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Y Lange

Publications and source records attributed to Y Lange.

At least 55 records · Page 3Linked to original sources
PubMed1982

Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane.

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.

Actins↗
PubMed1982

Interaction of cholesterol and lysophosphatidylcholine in determining red cell shape.

The effect of lysolecithin on the shape of human erythrocytes of varied cholesterol content was examined by scanning electron microscopy. Under the conditions of these experiments, all of the [14C]lysolecithin incubated with cells was shown to be located in the external membrane leaflet. The membrane lysolecithin required to induce echinocytosis (spiculation) in normal cells (0.8 mol cholesterol/mol phospholipid) was approximately 0.08-0.10 mumol/10(10) cells, which contributed 1.6-2.0 micrometer 2 or 1% of the cell surface area. This value is consistent with the premise that echinocytosis was caused by a slight differential expansion of the outer surface of the bilayer. The lysolecithin required for echinocytosis decreased as the membrane cholesterol content increased; from 0.14 mumol/10(10) cells at 0.5 mol cholesterol/mol phospholipid to 0.03 mumol/10(10) cells at 1.4 mol cholesterol/mol phospholipid. These data were interpreted in terms of a bilayer couple mechanism. Assuming that the two amphipaths acted additively, the amount of lysolecithin required to induce echinocytosis was used to estimate the partition of cholesterol between the two leaflets of the red cell membrane. A value of about 51:49% in favor of the outer leaflet was found at all cholesterol levels.

Cholesterol↗
PubMed1981

The rate of transmembrane movement of cholesterol in the human erythrocyte.

Cholesterol appears to be abundant on both sides of the human erythrocyte membrane, but its precise distribution and rate of transmembrane movement are undetermined. Although the cholesterol in the intact cell normally was resistant to cholesterol oxidase, enrichment of cells with exogenous cholesterol or preincubation at very low ionic strength rendered the entire cholesterol pool susceptibility to attack. Under these experimental conditions, all of the membrane cholesterol was oxidized in a strictly first order fashion with a half-time as short as 10 s at 37 degrees C. Since the enzyme had access only to the outer membrane surface, these data suggest that the transmembrane movement (flip-flop) of cholesterol is extremely rapid. From an error analysis, we estimate an upper bound on the half-time of the transmembrane movement of cholesterol of 3 s at 37 degrees C. A physiological function for rapid sterol flip-flop is suggested.

Cholesterol↗
PubMed1980

The effect of cholesterol and other intercalated amphipaths on the contour and stability of the isolated red cell membrane.

Three membrane properties were strikingly affected when the cholesterol of human erythrocytes, normally approximately 0.8 mol/mol of phospholipid (i.e. C/P approximately 0.8), was altered by equilibration with phospholipid dispersions of an appropriate cholesterol content. 1) While the sterol in intact red cell membranes of C/P less than or equal to 0.8 was resistant to cholesterol oxidase digestion, enrichment to C/P greater than or equal to 0.9 rendered the entire cholesterol pool sensitive to enzyme attack. Susceptibility to oxidation was reversed by removal of the excess cholesterol. Treatment of cells with 1 X 10(-4) M chlorpromazine also rendered the entire cholesterol pool susceptible to cholesterol oxidase. 2) Whereas ghosts with C/P less than or equal to 0.8 underwent invagination in low ionic strength, alkaline buffers to form inside-out vesicles, enrichment to C/P greater than or equal to 1.0 promoted right-side-out vesicle formation instead. This effect was mimicked by treatment with 2,4-dinitrophenol, an amphipath known to cause eversion of the membrane of red cells. In contrast, the presence of chlorpromazine, which promotes invagination of the intact red cell membrane, favored the formation of inside-out vesicles in ghosts. 3) The breakdown of ghosts into endocytic vesicles in dilute, alkaline media and the concomitant release and dissolution of the submembrane reticulum of spectrin and actin were retarded by excess cholesterol and promoted by its removal. This cholesterol effect was mimicked by exposing ghosts to chlorpromazine, but not dinitrophenol. Our data suggest that cholesterol may act physiologically both to stabilize the red cell membrane and to constrain its contour against invagination, and that red cell membrane cholesterol is maintained in vivo just below a critical level at which important organizational changes can occur.

Chlorpromazine↗
PubMed1979

The affinity of cholesterol for phosphatidylcholine and sphingomyelin.

Erythrocyte ghosts were incubated with sonicated vesicles and the uptake of cholesterol by vesicles allowed to proceed to equilibrium. The experiments were carried out for a series of phospholipids at different temperatures. The equilibrium partition of cholesterol between ghosts and single shelled vesicles provided a measure of the relative affinities of cholesterol for the different phospholipids studied. It was found that the affinity of cholesterol for dipalmitoyl phosphatidylcholine was the same as that for N-palmitoyl sphingomyelin both at temperatures above and below the gel to liquid crystalline transition temperature of these phospholipids.

Cholesterol↗
PubMed1978

Interaction of cholesterol, phospholipid and apoprotein in high density lipoprotein recombinants.

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of 3H cholesterol/14C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27 degrees C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1, or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. 160 X 55 A. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.

Apolipoproteins↗
PubMed1978

Transbilayer movement of cholesterol in phospholipid vesicles under equilibrium and non-equilibrium conditions.

1. The exchange of [3H] cholesterol between phospholipid: cholesterol vesicles and an excess of red cell ghosts is examined. 2. Using a number of different phophatidylcholines, only the cholesterol thought to be associated with the outer half of the bilayer (about 70 percent) is available for exchange, suggesting that at least at equilibrium the transbilayer movement of cholesterol or "flip-flop", occurs very slowly, if it occurs at all. 3. The rate of exchange of cholesterol between the vesicles and the ghosts is dependent on the nature of the fatty acid chain of the phospholipids, being a function of both the fatty acid chain length and the degree of unsaturation. 4. Under non-equilibrium conditions, when cholesterol is being both exchanged and depleted from the lipid vesicles to red cell ghosts, the previously non-exchangeable vesicle cholesterol becomes available for exchange, suggesting that under these conditions "flip-flop" can occur.

Biological Transport↗
PubMed1978

The exchangeability of human erythrocyte membrane cholesterol.

A new method has been used to determine what fraction of human erythrocyte cholesterol is available for exchange with plasma unesterified cholesterol. Erythrocytes labeled with 3H-cholesterol by this exchange process were incubated with sonicated phosphatidylcholine vesicles, giving rise to a net movement of cholesterol out of the cells. The specific activity of cholesterol taken up by the vesicles depended on the length of time of incubation. Initially the specific activity in the vesicles was greater than that in the cells, but after approximately 10% of cell cholesterol had been removed, the specific activity of subsequently removed cholesterol was equal to that of the remaining erythrocyte cholesterol. We conclude from these data that a) all of the cholesterol in the erythrocyte is exchangeable with plasma, and b) approximately 10% of erythrocyte cholesterol is in a more rapidly exchangeable pool than the remainder.

Cholesterol↗
PubMed1977

Characterization of mechanisms for transfer of cholesterol between human erythrocytes and plasma.

The removal from human erythrocytes of cholesterol (mass) and of [3H]cholesterol which had been introduced into the erythrocyte by exchange was studied. Removal was accomplished by incubating erythrocytes in plasma, the free cholesterol content of which had been lowered by the action of lecithin:cholesterol acyltransferase. It was shown that the exchange of cholesterol between erythrocytes and plasma and the net movement of cholesterol out of the membrane into plasma are characterized by the same rate constant and are driven by cholesterol to phospholipid ratios in cells and plasma. The apparent limitation on cholesterol depletion of erythrocytes observed in experiments of this type is explicable as the result of equilibrium between cholesterol in the membrane and in the plasma, an equilibrium reached when there is still cholesterol left in the cells. It is concluded from this study that all the exchangeable cholesterol in human erythrocytes is available for removal from the membrane.

Biological Transport↗
PubMed1977

Transmembrane movement of cholesterol in human erythrocytes.

We studied the exchange of cholesterol between radioactively labeled plasma and human erythrocytes. Results from experiments in which [3H]cholesterol and [14C]-cholesterol were exchanged sequentially into the cells and back out into unlabeled plasma, showed that transmembrane movement of cholesterol occurred with a half-time that was either less than 50 min or greater than 10 days. To obtain further information about the transmembrane movement of cholesterol, we used a technique [Jacobson, B. S. & Branton, D. (1977) Science 195, 302-304] for exposure of the cytoplasmic surface of erythrocyte membranes. This method involved the ionic attachment of erythrocytes to derivatized glass beads followed by disruption of the cells, leaving the beads covered by membrane with the cytoplasmic surface exposed [3H]Cholesterol was exchanged into intact erythrocytes which then were attached to beads. The beads with attached membrane were incubated with phospholipid-cholesterol vesicles and the exchange of cholesterol between the membrane cytoplasmic surface and vesicles was studied. We found that [3H]cholesterol was present at the cytoplasmic surface, indicating that transmembrane movement of cholesterol had occurred within the 2.5 hr required to complete the experiment. This result suggests that the more rapid rate of transmembrane cholesterol movement, inferred from the experiments described above, is the one that applies.

Biological Transport↗
PubMed1974

Ion diffusion selectivity in lecithin-water lamellar phases.

The diffusion coefficients of Na(+), Rb(+), and cl(-) were determined in lecithin-water lamellar phases at 18 degrees C as a function of phase hydration. Diffusion was measured within the phase with no transfer between phase and bulk aqueous medium. The relative diffusion coefficients of anion and cation depended strongly on phase hydration. At low water content, the diffusion coefficient of Cl(-) was greater than that of Na(+) or Rb(+) whereas at high water content both cations diffused faster than the anion. The change in relative diffusion coefficient occurred at 0.24 g water/g phase (24% water). The possibility that a change in conformation of the lecithin polar head occurs at a phase water content of 24% is considered. The diffusion coefficients of all three ions decreased at the water content where the relative diffusion rates inverted. Freeze fracture and polarizing microscopy studies were carried out to obtain information on phase structure. The latter study indicated that a change in long-range organization of the phase occured at 24% water. This change accounts for the decrease in the ion diffusion coefficients at this water content. The change in conformation of the choline phosphate group proposed as an explanation for the change in ion selectivity could lead to changes in long-range organization of the phase as a second order-effect.

Chlorides↗