bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of photoaffinity nucleotide analogs to determine the mechanism of ATP regulation of a membrane‐bound, cAMP‐activated protein kinase
Authors: Owens James R. , Haley Boyd E. 
Published: May 2005

AbstractUsing a radioactively tagged, photoaffinity analog of cAMP, 8‐azidoadenosine‐3′,5′‐cyclic monophosphate (8‐N3 cAMP), and [γ32P] ATP, the membranebinding properties of both the regulatory and catalytic subunits of the cAMP‐activated protein kinase of human erythrocyte membranes were investigated. [32P] 8‐N3 cAMP was used to locate and quantify regulatory subunits. Increased phosphorylation of specific membrane proteins by [γ32P] ATP was used to determine the presence of the catalytic subunit. The data support a mechanism which operates through a tight membrane‐bound regulatory subunit and a catalytic subunit that is released from the membrane when cAMP is present and the Mg · ATP concentration is below approximately 10 μM. The catalytic subunit is not required for the Mg · ATP inhibition of 8‐N3 cAMP binding. Experiments with a photoaffinity analog of ATP, 8‐azidoadenosine triphosphate (8‐N3ATP), support the hypothesis that ATP hydrolysis and phosphorylation are not involved in the regulation. The data indicate that the regulatory subunit contains an ATP regulatory site which inhibits 8‐N3 cAMP binding and the release of the catalytic subunit. These results indicate that the membrane‐bound type I enzyme (type IM) differs significantly from the soluble (type IS) enzyme studied in other tissues. These enzymes are compartmentalized by being in different cellular locations and are regulated differently by Mg · ATP.
Glycophorins A, B, and C: A family of sialoglycoproteins. Isolation and preliminary characterization of trypsin derived peptides
Authors: Furthmayr Heinz 
Published: May 2005

AbstractTwo new sialoglycoproteins, glycophorin B and glycophorin C, were isolated from erythrocyte membranes by extraction with lithium diiodosalicylate, partition in aqueous phenol, gel filtration in detergent, and preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The two proteins were characterized by amino acid and carbohydrate analysis, separation of tryptic peptides, and isolation and purification of the amino terminal glycopeptide from each polypeptide chain. Glycophorin B is found in two forms in electrophoretograms of normal erythrocyte membranes corresponding to monomer and dimer, as has been similarly described for glycophorin A. By using antibodies to a carboxy terminal determinant of glycophorin A, and direct staining of gels with antibodies and 125I‐protein A from Staph. aureus, as well as by two‐dimensional immunoelectrophoreis, only the two known forms of glycophorin A are detectable. The data confirm and extend the notion that the sialoglycoproteins in human red cells are dimeric molecules which are either preformed in the membrane or which can readily be generated in vitro. Only glycophorin A and glycophorin C are sensitive to trypsin while in situ in the intact red blood cells.
Phosphorylation and dephosphorylation of spectrin
Authors: Fairbanks Grant , Avruch Joseph , Dino Judith E. , Patel Vikram P. 
Published: May 2005

AbstractThe phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP‐independent, salt‐stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co‐fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane‐bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of 32Pi from [32P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [32P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions – notably, ATP and 2,3‐DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated (1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and (2) by membrane deformation that alters enzyme–spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to the resolution of major questions raised by previous work: the relationships between membrane‐bound and cytoplasmic forms of the enzymes, the nature of their physical interactions with the membrane, and the regulation of their activities in defined cell‐free systems.
Actin—membrane interactions: Association of G‐actin with the red cell membrane
Authors: Cohen Carl M. , Jackson Pamela L. , Branton Daniel 
Published: May 2005

AbstractChemically tritiated actin from rabbit skeletal muscle was used to investigate the association of G‐actin with the red cell membrane. The tritiated actin was shown to be identical to unmodified actin in its ability to polymerize and to activate heavy meromyosin ATPase. Using sealed and unsealed red cell ghosts we have shown that G‐actin binds to the cytoplasmic but not the extracellular membrane surface of ghosts. Inside‐out vesicles which have been stripped of endogenous actin and spectrin by low‐ionic‐strength incubation bind little G‐actin. However, when a crude spectrin extract containing primarily spectrin, actin, and band 4.1 is added back to stripped vesicles, subsequent binding of G‐actin can be increased up to 40‐fold. Further, this crude spectrin extract can compete for and abolish G‐actin binding to unsealed ghosts. Actin binding to ghosts increases linearly with added G‐actin and requires the presence of magnesium. In addition, actin binding is inhibited by cytochalasin B and DNAase I. Negative staining reveals an abundance of actin filaments formed when G‐actin is added to reconstituted inside‐out vesicles but none when it is added to unreconstituted vesicles. These observations indicate that added G‐actin binds to the red cell membrane via filament formation nucleated by some membrane component at the cytoplasmic surface.
An avidin‐biotinyl‐propranolol complex for β‐adrenergic receptor characterization
Authors: Meier Kathryn E. , Ruoho Arnold E. 
Published: May 2005

AbstractThe synthesis of biotinyl‐hexaglycyl‐NEDA (abbreviation:BGN), a biotinyl derivative of propranolol, is described. This bifunctional molecule binds with high affinity to the biotin‐binding protein, avidin. The duck erythrocyte was used as a model β‐receptor system. Formation of an avidin‐BGN‐β‐receptor complex was demonstrated in intact erythrocytes, in erythrocyte ghosts, and in the digitonin‐solubilized β‐receptor. The avidin‐BGN complex will be used for localization and purification of the β‐receptor.
A serological assay for the detection of cell surface receptors of nerve growth factor
Authors: Zimmermann Astrid , Sutter Arne , Samuelson John , Shooter Eric M. 
Published: May 2005

AbstractWhen single‐cell suspensions prepared from embroyonic day 8 (E8) chick sensory ganglia are incubated with nerve growth factor (NGF), anti‐NGF antiserum, and complement, an NGF‐dependent cytotoxic kill of 20 (±3)% of the ganglia cells is observed. This percentage is increased by a factor of two when only the neuronal cells are tested. No kill is observed on the nonneuronal cell population representing 50% of the ganglia dissociate. When E8 sensory ganglia cells are cultured in the presence of NGF following cytotoxic kill, the large, phase‐bright NGF‐reponsive neurons are missing from the culture. These results indicate that the cells recognized in the cytotoxicity assay have to carry NGF‐binding sites of type I, which is the one with the higher affinity of the two types of NGF‐binding sites (I and II) present on sensory ganglia cells. This conclusion is further supported by the following data: (a) half maximal cytotoxicity is reached already at a concentration of NGF which is below the KD of binding site I; (b) a washing step which removes all NGF bound to type II receptors while leaving a high percentage of type I receptors occupied has no effect on the percentage of ganglia cells killed.Using the cytotoxicity assay the presence of high‐affinity binding sites of type I can be demonstrated on sensory ganglia cells from E8 chick embryos but not from E4 embryos and not on liver and heart cells from E8 embryos. Further, type I receptor‐bearing cells were detectable in the brain using this assay. At E8, NGF receptors could be detected on cells of the forebrain and the tectum but not on brain stem cells. Cytotoxic kill of forebrain cells was found to be especially high at E8 and E9, and decreased by E10.
The spontaneous release of a high‐molecular‐weight aggregate containing immunoglobulin G from the surface of Ehrlich ascites tumor cells
Authors: Rittenhouse Harry G. , Ar Diane , Lynn Matthew D. , Denholm David K. 
Published: May 2005

AbstractThe spontaneous release of tumor cell antigens from the cell surface into the circulation has been proposed as a mechanism whereby tumors may escape the immune response of the host. In this study we have found that Ehrlich ascites tumor cells after removal from the host (mouse) spontaneously release significant amounts of cell surface components during incubation for 1 h in cold isotonic buffer. Immunodiffusion studies revealed that immunoglobulin G (IgG) and a complement component (C3) are included in this spontaneously released material. These surface‐bound humoral immune components are apparently released in the form of a high‐molecular‐weight aggregate (cell coat particle) as shown by ultracentrifugation and ultrafiltration experiments. Precipitation of IgG from the cell coat particle preparation with antibodies directed against mouse IgG followed by detergent gel electrophoresis of the immune precipitate revealed five major bands in addition to the heavy and light chains of IgG. These results suggest that host IgG is tightly bound to several other components at the cell surface, perhaps in the form of immune complexes.IgG is localized on the tumor cell surface in a highly heterogeneous pattern with the appearance of patches and caps in some cells as shown by immuno‐fluorescence analysis. The possibility that humoral immune components bind to the tumor cell surface and result in the shedding of high‐molecular‐weight aggregates of cell surface antigens into extracellular fluids is discussed.
Murine leukemia cell hybrids: The quantity of TL antigens expressed by parental and hybrid cells fails to correlate with their sensitivity to TL antibody and complement
Authors: Cohen Edward P. , Liang Weitze 
Published: May 2005

AbstractThe quantity of thymus‐leukemia (TL) antigens expressed by murine leukemia cells is significantly greater than that expressed by somatic hybrids of such cells. Based upon the results of 125I‐lactoperoxidase labeling and antibody absorption procedures, and corrected for size differences between the two cell types, the quantity of TL antigens expressed by RADA‐1 cells, a radiation‐induced murine leukemia cell line of strain A/J mice, is approximately 5.0 times greater than that of somatic hybrids of RADA‐1 and LM(TK)− cells. LM(TK)− cells are a thymidine kinase‐deficient TL(‐) mouse fibroblast cell line. The quantity of TL antigens expressed is related only in part to their susceptibility to lysis by TL antibodies and guinea pig complement (GPC). RADA‐1 cells resist lysis. The quantity of TL antigens expressed by RADA‐1 cells is analogous to that formed by nonneoplastic thymocytes obtained from F1 hybrids of two strains of TL(+) and TL(‐) mice; cells from both strains are sensitive to TL antiserum and GPC. ASL‐1 cells, a spontaneously occurring leukemia cell line of A/J mice, express TL antigens in significantly higher quantities than any of the cell types examined. Exposed to TL antisera, the quantity of TL antigens of ASL‐1 cells, but not that of hybrid cells, gradually diminishes. ASL‐1 cells convert over a 6‐h period of exposure to antibody and guinea pig complement (GPC) resistance; hybrid cells remain sensitive. However, ASL‐1 cells converted to TL antibody and GPC resistance continue for a time to express TL antigens in quantities similar to that of sensitive F1 thymocytes and resistant RADA‐1 cells. RADA‐1 X LM(TK)− hybrid cells, which are sensitive to TL antibodies and GPC, express the lowest quantities of TL antigens of any of the cell types examined. It is likely that differences in the quantities of TL antigens expressed by different cell lines reflect genetic mechanisms controlling TL antigen expression. The failure of TL antisera to affect the quantities of TL antigens expressed by hybrid cells is taken as an indication that genetic controls governing antigen expression may be distinguished from those involved in regulating responsiveness to specific antiserum.
Role of physiologic autoantibody in the removal of senescent human red cells
Authors: Kay Marguerite M. B. 
Published: May 2005

AbstractThe mechanism by which mononuclear phagocytes distinguish mature “self” from senescent “self” was investigated. Evidence is presented indicating that human mononuclear phagocytes distinguish senescent RBC from mature RBC on the basis of selective Ig attachment to the membranes of senescent cells. This Ig, eluted from senescent human RBC, was shown to be IgG and free of other Igs by immunodiffusion, immunoelectrophoresis, and polyacrylamide gel electrophoresis. The IgG was polyclonal with respect to light chains. The eluted IgG reattaches to homologous stored RBC, but not to mature autologous or allogeneic RBC, via the Fab region. It then initiates phagocytosis of these stored RBC by mononuclear phagocytes. Evidence suggests that the IgG is directed against altered membrane receptors. Thus, this IgG may be a “physiologic” autoantibody and contribute to the maintenance of homeostasis by performing regulatory functions.
A possible modulation of acetylcholine receptors of embryonic chick muscle cells by α‐bungarotoxin
Authors: Elson Hannah Friedman 
Published: May 2005

AbstractAcetylcholine receptors were assayed with α‐bugarotoxin on embryonic chick skeletal muscle growing in primary cell culture. Toxin was bound specifically to muscle cells and could be competed with D‐tubocurarine. Two dissociation constants were obtained by equilibrium binding: 7.2 × 10−9M and 2.7 × 10−7M at 25°C. Two sets of rate constants were also obtained from dissociation kinetics. There are five times more low affinity sites on cells than high affinity sites. The average density of high‐affinity receptors is about 200/μm2.A time course of toxin binding to receptors at 37°C vs 25°C in growth medium revealed that under conditions permitting growth and metabolism, toxin bound to cells was lost. The possibility that the growth medium was in‐activating toxin molecules was ruled out by showing that unbound toxin molecules in the medium were fully capable of binding to fresh cultures.
Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: The endocytic activation hypothesis
Authors: Fox C. Fred , Das Manjusri 
Published: May 2005

AbstractThe addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity 125I‐EGF, and the fate of the affinity labeled EGF‐receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF‐induced receptor internalization and EGF‐induced stimulation of 3H‐thymidine uptake into cellular DNA show that there is a direct correlation between EGF‐induced receptor internalization and EGF‐induced stimulation of DNA synthesis, but not between EGF binding and EGF‐induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF‐induced processes requires constant EGF‐induced internalization of receptor for a requisite 6–8 h period as an obligatory step in production of “second messenger” in the action of this hormone.
Detection of erythrocyte membrane structural abnormalities in lecithin: Cholesterol acyltransferase deficiency using a spin label approach
Authors: Maraviglia B. , Herring F. G. , Weeks G. , Godin D. V. 
Published: May 2005

AbstractThe membrane fluidity of erythrocytes from patients with Lecithin: cholesterol acyltransferase (LCT) deficiency was studied by means of electron spin resonance. The temperature dependence of the separation of the outer extrema of the spectra of 2‐(3‐carboxy‐propyl)‐4,4‐dimethyl, 2‐tridecyl‐3‐oxazolidinyloxyl spin probe was monitored for normal, presumed carrier and clinically affected subjects. The temperature profile of controls was significantly different form that of the presumed carriers and the clinically affected individuals. The results show that the compositional abnormalities previously noted in erythrocyte membranes from patients with LCAT deficiency are associated with alterations in the physicochemical state of the membrane. An investigation of the spectral lineshapes below 10°C allowed a distinction to be made at the membarne level between clinically affected subjects and clinically normal heterozygous carriers. Alterations in the temperature dependence of electron spin resonance parameters may provide a sensitive index of red cell membrane alterations in pathological states of generalized membrane involvement.
A high‐affinity ATP‐binding site on 30S dynein
Authors: Blum J. J. , Hayes A. 
Published: May 2005

AbstractThe enhancing effect of low concentrations (eg, 8 μM) of bis(4‐fluoro‐3‐nitrophenyl)sulfone (FNS) on 30S dynein ATPase activity is increased when 1 mM dithiothreitol (DTT) is present. The effect of FNS + DTT is optimal at pH 7.5. Activation of the latent ATPase activity of 30S dynein by FNS + DTT is partially prevented by 1–3 μM ATP. Adenylylimidodiphosphate (AMP‐PNP) is less effective than ATP, while β,γ‐methylene‐adenosine triphosphate (AMP‐PCP), though a much stronger inhibitor of ATPase activity than AMP‐PNP, does not protect against enhancement. These results demonstrate the presence of a high‐affinity ATP‐binding site on 30S dynein.
Threshold effects on the lectin‐mediated aggregation of synthetic glycolipid‐containing liposomes
Authors: Rando Robert R. , Bangerter Faan Wen 
Published: May 2005

AbstractCholesterol analogs containing sugar residues linked by spacer groups to the cholesterol O can be incorporated into egg yolk lecithin small unilamellar liposomes. The synthetic glycolipid analogs distribute evenly on both sides of the bilayer. These liposomes are aggregated by the appropriate lectin. For example, when the sugar residue is a β‐galactoside the liposomes are aggregated by ricin and when it is an α‐mannoside they are aggregated by Con A. The lectin‐mediated aggregation of these liposomes is reversed by the addition of the appropriate sugar. The rates but not the extents of aggregation of these liposomes are highly sensitive to the amount of glycolipid incorporated. Below approximately 5% glycolipid incorporation the rate of the lectin‐mediated aggregation of these liposomes is exceedingly slow, whereas above this level rapid aggregation proceeds. At all concentrations studied the synthetic glycolipids are incorporated in a unimodal fashion so that the observed threshold effects cannot be based on possible differences in the manner in which the glycolipids are incorporated at different concentrations. This conclusion is based on (1) studies with galactose oxidase that show that the percentage of galactose oxidation in a liposome prepared from a galactosyl‐containing glycolipid is independent of glycolipid concentration, and (2) studies on the aggregation of liposomes containing mixed glycolipids in which the glycolipids are shown to behave independently. The importance of a critical density of membrane‐bound receptors in order for aggregation to occur is discussed.
Biosynthesis of membrane glycoproteins in rat hepatoma tissue culture cells
Authors: Baumann Heinz 
Published: May 2005

AbstractThe early steps in the biosynthesis of glycoproteins associated with the plasma membranes of rat hepatoma tissue culture cells has been analyzed. By measuring the effect of tunicamycin on the incorporation of [3H] mannose and [3H] fucose into cell glycoproteins, it was determined that an interval of about 1 h was required to transfer the glycoprotein from the site of mannosylation to the site of fucosylation. This result was corroborated by an analysis of the time required for the appearance of either mannose or fucose‐labeled glycoproteins at the cell surface. The separation of membrane glycoproteins by a two‐dimensional gel system allowed the visualization of the modifications leading to both size and charge heterogeneity of these proteins. By following the changes in electrophoretic mobility introduced into membrane glycoproteins during a chase period after a pulse labeling, the time course of these molecular alterations could be estimated. Several glycoproteins have apparently higher rates of synthesis than the bulk of membrane‐associated glycoproteins. Most of these glycoproteins were released within 2 h after biosynthesis from the intracellular membrane fraction and appear after 3 h in the medium. In addition to the glycoproteins that contain both mannose and fucose and that show a high degree of charge heterogeneity, there are other membrane‐bound species that are not noticeably modified by the in corporation of fucose or sialic acids. These glycoproteins could represent constituents limited to the internal membrane system of the HTC cell.