemical changes, such as generation of diacylglycerol and fatty acids, were found to have no effect on Ca2+ crenation or its reversal, or upon transbilayer distribution of any phospholipid. These findings suggest that Ca2+ induces phospholipid redistribution, possibly by direct interaction with the lipid bilayer and, further, that metabolic recovery from Ca2+ crenation reflects selective retransport of phosphatidylserine to the membrane inner monolayer.

    View details for Web of Science ID A1994NT32800039

    View details for PubMedID 8003498

  • OXIDATIVE INTERACTIONS BETWEEN THE ERYTHROCYTE-MEMBRANE AND PHOSPHATIDYLCHOLINE VESICLES JOURNAL OF BIOLOGICAL CHEMISTRY Yang, E. Y., Huestis, W. H. 1994; 269 (20): 14518-14524

    Abstract

    Sonicated unilamellar phosphatidylcholine vesicles induce hemoglobin oxidation in erythrocytes and resealed membrane fragments (buds) at pH 5.5. No such oxidation was observed in vesicle-bud mixtures at pH 7.4, in cells or buds suspended in pH 5.5 buffer, or in cells incubated with multilamellar lipid vesicles at pH 5.5. In buds, vesicle-induced hemoglobin oxidation was accompanied by lipid peroxidation and formation of covalent high molecular weight protein aggregates. The causative relationships among the oxidative events were examined using selective antioxidants and membrane fragments in which the cytoplasmic domain of band 3 was cleaved. Protein cross-linking and lipid peroxidation were found to be independent events, but both were found only in concert with heme oxidation. Further, vesicle-induced hemoglobin oxidation was found to be correlated with quasi-stable adsorption of intact vesicles to cells; intercalation of foreign lipid into the cell bilayer was not required. The inability of multilamellar vesicles to induce these low pH oxidative effects suggests a steric limitation on this cell-vesicle association. The results suggest that membrane component reorganization and patching induced by low pH may enhance vesicle adsorption, which in turn initiates oxidative damage.

    View details for Web of Science ID A1994NM06500034

    View details for PubMedID 8182058

  • HYDROGEN-PEROXIDE OXIDATION INDUCES THE TRANSFER OF PHOSPHOLIPIDS FROM THE MEMBRANE INTO THE CYTOSOL OF HUMAN ERYTHROCYTES BIOCHEMISTRY Brunauer, L. S., MOXNESS, M. S., Huestis, W. H. 1994; 33 (15): 4527-4532

    Abstract

    The effects of oxidative damage on membrane phospholipid organization were examined in human erythrocytes. Exposure to H2O2 induced shape changes in these cells; normal discocytes became echinocytic, and stomatocytes generated by foreign phosphatidylserine incorporation reverted to discoid morphology. H2O2 treatment also inhibited phosphatidylserine transport from the outer to inner membrane monolayer, consistent with earlier reports on oxidative sensitivity of the aminophospholipid translocator. The morphological changes are consistent with movement of inner monolayer lipids to the outer monolayer, as might be expected if aminophospholipid sequestration is compromised. However, lipid extraction and prothrombinase activation assays showed no increased exposure of phosphatidylserine on the cell surface. Instead, phosphatidylserine was found associated with the cytosolic fraction of H2O2-treated cells. These observations suggest that oxidative damage alters the lipid organization of erythrocyte membranes, not by randomizing the lipid classes within the bilayer, but by inducing extraction of inner monolayer components into the cytosol.

    View details for Web of Science ID A1994NH49300013

    View details for PubMedID 8161507

  • PHOSPHATIDYLSERINE HEADGROUP DIASTEREOMERS TRANSLOCATE EQUIVALENTLY ACROSS HUMAN ERYTHROCYTE-MEMBRANES BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Hall, M. P., Huestis, W. H. 1994; 1190 (2): 243-247

    Abstract

    The natural chiral phospholipid substrates for the plasma membrane aminophospholipid translocator are L-alpha-phosphatidyl-L-serine and L-alpha-phosphatidylethanolamine. The glyceric D-stereoisomers of these lipids, D-alpha-phosphatidyl-L-serine and D-alpha-phosphatidylethanolamine, are not translocated (Martin, O.C. and Pagano, R.E. (1987) J. Biol. Chem. 262, 5890-5898). We have synthesized a diastereomer of phosphatidylserine, L-alpha-phosphatidyl-D-serine, to study the effects of headgroup stereochemistry on translocation. The diastereomer was synthesized as the dilauroyl (12:0) species, and the translocation was monitored by human erythrocyte morphology changes at 25 degrees C and 37 degrees C. Unlike other phosphatidylserine stereoisomers, L-alpha-phosphatidyl-D-serine is translocated to the same degree as the natural L,L-isomer. Incorporation of apparently equal amounts of the L,D- and L,L-diastereomers does produce minor quantitative differences in the cell morphological response, possibly as a result of differences in lipid packing of the two isomers.

    View details for Web of Science ID A1994ND19200006

    View details for PubMedID 8142422

  • PROTEIN-KINASE-C AS A MEASURE OF TRANSBILAYER PHOSPHATIDYLSERINE ASYMMETRY ANALYTICAL BIOCHEMISTRY Daleke, D. L., Huestis, W. H., Newton, A. C. 1994; 217 (1): 33-40

    Abstract

    The endogenous phosphatidylserine of normal erythrocytes is confined to the cytoplasmic leaflet of the membrane. However, under pathologic conditions transmembrane asymmetry can be altered and cytofacial phosphatidylserine may appear on the cell surface. A sensitive alternative method for the measurement of the exposed phosphatidylserine content of erythrocyte membrane was developed using the activation of exogenous protein kinase C. Erythrocytes containing exogenous phosphatidylcholine incorporated into the outer membrane monolayer do not stimulate protein kinase C activity more than untreated cells. In contrast, red cells that have exogenous phosphatidylserine incorporated into their membrane outer monolayer, by prior inhibition of the aminophospholipid transporter, stimulate protein kinase C significantly more than red cells in which exogenous phosphatidylserine is allowed to translocate to the inner membrane monolayer. Kinase activation is comparable for normal cells and cells not exposed to lipid in which the aminophospholipid transporter is inhibited with sulfhydryl reagents (diamide or N-ethylmaleimide). However, Ca(2+)-loading results in an increase in activation of protein kinase C over control cells, consistent with previous reports that Ca2+ induces the exposure of erythrocyte and platelet phosphatidylserine. By reference to protein kinase C activation by phosphatidylserine in model systems, the quantity of phosphatidylserine on the cell surface may be estimated. Thus, protein kinase C activation affords a sensitive and specific measure of phosphatidylserine in the outer monolayer of biological membranes.

    View details for Web of Science ID A1994MW32100006

    View details for PubMedID 8203737

  • MECHANISM OF INTERMEMBRANE PHOSPHATIDYLCHOLINE TRANSFER - EFFECTS OF PH AND MEMBRANE CONFIGURATION BIOCHEMISTRY Yang, E. Y., Huestis, W. H. 1993; 32 (45): 12218-12228

    Abstract

    The mechanism of phospholipid transfer between membranes has been studied as a function of the configuration and concentration of donor and recipient membranes. The study was motivated by the observation that dimyristoylphosphatidylcholine transfers from sonicated vesicles to erythrocytes at a 4-fold faster rate at pH 5.5 than at pH 7.4. It is unexpected that the solubility of phosphatidylcholine should be affected by pH changes in this range; indeed, the more hydrophilic homolog dilauroylphosphatidylcholine transfers at closely similar rates at pH 5.5 and 7.4. The behavior of the more hydrophobic lipid is not consistent with transfer solely as a monomer passing through the aqueous phase. The effects of membrane proximity on phospholipid transfer were examined in dilution experiments employing intact erythrocytes, resealed ghosts, erythrocyte membrane buds, and sonicated vesicles as both donor and recipient membranes. For both hydrophobic and less hydrophobic lipids, the kinetics of intermembrane transfer were affected significantly by dilution at constant donor:recipient ratios. The results were fit to a kinetic model containing contributions from both through-solution monomer transfer and transient collisional transfer. The model predicts that the mechanism of intermembrane transfer varies with experimental conditions such as membrane concentration, donor and acceptor membrane area, and surface curvature. Through-solution monomer transfer predominates for less hydrophobic lipids at all values of pH and membrane concentration, and for more hydrophobic lipids at very high membrane dilutions. Transient collisional transfer contributes significantly to the rate for relatively hydrophobic lipids in concentrated donor-acceptor systems, an effect that is particularly evident at pH values below 6. The size and surface configuration of donor and recipient membranes also alter the relative contributions of through-solution and collisional transfer.

    View details for Web of Science ID A1993MG89300035

    View details for PubMedID 8218299

  • HUMAN ERYTHROCYTE-MEMBRANE LIPID ASYMMETRY - TRANSBILAYER DISTRIBUTION OF RAPIDLY DIFFUSING PHOSPHATIDYLSERINES BIOCHEMISTRY Loh, R. K., Huestis, W. H. 1993; 32 (43): 11722-11726

    Abstract

    Human erythrocytes were incubated with sonicated vesicles composed of diheptanoyl-, dioctanoyl-, didecanoyl-, or dimyristoylphosphatidylserine, and the transbilayer distribution of the incorporated foreign lipid was examined by monitoring changes in cell morphology (Daleke & Huestis (1989) J. Cell. Biol. 108, 1375). Cells incubated with all phosphatidylserine homologs crenated initially and then reverted to discoid and stomatocytic morphology. Cells exposed to didecanoyl- or dimyristoylphosphatidylserine retained stable stomatocytic morphology during more than 10 h of incubation at 37 degrees C. Cells exposed to the diheptanoyl or dioctanoyl homologs reverted from stomatocytes to discocytes within 1-4 h. This reversion was more rapid for the shorter acyl chain diheptanoylphosphatidylserine. Reversion was accelerated in both cases by vanadate, an inhibitor of the aminophospholipid translocator. Heat denaturation of cytoskeletal proteins had no effect on phosphatidylserine-induced stomatocytosis or on the reversion to discoid shape of cells exposed to the short-chained homologs. These observations suggest that the aminophospholipid transporter rather than cytofacial lipid binding sites plays the primary role in maintenance of phosphatidylserine asymmetry in the erythrocyte membrane bilayer.

    View details for Web of Science ID A1993ME65600031

    View details for PubMedID 8218241

  • EFFECTS OF EXOGENOUS PHOSPHOLIPIDS ON PLATELET ACTIVATION BIOCHIMICA ET BIOPHYSICA ACTA Brunauer, L. S., Huestis, W. H. 1993; 1152 (1): 109-118

    Abstract

    Intercalation of amphipaths into the plasma membrane of platelets has a marked effect on their morphology. Incubation of platelets with phosphatidylcholines (PC) results in rounding of the platelet body and speculation, while incubation with aminophospholipids such as dilauroylphosphatidylserine (DLPS) results in a biphasic shape change consistent with the bilayer couple model (Sheetz, M.P. and Singer, S.J. (1982) Proc. Natl. Acad. Sci. USA 71, 4457-4461) and with the activity of an aminophospholipid translocator facilitating transverse bilayer diffusion (Daleke, D.L. and Huestis, W.H. (1985) Biochemistry 24, 5406-5416). The present study extends this work to investigate the effects of PC and PS on platelet responses to a natural agonist, thrombin. PC incorporation produces a concentration-dependent progression of shape changes, beginning with surface ruffling and development of fine spicules, followed by sphering of the cell body, and ending with the apparent loss of spicules. PC reduces platelet responses to thrombin only under conditions that promote membrane vesiculation, seen morphologically as a loss of spicules and biochemically as a loss of 14C-PC labeled membrane. PS homologues of varying acyl chain composition induce concentration- and time-dependent platelet sphering. Incorporation of PS inhibits thrombin-induced platelet shape change, granule secretion, and protein phosphorylation. Inhibition of these responses requires transit of the exogenous PS to the cytofacial leaflet of the membrane bilayer.

    View details for Web of Science ID A1993MB69600015

    View details for PubMedID 8399288

  • DITHIOTHREITOL STIMULATES THE ACTIVITY OF THE PLASMA-MEMBRANE AMINOPHOSPHOLIPID TRANSLOCATOR BIOCHIMICA ET BIOPHYSICA ACTA TRUONG, H. T., Daleke, D. L., Huestis, W. H. 1993; 1150 (1): 57-62

    Abstract

    Metabolic depletion induces human erythrocytes to crenate, a shape change that is reversed when ATP is regenerated by nutrient supplementation. In the presence of the sulfhydryl reducing agent dithiothreitol (DTT), this shape reversal is exaggerated, proceeding beyond normal discoid morphology to stomatocytic forms. DTT-induced stomatocytosis does not correlate consistently with alterations in cell ATP, spectrin phosphorylation, or phosphoinositide metabolism (Truong, H.-T.N., Ferrell, J.E., Jr. and Huestis, W.H. (1986) Blood 67, 214-221). The effect of DTT on outer-to-inner-monolayer transport of aminophospholipids was examined by monitoring shape changes induced by dilauroylphosphatidylserine (DLPS). Stomatocytosis induced by transport of this exogenous lipid to the membrane inner monolayer is accelerated and exaggerated by DTT. The effect of DTT on DLPS translocation is reversible and temperature dependent, consistent with the intervention of reducing agents in the activity of the aminophospholipid translocator. These findings bear on the relationship between cell redox status and shape regulation.

    View details for Web of Science ID A1993LP97100008

    View details for PubMedID 8334138

  • HUMAN ERYTHROCYTE SHAPE REGULATION - INTERACTION OF METABOLIC AND REDOX STATUS BIOCHIMICA ET BIOPHYSICA ACTA TRUONG, H. T., Daleke, D. L., Huestis, W. H. 1993; 1150 (1): 51-56

    Abstract

    The echinocyte-to-discocyte shape recovery of metabolically depleted erythrocytes is compromised by sulfhydryl reducing agents (Truong, H.-T.N., Ferrell, J.E., Jr. and Huestis, W.H. (1986) Blood 67, 214-221). In the presence of dithiothreitol (DTT) and sugars, crenated cells recover normal discoid shape transiently, but then develop the invaginations and intracellular inclusions of stomatocytes. The stomatogenic effects of DTT were investigated in erythrocytes recovering from crenation induced by several independent mechanisms. Cells crenated by direct manipulation of the membrane bilayer (lysophosphatidylcholine incorporation) recovered discoid shape similarly in the presence and absence of the reducing agent. In contrast, resealed ghosts and cells crenated by Mg2+ depletion or Ca2+ loading did not maintain stable discoid morphology in the presence of DTT, proceeding further to form stomatocytes. Thus cell crenation by expedients that involve cellular metabolic processes develop a redox-related morphological instability that is not found in amphipath-crenated cells.

    View details for Web of Science ID A1993LP97100007

    View details for PubMedID 8334137

  • STRUCTURE AND ORIENTATION OF A BILAYER-BOUND MODEL TRIPEPTIDE - A H-1-NMR STUDY JOURNAL OF PHYSICAL CHEMISTRY Brown, J. W., Huestis, W. H. 1993; 97 (12): 2967-2973
  • ERYTHROCYTE MORPHOLOGY REFLECTS THE TRANSBILAYER DISTRIBUTION OF INCORPORATED PHOSPHOLIPIDS JOURNAL OF CELL BIOLOGY Daleke, D. L., Huestis, W. H. 1989; 108 (4): 1375-1385

    Abstract

    The transbilayer distribution of exogenous phospholipids incorporated into human erythrocytes is monitored through cell morphology changes and by the extraction of incorporated 14C-labeled lipids. Dilauroylphosphatidylserine (DLPS) and dilauroylphosphatidylcholine (DLPC) transfer spontaneously from sonicated unilamellar vesicles to erythrocytes, inducing a discocyte-to-echinocyte shape change within 5 min. DLPC-induced echinocytes revert slowly (t1/2 approximately 8 h) to discocytes, but DLPS-treated cells revert rapidly (10-20 min) to discocytes and then become invaginate stomatocytes. The second phase of the phosphatidylserine (PS)-induced shape change, conversion of echinocytes to stomatocytes, can be inhibited by blocking cell protein sulfhydryl groups or by depleting intracellular ATP or magnesium (Daleke, D. L., and W. H. Huestis. 1985. Biochemistry. 24:5406-5416). These cell shape changes are consistent with incorporation of phosphatidylcholine (PC) and PS into the membrane outer monolayer followed by selective and energy-dependent translocation of PS to the membrane inner monolayer. This hypothesis is explored by correlating cell shape with the fraction of the exogenous lipid accessible to extraction into phospholipid vesicles. Upon exposure to recipient vesicles, DLPC-induced echinocytes revert to discoid forms within 5 min, concomitant with the removal of most (88%) of the radiolabeled lipid. On further incubation, 97% of the foreign PC transfers to recipient vesicles. Treatment of DLPS-induced stomatocytes with acceptor vesicles extracts foreign PS only partially (22%) and does not affect cell shape significantly. Cell treated with inhibitors of aminophospholipid translocation (sulfhydryl blockers or intracellular magnesium depletion) and then incubated with either DLPS or DLPC become echinocytic and do not revert to discocytic or stomatocytic shape for many hours. On treatment with recipient vesicles, these echinocytes revert to discocytes in both cases, with concomitant extraction of 88-99% of radiolabeled PC and 86-97% of radiolabeled PS. The accessibility of exogenous lipids to extraction is uniformly consistent with the transbilayer lipid distribution inferred from cell shape changes, indicating that red cell morphology is an accurate and sensitive reporter of the transbilayer partitioning of incorporated exogenous phospholipids.

    View details for Web of Science ID A1989T953300018

    View details for PubMedID 2925790

  • VESICLE-TO-CELL PROTEIN TRANSFER - INSERTION OF BAND-3, THE ERYTHROCYTE ANION TRANSPORTER, INTO LYMPHOID-CELLS BIOCHEMISTRY Newton, A. C., Huestis, W. H. 1988; 27 (13): 4655-4659

    Abstract

    Band 3, the erythrocyte anion transporter, transfers spontaneously between human red cells and model membranes. During incubation of intact erythrocytes with sonicated dimyristoylphosphatidylcholine vesicles, the transporter inserts in functional form and native orientation into the liposome bilayer, with the cytoplasmic segment of the protein contacting the lumen of the vesicle [Newton, A. C., Cook, S. L., & Huestis, W. H. (1983) Biochemistry 22, 6110-6117; Huestis, W. H., & Newton, A. C. (1986) J. Biol. Chem. 261, 16274-16278]. When band 3-vesicle complexes are incubated with erythrocytes whose native band 3 has been inhibited irreversibly, reverse transfer of the protein restores anion transport capacity to the cells [Newton, A. C., Cook, S. L., & Huestis, W. H. (1983) Biochemistry 22, 6110-6117]. Here we report the vesicle-mediated transfer of band 3 to human peripheral blood lymphocytes and to cultured murine lymphoma cells (BL/VL3). Subsequent to incubation with protein-vesicle complexes, both lymphoid cell types exhibit a 2-4-fold increase in the rate of chloride uptake. This enhanced permeability is inhibited greater than or equal to 98% by the exofacial band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, consistent with right-side-out insertion of functional band 3 into the lymphoid cell membrane.

    View details for Web of Science ID A1988P028800011

    View details for PubMedID 3167008

  • LYMPHOMA VESICLE INTERACTIONS - VESICLE ADSORPTION, MEMBRANE FRAGMENTATION, AND INTERMEMBRANE PROTEIN TRANSFER BIOCHEMISTRY Newton, A. C., Huestis, W. H. 1988; 27 (13): 4645-4655

    Abstract

    Sonicated dimyristoylphosphatidylcholine vesicles interact with cultured murine lymphoma (BL/VL3) to generate complexes of vesicle and cell membrane components. Cell-free supernatants harvested after cell-vesicle incubations contain three distinct lipid species that can be separated by density gradient centrifugation. Analysis of protein and lipid composition and assays for cell and vesicle lumen contents reveal that the densest of the three lipid species comprises sealed plasma membrane fragments complexed with vesicles, while the least dense species is indistinguishable from pure phospholipid vesicles. The third, intermediate density species consists of topologically intact vesicles with associated plasma membrane proteins but without detectable cell lipids or cytoplasmic components. The membrane fragmentation and cell-to-vesicle protein transfer observed during lymphoma-vesicle incubations are examined as functions of cell and vesicle concentrations and incubation time.

    View details for Web of Science ID A1988P028800010

    View details for PubMedID 3167007

  • MEMBRANE BILAYER BALANCE AND PLATELET SHAPE - MORPHOLOGICAL AND BIOCHEMICAL RESPONSES TO AMPHIPATHIC COMPOUNDS BIOCHIMICA ET BIOPHYSICA ACTA Ferrell, J. E., Mitchell, K. T., Huestis, W. H. 1988; 939 (2): 223-237

    Abstract

    Activated platelets adopt a characteristic spiculate morphology. A wide variety of anionic and zwitterionic amphipathic compounds were found to effect a similar shape change and to cause the open canalicular system to become less prominent. Several cationic amphipaths reversed thrombin-, PAF-, and amphipath-induced spiculation and restored the discoid shape. Higher concentrations of cationic amphipaths caused the cells to assume spheroid and indented forms, and caused the canalicular system to appear more prominent. Three amphipaths were studied further to address possible mechanisms underlying their morphological effects. Dilauroylphosphatidylcholine was found to induce spiculation without causing the changes in protein phosphorylation and inositide metabolism generally associated with platelet activation. Two other amphipaths, chlorpromazine (which induced sphering) and dilauroylphosphatidylserine (which caused spiculation followed by sphering) caused specific changes in protein and/or lipid phosphorylation, which may be responsible for some, but not all, of the morphological effects of these compounds. To account for these findings, we propose that platelet shape can be influenced by changes in the plasma membrane bilayer balance. Agents that bind to the membrane outer monolayer are accommodated by spiculation; those that bind to the inner monolayer are accommodated by sphering.

    View details for Web of Science ID A1988N073500006

    View details for PubMedID 3355815

  • INTERMEMBRANE PROTEIN TRANSFER - BAND-3, THE ERYTHROCYTE ANION TRANSPORTER, TRANSFERS IN NATIVE ORIENTATION FROM HUMAN RED-BLOOD-CELLS INTO THE BILAYER OF PHOSPHOLIPID-VESICLES JOURNAL OF BIOLOGICAL CHEMISTRY Huestis, W. H., Newton, A. C. 1986; 261 (34): 6274-6278
  • SEPARATION OF PHOSPHOINOSITIDES AND OTHER PHOSPHOLIPIDS BY TWO-DIMENSIONAL THIN-LAYER CHROMATOGRAPHY ANALYTICAL BIOCHEMISTRY Mitchell, K. T., Ferrell, J. E., Huestis, W. H. 1986; 158 (2): 447-453

    Abstract

    A simple, rapid, two-dimensional TLC system is presented which resolves the four phosphoinositide cycle phospholipids as well as all commonly encountered major and minor phospholipids. Ca2+-free lipid samples are loaded onto silica gel HL plates and developed first in 48:40:7:5 chloroform:methanol:water:concentrated ammonia, and then in 55:25:5 chloroform:methanol:formic acid. The method was applied successfully to human erythrocytes, human platelets, and BL/VL3 murine lymphoma cells.

    View details for Web of Science ID A1986F070500033

    View details for PubMedID 3028208

  • EFFLUX OF DIPICOLINIC ACID FROM HUMAN-ERYTHROCYTES, SEALED MEMBRANE-FRAGMENTS, AND BAND-3-LIPOSOME COMPLEXES - A FLUORESCENCE PROBE FOR THE ERYTHROCYTE ANION TRANSPORTER ANALYTICAL BIOCHEMISTRY Newton, A. C., Huestis, W. H. 1986; 156 (1): 56-60

    Abstract

    The greatly enhanced fluorescence of Tb3+ when complexed with dipicolinic acid affords a simple and highly sensitive method for monitoring continuous anion flux through the erythrocyte anion transporter, band 3. Dipicolinic acid (pyridine-2,6-dicarboxylic acid) is entrapped in human erythrocytes and other band 3-membrane preparations. Efflux of dipicolinic acid from preequilibrated systems into Tb3+-containing medium is monitored fluorometrically. Dipicolinic acid efflux is demonstrated in intact erythrocytes, sealed red cell membrane fragments, and band 3-liposome complexes. In each system, dipicolinic acid efflux is blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid. The technique is highly sensitive and reproducible, and is applicable to hemoglobin-containing and hemoglobin-free systems of widely varying surface area to volume ratios.

    View details for Web of Science ID A1986D132300009

    View details for PubMedID 3740418

  • SULFHYDRYL REDUCING AGENTS AND SHAPE REGULATION IN HUMAN-ERYTHROCYTES BLOOD TRUONG, H. T., Ferrell, J. E., Huestis, W. H. 1986; 67 (1): 214-221

    Abstract

    Metabolic crenation of red cells is reversible; on addition of nutrients, echinocytes recover the normal discoid shape. When the shape recovery takes place in the presence of reducing agents such as dithiothreitol (DTT), morphological change continues until the cells are stomatocytic. The degree of stomatocytosis varies, depending on the cell morphology when the nutrients and reducing agent are added. DTT has minimal effect on the shape of normal discocytes, but in its presence, mildly echinocytic cells become slightly cupped and advanced-stage echinocytes become severely stomatocytic. DTT must be present continuously for development and retention of stomatocytosis; echinocytes preincubated with or metabolically depleted in DTT do not become stomatocytic when supplemented in the absence of DTT, and DTT-induced stomatocytes revert to discocytes when the reducing agent is removed. DTT has no effect on adenosine triphosphate synthesis or equilibrium cell glutathione levels, and the induced stomatocytosis is not inhibited by excluding oxygen from cells during depletion. Spectrin phosphorylation and phosphate turnover are not affected by DTT. The echinocyte-to-discocyte transformation coincides with phosphorylation of membrane inner monolayer lipids (diacylglycerol to phosphatidic acid and phosphatidylinositol to phosphatidylinositol-4,5-bisphosphate). Overphosphorylation of these phospholipids is not responsible for the exaggerated shape recovery seen with reducing agents; phosphorylation of inner monolayer lipids proceeds identically in the presence and absence of DTT.

    View details for Web of Science ID A1986AYB4500034

    View details for PubMedID 3000479

  • MEMBRANE BILAYER BALANCE AND ERYTHROCYTE SHAPE - A QUANTITATIVE ASSESSMENT BIOCHEMISTRY Ferrell, J. E., Lee, K. J., Huestis, W. H. 1985; 24 (12): 2849-2857

    Abstract

    When human erythrocytes are incubated with certain phospholipids, the cells become spiculate echinocytes, resembling red cells subjected to metabolic starvation or Ca2+ loading. The present study examines (1) the mode of binding of saturated phosphatidylcholines and egg lysophosphatidylcholine to erythrocytes and (2) the quantitative relationship between phospholipid incorporation and red cell shape. We find that the phospholipids studied become intercalated into erythrocyte membranes, not simply adsorbed to the cell surface. Spin-labeling and radiolabeling data show that the incorporation of (4 +/- 1) X 10(6) molecules of exogenous phosphatidylcholine per cell converts discocytes to stage 3 echinocytes with about 35 conical spicules. This amount of lipid incorporation is estimated to expand the red cell membrane outer monolayer by 1.7% +/- 0.6%. Calculations of the inner and outer monolayer surface areas of model discocytes and stage 3 echinocytes yield an estimated difference of 0.7% +/- 0.2%.

    View details for Web of Science ID A1985AKK7800006

    View details for PubMedID 2990533

  • SELECTIVE ABSORPTION OF ULTRAVIOLET-LASER ENERGY BY HUMAN ATHEROSCLEROTIC PLAQUE TREATED WITH TETRACYCLINE AMERICAN JOURNAL OF CARDIOLOGY MURPHYCHUTORIAN, D., Kosek, J., Mok, W., Quay, S., HUESTIS, W., MEHIGAN, J., Profitt, D., Ginsburg, R. 1985; 55 (11): 1293-1297

    Abstract

    Tetracycline is an antibiotic that absorbs ultraviolet light at 355 nm and preferentially binds to atherosclerotic plaque both in vitro and in vivo. Tetracycline-treated human cadaveric aorta was compared with untreated aorta using several techniques: absorptive spectrophotometry, which demonstrated a distinct absorptive peak at 355 nm in tetracycline-treated plaque that was absent in treated normal vessel; ultraviolet microscopy, which showed that treated atheroma acquired the characteristic fluorescence of tetracycline under ultraviolet light; and tissue uptake of radiolabeled tetracycline, which showed 4-fold greater uptake by atheroma than by normal vessel. In addition, intravenous tetracycline administered to patients undergoing vascular surgery demonstrated characteristic fluorescence in surgically excised diseased arteries. Because of tetracycline's unique properties, we exposed tetracycline-treated and untreated aorta to ultraviolet laser radiation at a wavelength of 355 nm. We found enhanced ablation of tetracycline-treated atheroma compared with untreated atheroma. The plaque ablation caused by ultraviolet laser radiation was twice as extensive in tetracycline-treated vs nontreated plaque (2.2 +/- 0.25 mm vs 1.3 +/- 0.55 mm, p less than 0.017). This study demonstrates the potential of tetracycline plaque enhancement for the selective destruction of atheroma by ultraviolet laser radiation.

    View details for Web of Science ID A1985AGP2000009

    View details for PubMedID 3993559

  • INCORPORATION AND TRANSLOCATION OF AMINOPHOSPHOLIPIDS IN HUMAN-ERYTHROCYTES BIOCHEMISTRY Daleke, D. L., Huestis, W. H. 1985; 24 (20): 5406-5416

    Abstract

    Cell morphology changes are used to examine the interaction of exogenous phosphatidylserine and phosphatidylethanolamine with human erythrocytes. Short-chain saturated lipids transfer from liposomes to cells, inducing shape changes that are indicative of their incorporation into, and in some cases translocation across, the cell membrane bilayer. Dioleoylphosphatidylserine and low concentrations of dilauroyl- and dimyristoylphosphatidylserine induce stomatocytosis. At higher concentrations, dilauroylphosphatidylserine and dimyristoylphosphatidylserine induce a biphasic shape change: the cells crenate initially but rapidly revert to a discocytic and eventually stomatocytic shape. The extent of these shape changes is dose dependent and increases with increasing hydrophilicity of the phospholipid. Cells treated with dilauroylphosphatidylethanolamine and bovine brain lysophosphatidylserine exhibit a similar biphasic shape change but revert to discocytes rather than stomatocytes. These shape changes are not a result of vesicle--cell fusion nor can they be accounted for by cholesterol depletion. The reversion from crenated to stomatocytic forms is dependent on intracellular ATP and Mg2+ concentrations and the state of protein sulfhydryl groups. The present results are consistent with the existence of a Mg2+- and ATP-dependent protein in erythrocytes that selectively translocates aminophospholipids to the membrane inner monolayer engendering aminophospholipid asymmetry.

    View details for Web of Science ID A1985ARS3600019

    View details for PubMedID 4074704

  • LIPID TRANSFER BETWEEN PHOSPHATIDYLCHOLINE VESICLES AND HUMAN-ERYTHROCYTES - EXPONENTIAL DECREASE IN RATE WITH INCREASING ACYL CHAIN-LENGTH BIOCHEMISTRY Ferrell, J. E., Lee, K. J., Huestis, W. H. 1985; 24 (12): 2857-2864

    Abstract

    The rate of phospholipid transfer from sonicated phospholipid vesicles to human erythrocytes has been studied as a function of membrane concentration and lipid acyl chain composition. Phospholipid transfer exhibits saturable first-order kinetics with respect to both cell and vesicle membrane concentrations. This kinetic behavior is consistent either with transfer during transient contact between cell and vesicle surfaces (but only if the fraction of the cell surface susceptible to such interaction is small) or with transfer of monomers through the aqueous phase. The acyl chain composition of the transferred phospholipid affects the transfer kinetics profoundly; for homologous saturated phosphatidylcholines, the rate of transfer decreases exponentially with increasing acyl chain length. This behavior is consistent with passage of phospholipid monomers through a polar phase, which might be the bulk aqueous phase( as in the monomer transfer model) or the hydrated head-group regions of a cell-vesicle complex (transient collision model). Collisional transfer also predicts that intercell transfer of phospholipids should be slow compared to cell-vesicle transfer, as surface charge and steric effects should prevent close apposition of donor and acceptor membranes. This is not found; dilauroylphosphatidylcholine transfers rapidly between red cells. Thus, the observed relationship between acyl chain length and intermembrane phospholipid transfer rates likely reflects the energetics of monomer transfer through the aqueous phase.

    View details for Web of Science ID A1985AKK7800007

    View details for PubMedID 4016076

  • PHOSPHOINOSITIDE METABOLISM AND THE MORPHOLOGY OF HUMAN-ERYTHROCYTES JOURNAL OF CELL BIOLOGY Ferrell, J. E., Huestis, W. H. 1984; 98 (6): 1992-1998

    Abstract

    ATP-depleted human erythrocytes lose their smooth discoid shape and adopt a spiny, crenated form. This shape change coincides with the conversion of phosphatidylinositol-4,5-bisphosphate to phosphatidylinositol and phosphatidic acid to diacylglycerol. Both crenation and lipid dephosphorylation are accelerated by iodoacetamide, and both are reversed by nutrient supplementation. The observed changes in lipid populations should shrink the membrane inner monolayer by 0.6%, consistent with estimates of bilayer imbalance in crenated cells. These observations suggest that metabolic crenation arises from a loss of inner monolayer area secondary to the degradation of phosphatidylinositol-4,5-bisphosphate and phosphatidic acid. A related process, crenation after Ca2+ loading, appears to arise from a loss inositides by a different pathway.

    View details for Web of Science ID A1984SV47200008

    View details for PubMedID 6327723

  • SHAPE CHANGES IN GOOSE ERYTHROCYTES BIOCHIMICA ET BIOPHYSICA ACTA Nikinmaa, M., Huestis, W. H. 1984; 773 (2): 317-320

    Abstract

    Goose erythrocytes were subjected to agents and treatments that produce echinocytosis in human erythrocytes. In the presence of the ionophore A23187 and calcium at greater than micromolar concentrations, goose red cells retained their normal ellipsoidal symmetry, but developed extensive semiregular membrane wrinkles or corrugations. Metabolic NTP (nucleoside triphosphate) depletion, induced either by iodoacetamide or by incubating the cells without a substrate, initially produced a similar cell corrugation, but after prolonged incubation most cells became spherical with the nucleus displaced to the cell periphery. The echinocytic agents indomethacin and dimyristoylphosphatidylcholine had no effect on the gross morphology of goose erythrocytes.

    View details for Web of Science ID A1984TB62700016

    View details for PubMedID 6428453

  • ADRENERGIC SWELLING OF NUCLEATED ERYTHROCYTES - CELLULAR MECHANISMS IN A BIRD, DOMESTIC GOOSE, AND 2 TELEOSTS, STRIPED BASS AND RAINBOW-TROUT JOURNAL OF EXPERIMENTAL BIOLOGY Nikinmaa, M., Huestis, W. H. 1984; 113 (NOV): 215-224
  • TRANSFER OF BAND-3, THE ERYTHROCYTE ANION TRANSPORTER, BETWEEN PHOSPHOLIPID-VESICLES AND CELLS - APPENDIX - ANALYSIS OF CHLORIDE INFLUX BIOCHEMISTRY Newton, A. C., Cook, S. L., Huestis, W. H., Ferrell, J. E. 1983; 22 (26): 6110-6117
  • CALCIUM DOES NOT MEDIATE THE SHAPE CHANGE THAT FOLLOWS ATP DEPLETION IN HUMAN-ERYTHROCYTES BIOCHIMICA ET BIOPHYSICA ACTA Ferrell, J. E., Huestis, W. H. 1982; 687 (2): 321-328

    Abstract

    Crenation, the shape change that follows ATP depletion in human erythrocytes, also follows ionphore-mediated Ca2+-loading. Experiments designed to test whether Ca2+ mediates metabolic crenation showed that: (1) an influx of extracellular Ca2+ is not required for metabolic crenation; (2) metabolic crenation is accompanied by a 70% increase in 86Rb+ permeability, a change much smaller than the increase expected if crenating concentrations of Ca2+ were released from bound intracellular pools; (3) A23187 plus EGTA, a treatment that depletes intracellular Ca2+ and stops Ca2+ crenation, does not affect metabolic crenation; (4) calmodulin inhibitors do not slow metabolic crenation. We conclude that Ca2+ does not mediate metabolic crenation. Albumin washes reverse Ca2+ crenation and metabolic crenation involve the accumulation of some amphiphilic species (e.g., lysolipid or diacylglycerol) in the cell membrane outer monolayer, and that ATP depletion induces a second crenating process which might be a reorganization of the cytoskeleton.

    View details for Web of Science ID A1982NQ06400026

    View details for PubMedID 6807344

  • CHOLINERGIC STIMULATION OF GLUCOSE-TRANSPORT IN HUMAN-ERYTHROCYTES BIOCHIMICA ET BIOPHYSICA ACTA Nelson, M. J., Huestis, W. H. 1982; 685 (3): 279-282

    Abstract

    The effects of cholinergic stimulation on glucose equilibrium exchange rate have been studied in human erythrocytes. Carbamylcholine increases in V of equilibrium exchange by 20% but has no significant effect on Km. The cholinergic effect is abolished by the muscarinic antagonist atropine or by alterations in intracellular calcium concentrations induced by the calcium ionophore A23187.

    View details for Web of Science ID A1982NF39300007

    View details for PubMedID 6802180

  • CALMODULIN-DEPENDENT SPECTRIN KINASE-ACTIVITY IN RESEALED HUMAN-ERYTHROCYTE GHOSTS BIOCHIMICA ET BIOPHYSICA ACTA Nelson, M. J., Daleke, D. L., Huestis, W. H. 1982; 686 (2): 182-188

    Abstract

    Membrane protein phosphorylation has been studied in resealed human erythrocyte ghosts by measuring the incorporation of 32P into spectrin and band 3. Norepinephrine- and Ca2+-stimulated phosphate incorporation was diminished in ghosts depleted of calmodulin. Ghosts prepared with endogenous calmodulin showed Ca2+- and norepinephrine-stimulated protein phosphorylation only when the ghosts had been resealed in the presence of (gamma-32P)ATP. Ghosts resealed with or without calmodulin in the presence of unlabeled ATP showed no net gain or loss of 32P when exposed to norepinephrine or a Ca2+-specific ionophore. These observations suggest that Ca2+ and norepinephrine stimulation of membrane protein phosphorylation is mediated by calmodulin-dependent spectrin kinase activity, and not by increased turnover of spectrin ATPase or by inhibition of phosphospectrin phosphatase.

    View details for Web of Science ID A1982NL64700006

    View details for PubMedID 6805511

  • PREPARATION OF A NOVEL C-13-LABELED HEME PROTEIN BIOCHIMICA ET BIOPHYSICA ACTA Nelson, M. J., Huestis, W. H. 1980; 623 (2): 467-470

    Abstract

    Protohemin chloride 90% enriched with 13C in the terminal carbons of the 2-nd and 4-position vinyl side-chains has been synthesized. The 13C-NMR spectrum of carboxymyoglobin reconstituted with the 13C-enriched hemin revealed two signals of nearly equal intensity at 117.6 and 113.4 ppm downfield of tetramethylsilane. The inequivalence of these vinyl carbons must reflect differences in environment imposed by the protein.

    View details for Web of Science ID A1980JZ85700026

    View details for PubMedID 7397228

  • "α-Adrenergic stimulation of human erythrocyte membrane protein phosphorylation BBA Biomembranes J, N. M., Ferrell, J. E., Huestis, W. H. 1980; 558: 136 - 140
  • CELL TO VESICLE TRANSFER OF INTRINSIC MEMBRANE-PROTEINS - EFFECT OF MEMBRANE FLUIDITY BIOCHEMISTRY Cook, S. L., Bouma, S. R., Huestis, W. H. 1980; 19 (20): 4601-4607

    View details for Web of Science ID A1980KJ73800010

    View details for PubMedID 7426618

  • EVIDENCE THAT CALCIUM ACTS AS AN INTRACELLULAR MESSENGER FOR ADRENERGIC RESPONSES IN HUMAN-ERYTHROCYTES BIOCHIMICA ET BIOPHYSICA ACTA Nelson, M. J., Huestis, W. H. 1980; 600 (2): 398-405

    Abstract

    Adrenergic stimulation of membrane protein phosphorylation has been studied in human erythrocytes. The adrenergic enhancement in phosphorylation of band 2 could be mimicked by the calcium-specific ionophore A23187 in the presence of 10 micron extracellular calcium. Experiments with the potassium ionophore, valinomycin, showed that potassium efflux was not the primary effector of the response. Trifluoperazine, an inhibitor of the Ca2+-dependent regulatory protein, calmodulin, inhibited phosphorylation stimulation by either norepinephrine or the calcium ionophore. The norepinephrine response was observed in the absence of extracellular calcium, implicating Ca2+ released from cellular bound pools in mediating the response.

    View details for Web of Science ID A1980KG49300014

    View details for PubMedID 6773571

  • MECHANISM OF ERYTHROCYTE LYSIS BY LYSOPHOSPHATIDYLCHOLINE BIOCHIMICA ET BIOPHYSICA ACTA Bierbaum, T. J., Bouma, S. R., Huestis, W. H. 1979; 555 (1): 102-110

    Abstract

    Lysophosphatidylcholine micelles liberate several cell surface polypeptides from erythrocyte membranes, inducing a sodium-selective permeability defect which leads to colloid osmotic lysis. Evidence is presented to support the hypothesis that at the lowest lytic lysophospholipid concentrations, selective disruption of membrane protein function, rather than gross structural reorganization of the membrane, is the primary lytic mechanism.

    View details for Web of Science ID A1979HG10600009

    View details for PubMedID 476093

  • BROMOTRIFLUOROACETONE ALKYLATES HEMOGLOBIN AT CYSTEINE BETA-93 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Huestis, W. H., Raftery, M. A. 1978; 81 (3): 892-899

    View details for Web of Science ID A1978EW51300028

    View details for PubMedID 666799

  • SELECTIVE EXTRACTION OF MEMBRANE-BOUND PROTEINS BY PHOSPHOLIPID VESICLES JOURNAL OF BIOLOGICAL CHEMISTRY Bouma, S. R., Drislane, F. W., Huestis, W. H. 1977; 252 (19): 6759-6763

    Abstract

    Extraction of membrane proteins from erythrocytes into sonicated phosphatidylcholine vesicles is described. In a process involving phospholipid and neutral lipid exchange, cell membrane proteins associate with the vesicles and can be separated from the cells by centrifugation. The protein transfer appears to be reversible; phospholipid vesicles mediate the delivery of small amounts of previously extracted protein into cell membranes. Prior to extraction, all but one of the proteins are accessible to lactoperoxidase iodination, and lipid analysis indicates that primarily the outer monolayer of the cell is involved in phospholipid exchange. Among the extracted proteins is acetylcholinesterase which is removed much more efficiently by this procedure than by concentrated salt solutions. The most abundant proteins of the erythrocyte membrane are not represented in the vesicle extract.

    View details for Web of Science ID A1977DX51300032

    View details for PubMedID 893440

  • SODIUM-SPECIFIC MEMBRANE-PERMEABILITY DEFECT INDUCED BY PHOSPHOLIPID VESICLE TREATMENT OF ERYTHROCYTES JOURNAL OF BIOLOGICAL CHEMISTRY Huestis, W. H. 1977; 252 (19): 6764-6768

    Abstract

    Treatment of human erythrocytes with phospholipid vesicles induces a selective membrane permeability defect which leads to osmotic lysis. The defective cells exhibit a massive sodium ion leak while maintaining normal impermeability to other cations, anions, and neutral small molecules. The sodium ion influx and resulting hemolysis may be inhibited by increased pH, by tetrodotoxin, and by reintroduction of vesicle-extracted proteins into the cell. These characteristics suggest that phospholipid vesicle treatment destroys the cell by disrupting a membrane protein system involved in regulation of cation permeability.

    View details for Web of Science ID A1977DX51300033

    View details for PubMedID 19477

  • PRELIMINARY CHARACTERIZATION OF ACETYLCHOLINE-RECEPTOR IN HUMAN ERYTHROCYTES JOURNAL OF SUPRAMOLECULAR STRUCTURE Huestis, W. H. 1976; 4 (3): 355-365

    Abstract

    The response of human erythrocytes to cholinergic ligands was studied with an electron spin resonance assay. The membrane response to carbamyl choline was found to be antagonized by atropine and, in the absence of calcium, by tetrodotoxin. Experiments with resealed ghosts showed that the membrane response to carbamyl choline required ATP and calcium. Reductive alkylation of intact cells eliminated the cholinergic response, but the presence of saturating amounts of carbamyl choline protected the putative receptor against inactivation. Affinity labeling was used to demonstrate an apparent molecular weight of 41,000 for the carbamyl choline-binding species. A lipid vesicle extraction technique was used to induce a specific cation permeability defect in intact cells. Preliminary investigation of this phenomenon is described.

    View details for Web of Science ID A1976BP46300005

    View details for PubMedID 4659

  • A single-cell system for study of cholinergic receptor function Proceedings of the Dahlem Workshop on Hormone and Antihormone Action at the Target Cell H, H. W. edited by Clark, J. H. 1976
  • CONFORMATION AND COOPERATIVITY IN HEMOGLOBIN BIOCHEMISTRY Huestis, W. H., Raftery, M. A. 1975; 14 (9): 1886-1892

    Abstract

    19-F and 31-P nuclear magnetic resonance (NMR) spectroscopy have been used to study the ligand binding process in human hemoglobin. 19-F nuclear magnetic resonance studies of hemoglobin specifically trifluoroacetonylated at cysteine-beta93 have permitted observation and characterization of molecular species containing two and three ligands. The behavior of these intermediate species in response to changes in pH and organic phosphate concentration is not completely consistent with any of the current theories of allostery. A model consistent with the 19-F and 31-P NMR data is proposed.

    View details for Web of Science ID A1975AA79700013

    View details for PubMedID 235969

  • FUNCTIONAL ACETYLCHOLINE-RECEPTOR IN HUMAN ERYTHROCYTE BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Huestis, W. H., MCCONNEL, H. M. 1974; 57 (3): 726-732
  • MOLECULAR-CONFORMATION AND COOPERATIVITY IN HEMOGLOBIN ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Raftery, M. A., Huestis, W. H. 1973; 222 (DEC31): 40-55

    View details for Web of Science ID A1973S230200004

    View details for PubMedID 4522436

  • Characterization of intermediate states in the ligation of hemoglobin Biochemistry H, H. W., Raftery, M. A. 1973; 12: 2531 - 2535
  • Comparison of the functional properties of trifluoroacetonyled hemoglobin and native hemoglobin Biochemistry T, L. T., Huestis, W. H., Raftery, M. A. 1973; 12: 2535 - 2539
  • Investigation of some transition metal complexes of hydrogen cyanamide J. Inorg. Nucl.Chem. C, W. W., Huestis, W. H., Theyson, T. W. 1972; 39: 2358 - 2362
  • F-19-NMR STUDIES OF OXYGEN BINDING TO HEMOGLOBIN BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Huestis, W. H., Raftery, M. A. 1972; 49 (5): 1358-?

    View details for Web of Science ID A1972O216400028

    View details for PubMedID 4645548

  • 31P-nmr studies of the release of diphosphoglyceric acid on carbon monoxide binding to hemoglobin Biochem. Biophys. Res. Commun. H, H. W., Raftery, M. A. 1972; 49: 428 - 433
  • The binding of n-butyl isocyanide to human hemoglobin Biochem. Biophys. Res. Commun. H, H. W., Raftery, M. A. 1972; 48: 678 - 683
  • OBSERVATION OF COOPERATIVE IONIZATIONS IN HEMOGLOBIN PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Huestis, W. H., Raftery, M. A. 1972; 69 (7): 1887-?

    Abstract

    (19)F-Nuclear magnetic resonance studies of specifically fluorinated hemoglobin derivatives have been used to determine the apparent pK(a) of the histidine beta146 imidazole in deoxyhemoglobin. The titration of this residue was found to be abnormally sharp, particularly in the presence of diphosphoglyceric acid. The explanation advanced for this unusual titration curve may have implications for the mechanism of cooperative ligand binding. The possible role of such ionizations is discussed in light of some chemical evidence that the cooperative binding process is governed to a greater extent by internal nonpolar forces than by electrostatic interactions of exposed groups.

    View details for Web of Science ID A1972N026000055

    View details for PubMedID 4505667

  • STUDY OF COOPERATIVE INTERACTIONS IN HEMOGLOBIN USING FLUORINE NUCLEAR MAGNETIC-RESONANCE BIOCHEMISTRY Huestis, W. H., Raftery, M. A. 1972; 11 (9): 1648-?

    View details for Web of Science ID A1972M255400018

    View details for PubMedID 5028108

  • USE OF F-19-NUCLEAR MAGNETIC-RESONANCE SPECTROSCOPY FOR DETECTION OF PROTEIN CONFORMATION CHANGES - APPLICATION TO LYSOZYME, RIBONUCLEASE AND HEMOGLOBIN COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Raftery, M. A., Huestis, W. H., Millett, F. 1971; 36: 541-?
  • Use of fluorine-19 nuclear magnetic resonance to study conformation changes in specifically modified ribonuclease S Biochemistry H, H. W., Raftery, M. A. 1971; 10: 1181 - 1186