The binding in vitro of modified LDL to the intermediate filament protein vimentin

Membrane-associated proteins with specific binding properties to modified LDL were investigated in J774 macrophages and Mono Mac 6 sr cells. Ligand blotting of membrane proteins revealed a 54-kDa protein which bound oxidized and acetylated but not native LDL. The 54-kDa protein, isolated by 2D-PAGE, was identified as vimentin. (125)I-AcLDL bound to purified vimentin and desmin in a saturable manner, with an approximate K(d) of 1.7 x 10(-7) M (89 microgram/ml) and 8.0 x 10(-8) M (41 microgram/ml), respectively. Blots of vimentin mutant proteins with deletions in the positively charged N-terminal head domain showed that amino acids 26-39 are essential for the binding of AcLDL by vimentin. Taken together, our data indicate that vimentin binds modified LDL, but not native LDL, in a specific and saturable manner. Vimentin filaments extend throughout the cytoplasm as far as the inner surfaces of plasma and vesicular membranes. Vimentin may thus play a role in membrane-associated steps involved in the intracellular processing of oxidized LDL, contributing to its unregulated uptake and intracellular retention by cells of the atherogenic plaque.     

Structural requirements for binding to the sugar-transport system of the human erythrocyte

The structural requirements for binding to the glucose/sorbose-transport system in the human erythrocyte were explored by measuring the inhibition constants, K(i), for specifically substituted analogues of d-glucose when l-sorbose was the penetrating sugar. Derivatives in which a hydroxyl group in the d-gluco configuration was inverted, or replaced by a hydrogen atom, at C-1, C-2, C-3, C-4 or C-6 of the d-glucose molecule, all bound to the carrier, confirming that no single hydroxyl group is essential for binding to the carrier. The binding and transport of 1-deoxy-d-glucose confirmed that the sugars bind in the pyranose form. The relative inhibition constants of d-glucose and its deoxy, epimeric and fluorinated analogues are consistent with the combination of beta-d-glucopyranose with the carrier by hydrogen bonds at C-1, C-3, probably C-4, and possibly C-6 of the sugar. Both polar and non-polar substituents at C-6 enhance the affinity of d-glucose derivatives relative to d-xylose, and d-galactose derivatives relative to l-arabinose, and it is suggested that the carrier region around C-6 of the sugar may contain both hydrophobic and polar binding groups. The spatial requirements at C-1, C-2, C-3, C-4 and C-6 were explored by comparing the relative binding of d-glucose and its halogeno and O-alkyl substituents. The carrier protein closely approaches the sugar except at C-3 in the d-gluco configuration, C-4 and C-6. d-Glucal was a good inhibitor, showing that a strict chair form is not essential for binding. 3-O-(2',3'-Epoxypropyl)-d-glucose, a potential substrate-directed alkylating agent, bound to the carrier, but did not inactivate it.     

Reversible DIDS binding to band 3 protein in human erythrocyte membranes

Reversible binding of DIDS [4,4'-diisothiocyanato-2,2'-stilbenedisulphonate] to Band 3 protein, the anion exchanger located in erythrocyte plasma membrane, was studied in human erythrocytes. For this purpose, the tritiated form of DIDS ([3H]DIDS) has been synthesized and the filtering technique has been used to follow the kinetics of DIDS binding to the sites on Band 3 protein. The obtained results showed monophasic kinetics both for dissociation and association of the 'DIDS--Band 3' complex at 0 degree C in the presence of 165 mM KCl outside the cell (pH 7.3). A pseudo-first order association rate constant k+1 was determined to be (3.72 +/- 0.42) x 10(5) M-1 s-1, while the dissociation rate constant K-1 was determined to be (9.40 +/- 0.68) x 10(-3) s-1. The dissociation constant KD, calculated from the measured values of k-1 and k+1, was found to be 2.53 x 10(-8) M. The standard thermodynamics parameters characterizing reversible DIDS binding to Band 3 protein at 0 degree C were calculated. The mean values of the activation energies for the association and dissociation steps in the DIDS binding mechanism were determined to be (34 +/- 9) kJ mole-1 and (152 +/- 21) kJ mole-1, respectively. The results provide, for the first time, evidence for the reversibility of DIDS binding to Band 3 protein at 0 degree C. The existence of a stimulatory site is suggested, nearby the transport site on the Band 3 protein. The binding of an anion to this site can facilitate (through electrostatic repulsion interaction between two anions) the transmembrane movement of another anion from the transport site.     

Reversible DIDS binding to Band 3 protein in human erythrocyte membranes

Reversible binding of DIDS [4,4'-diisothiocyanato-2,2'-stilbenedisulphonate] to Band 3 protein, the anion exchanger located in erythrocyte plasma membrane, was studied in human erythrocytes. For this purpose, the tritiated form of DIDS ([³H]DIDS) has been synthesized and the filtering technique has been used to follow the kinetics of DIDS binding to the sites on Band 3 protein. The obtained results showed monophasic kinetics both for dissociation and association of the 'DIDS-Band 3' complex at 0° C in the presence of 165 mM KCl outside the cell (pH 7.3). A pseudo-first order association rate constant k₊₁     

Temperature-dependent modes for the binding of the polyene antibiotic amphotericin B to human erythrocyte membranes. A circular dichroism study

The interaction of amphotericin B with isolated human erythrocyte ghosts was monitored by circular dichroism at 37 degrees C and 15 degrees C. Although different, these spectra were not concentration dependent over a concentration range covering the inducement of K+ leakage and hemolysis, which suggests the existence of only one bound amphotericin B species. At 15 degrees C, the spectra indicate that amphotericin B is complexed with membrane cholesterol; the complex formation is saturable but not cooperative. At 37 degrees C new spectra are observed, and their existence is conditioned by the presence of membrane proteins. The binding is cooperative but not saturable. The amphotericin B right side-out vesicles complexation is temperature as well as ionic strength dependent: at high ionic strength it is the same as with ghosts, with the same temperature dependence. At low ionic strength it is characteristic of an interaction with cholesterol, regardless of temperature. In the large unilamellar vesicles reconstituted from the total lipid extracts of erythrocyte membranes, amphotericin B is complexed with cholesterol, regardless of temperature and ionic strength. These results indicate that there are two different modes of amphotericin B complexation with erythrocyte membranes, reversible one in the other, depending on the molecular organization of the membrane and the presence of membrane proteins.     

Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes

Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes was studied by incubating the membranes at different pH and temperatures and determining the bound bilirubin. At all pH values, the amount of membrane-bound bilirubin increased with the increase in bilirubin-to-albumin molar ratios (B/As), being highest at lower pH values in all cases. Further, linear increase in bound bilirubin with the increase in bilirubin concentration in the incubate was observed at a constant B/A and at all pH values. However, the slope value increased with the decrease in pH suggesting more bilirubin binding to membranes at lower pH values. Increase in bilirubin binding at lower pH can be explained on the basis of increased free bilirubin concentration as well as more conversion of bilirubin dianion to monoanion. Temperature dependence of bilirubin binding to membranes was observed within the temperature range of 7 degrees -60 degrees C, showing minimum binding at 27 degrees C and 37 degrees C which increased on either side. Increase in bilirubin binding at temperatures lower than 20 degrees C and higher than 40 degrees C can be ascribed to the change in membrane topography as well as bilirubin-albumin interaction.     

Characterization of immunoglobulin binding to isolated human erythrocyte membranes: evidence for selective, temperature-induced binding of naturally occurring autoantibodies to the cytoskeleton

Human plasma contains naturally occurring autoantibodies to the predominant components of the erythrocyte membrane: band 3 and spectrin bands 1 and 2 of the cytoskeleton. The titer of cytoskeletal plasma autoantibodies increases in various hemolytic conditions, suggesting that opsonization of the cytoskeleton may play an important role in the clearance of hemolyzed (not senescent) erythrocytes from the circulation. In this study, we use Alexa Fluor 488 goat anti-human IgG conjugate (Molecular Probes, Eugene, OR, USA), to characterize plasma immunoglobulin binding to erythrocyte membranes from osmotically hemolyzed cells ('ghosts'). The results show that exposure of ghosts to plasma results in 4-fold more immunoglobulin binding to the cytoskeleton than is bound to the proteins contained within the lipid bilayer. Preincubation of the ghosts at 37 degrees C causes 8-fold more immunoglobulin binding to the cytoskeleton compared to bilayer proteins. This temperature-induced change resulted from selective immunoglobulin binding to the cytoskeleton, with no change in immunoglobulin binding to bilayer proteins. However, the rate of increase in cytoskeletal antigenicity at 37 degrees C did correlate with the rate of a conformational change in band 3, a transmembrane protein which serves as a major membrane attachment site for the cytoskeleton. The results of this study suggest that the cytoskeleton is the primary target in the opsonization of hemolyzed erythrocyte membranes by naturally occurring plasma autoantibodies. The conformational changes which occur in ghosts at 37 degrees C are associated with selective exposure of new immunoglobulin binding sites on the cytoskeleton, and with a change in the structure of band 3. We propose a model suggesting that opsonization of the cytoskeleton occurs prior to the decomposition of hemolyzed erythrocytes at 37 degrees C.     

Distribution of insulin receptors in human erythrocyte membranes. Insulin binding to sealed right-side-out and inside-out human erythrocyte vesicles

Analyses of insulin binding to human erythrocytes and to resealed right-side-out and inside-out erythrocyte membrane vesicles have revealed that high affinity insulin binding receptors are present on both sides of the erythrocyte membranes. Insulin binding to human erythrocytes was examined with the use of a binding assay designed to minimize the potential errors arising from the low binding capacity of this cell type and from non-specific binding in the assay. Scatchard analysis of equilibrium binding to the cells revealed a class of high affinity sites with a dissociation constant (Kd) of (1.5 +/- 0.5) X 10(-8) M and a maximum binding capacity of 50 +/- 5 sites per cell. Interestingly, both resealed right-side-out and inside-out membrane vesicles exhibited nearly identical specific sites for insulin binding. At the high affinity binding sites, for both right-side-out and inside-out vesicles, the dissociation constant (Kd) was (1.5 +/- 0.5) X 10(-8) M, and the maximum binding capacity was 17 +/- 3 sites per cell equivalent. These findings suggest that insulin receptors are present on both sides of the plasma membrane and are consistent with the participation of the erythrocyte insulin receptors in an endocytic/recycling pathway which mediates receptor-ligand internalization/externalization.     

Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes.

Saturable bilirubin binding to human erythrocyte membranes was measured before and after digestion with neuraminidase and phospholipases. Neuraminidase-treated erythrocyte membranes did not show any change in their binding properties, indicating that gangliosides could be excluded as candidates for saturable bilirubin-binding sites on erythrocyte membranes. Although bilirubin-binding properties of the membranes did not change after phospholipase D digestion, either, phospholipase C treatment greatly enhanced bilirubin binding. Thus it is suggested that a negatively charged phosphoric acid moiety of phospholipids on the membrane surface may play a role to prevent a large amount of bilirubin from binding to the membranes. Further saturable bilirubin binding to inside-out sealed erythrocyte membrane vesicles showed values comparable with those of the right-side-out sealed membranes, suggesting that the bilirubin-binding sites may be distributed on both outer and inner surfaces of the membranes, or may exist in the membranes where bilirubin may be accessible from either side.     

The binding in vitro of the intermediate filament protein vimentin to synthetic oligonucleotides containing telomere sequences

The ability of the intermediate filament subunit protein vimentin to bind synthetic oligonucleotide telomere models containing repeat sequences from Oxytricha (T4G4), Saccharomyces (TGTGTG3), or Tetrahymena (T2G4) was investigated in vitro with a filter binding assay and a gel overlay assay. At low ionic strength, vimentin bound these oligonucleotides with high affinity. At higher ionic strength, the vimentin-oligonucleotide complex was less stable, such that approximately 30% of the initial binding remained at 150 mM KCl. One mole of vimentin tetramer bound approximately 1 mol of telomere oligonucleotide. Vimentin bound well oligonucleotides containing either a random duplex or random 3'-overhang, but showed a reduced affinity for a blunt-ended oligonucleotide. A control random sequence oligonucleotide was not bound by vimentin. The oligonucleotide-binding site of vimentin was shown to be localized in the non-alpha-helical N-terminal domain by assays employing purified proteolytic fragments of vimentin. Preliminary results in the gel overlay assay show that other members of the intermediate filament family, nuclear lamins A-C, all bind the synthetic oligonucleotide containing the telomere repeat sequence of Oxytricha.     

Role of lysine residues in the binding of glyceraldehyde-3-phosphate dehydrogenase to human erythrocyte membranes

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) binds reversibly to human erythrocyte membranes. Several specific amino acid residues involved in the enzyme-membrane contact region have already been identified. These include tyrosine 46 and threonine 150. Covalent modification of lysines 212 and 191 with pyridoxal phosphate results in a decreased affinity of the enzyme for erythrocyte membranes if the enzyme-linked pyridoxal phosphate is not reduced prior to binding. Reduction of the pyridoxal phosphate-lysine complex completely inhibits the binding of the enzyme to erythrocyte membranes. These results suggest a role for lysines 212 and 191 in the interaction of glyceraldehyde-3-phosphate with human erythrocyte membranes.     

A reappraisal of the binding of cytosolic enzymes to erythrocyte membranes

Several cytosolic proteins have been shown to be associated with hypotonic erythrocyte ghosts via electrostatic interactions with the anion transport band 3 protein. This article considers the problems of demonstrating binding under physiological conditions and reviews the evidence for the relevance of enzyme binding to the membrane for the regulation of glycolysis. The hypotheses for the existence of topological and sequential multienzyme complexes of the glycolytic enzymes in erythrocytes are also discussed.     

Zebrafish: a model system for the study of human disease

The zebrafish (Danio rerio) is a powerful model organism for the study of vertebrate biology, being well suited to both developmental and genetic analysis. Large-scale genetic screens have identified hundreds of mutant phenotypes, many of which resemble human clinical disorders. The creation of critical genetic reagents, coupled with the rapid progress of the zebrafish genome initiative directed by the National Institutes of Health, are bringing this model system to its full potential for the study of vertebrate biology, physiology and human disease.     

Effects of the cytosol fraction and ATP on the kinetics of cAMP binding to human erythrocyte membranes

The cytosol fraction of human erythrocytes as well as 1 mM ATP decreased the rate constant (Kl) of association of [3H]cAMP with human erythrocyte membranes. However, the effect of 1 mM ATP on the association kinetics was much greater than that of the cytosol fraction. Accordingly, the cytosol fraction and 1 mM ATP increased the rate constant (k-l) of dissociation of bound [3H]cAMP from the human erythrocyte membranes. However, the cytosol fraction affected the dissociation kinetics to a far greater extent than 1 mM ATP. Thus, although both cytosol and 1 mM ATP decreased the affinity of [3H]cAMP for binding sites on human erythrocyte membranes at equilibrium, the detailed mode of their action on the membrane [3H]cAMP binding sites appears to differ.     

竹红菌甲素对红细胞膜上几种酶光敏失活作用的研究

Hypocrellin A (HA)-sensitized photoinactivation of enzymes in human erythrocyte membrane, including AchE, GPDH, Na(+)-K+ ATPase, Ca2(+)-Mg2+ ATPase were studied in this paper. The sensitivity of these four enzymes inactivated by HA and light are as following order: Ca2(+)-Mg2+ ATPase greater than Na(+)-K+ ATPase greater than GPDH greater than AchE. The relationship among ATPase inactivation, sulfhydryl photoinactivation and lipid peroxidation was also investigated. Results show that SH group photooxidation probably is one of the major reasons of enzyme inactivation whereas lipid peroxidation has little effect. The isolated GPDH was less sensitive than that membrane-bound, GSH, NAD acted protectively on GPDH and ATPase respectively. The evidence of electrophoresis and protein intrinsic fluorescence showed that protein structure did not change significantly even though most activity had lost in case of GPDH.