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Y Lange

Publications and source records attributed to Y Lange.

At least 37 records · Page 2Linked to original sources
PubMed1990

Zymosterol is located in the plasma membrane of cultured human fibroblasts.

Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. 1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. 2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. 3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool.

Acetates↗
PubMed1989

Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts.

The literature suggests that cholesterol and sphingomyelin might be essentially confined to plasma membranes in mammalian cells; however, this premise has thus far escaped a direct test. We explored the issue in three ways. First, we fractionated whole homogenates of cultured human fibroblasts by equilibrium sucrose density gradient centrifugation. We found that the profiles of cholesterol and sphingomyelin were indistinguishable from those of two plasma membrane markers, 5' nucleotidase and [3H]galactose, which was conjugated to the surface of intact cells from an exogenous donor by galactosyltransferase. Second, we determined the relative surface areas of intact cells from their uptake of 1-(4-trimethyl-amino)phenyl-6-phenylhexa-1,3,5-triene, a cationic fluorescent dye which partitions into but does not cross plasma membranes. Relative to human red cell ghosts, the apparent surface area of the fibroblasts was 17,500 microns2/cell while for canine hepatocytes, the value was 11,500 microns2/cell. The relative ratios of cell cholesterol to dye binding (hence, surface area) were quite similar in ghosts, fibroblasts, and liver cells; namely 1.0, 1.12, and 0.67, respectively. Finally, we found that the specific ratios of both cholesterol and sphingomyelin to 5' nucleotidase were only 10% less in gradient-purified plasma membranes than in whole homogenates. Similar results were obtained using an entirely different method of purification: two-phase aqueous partition. The cholesterol and sphingomyelin in fractions rich in other membranes was closely proportional to their 5' nucleotidase content, suggesting that the presence of these lipids reflected contamination by plasma membrane fragments. The 5' nucleotidase/phospholipid ratio in the purified plasma membrane fraction was roughly twice that in whole cells. We conclude that the compartment marked by 5' nucleotidase in cultured human fibroblasts contains approximately 90% of the two named lipids and half the cell phospholipid phosphorus.

Cell Compartmentation↗
PubMed1988

An activation-collision mechanism for cholesterol transfer between membranes.

We report the results of experiments which show that cholesterol transfer between membranes cannot proceed by aqueous diffusion, as widely held, but must involve a more complex mechanism. (a) The rate of transfer of [3H]cholesterol from red blood cells was found to vary inversely with the size of the acceptor particle (ghosts, vesicles of ghosts, liposomes, and plasma lipoproteins). (b) The transfer of [3H]cholesterol from red blood cells to ghosts was accelerated by the presence of plasma, even though the plasma competed with the ghosts as an acceptor. (c) The rate of transfer of [3H]cholesterol from red blood cells to ghosts decreased to zero with increasing dilution but was not simply second-order. (d) The cholesterol in retinal rod disc membranes is not at equilibrium with plasma lipoproteins in that disc cholesterol increased when the homogenates were incubated in vitro with plasma. (e) The kinetics of cholesterol transfer cannot be limited by unstirred layer effects since the transfer of lysolecithin in the same system was faster than that of cholesterol by 3 orders of magnitude. The simplest model compatible with all the data suggests a two-step pathway involving a first-order followed by a second-order process. The first step could be a unimolecular activation event, perhaps the movement of the sterol in the donor particle to a more exposed (hydrated) position. In the second step, the activated sterol would be transferred during transient collisions between donor and acceptor particles. When collision is not rate-limiting, the overall process would appear to be simply first-order, hence kinetically indistinguishable from the aqueous diffusion mechanism. The activation-collision model thus not only rationalizes our data but is also consistent with the simpler kinetics previously reported for the transfer of both membrane phospholipids and sterols.

Algorithms↗
PubMed1988

Topographic heterogeneity in cholesterol biosynthesis.

We have examined the membrane topography of cholesterol biosynthesis in cultured human fibroblasts. We fed the cells with radioacetate and then interrupted the biosynthetic pathway so as to trap labeled intermediates in their subcellular locations. We analyzed homogenates of human fibroblasts labeled biosynthetically from radioacetate by centrifugation to equilibrium on sucrose gradients. The following two methods were used to interrupt cholesterol biosynthesis: incubation at 10 degrees C and treatment with 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, a specific inhibitor of oxidosqualene cyclase. Incubation at 10 degrees C caused the accumulation of radiolanosterol at the expense of cholesterol. The lanosterol appeared predominantly at an unusually buoyant density (20% (w/w) sucrose; d = 1.08 g/cm3) as well as at the density normally labeled at 37 degrees C (30% sucrose; d = 1.13 g/cm3). 4,4,10 beta-Trimethyl-trans-decal-3 beta-ol treatment caused the accumulation of labeled squalene and squalene 2,3-oxide. Reversal of the block permitted the label to progress rapidly as a wave into lanosterol and ultimately into cholesterol. The profiles of the three precursors did not coincide, suggesting that they were mostly in different membranes. Squalene was uniquely confined to a density of 1.18 g/cm3 (40% sucrose) while squalene 2,3-oxide appeared in peaks of density 1.08 g/cm3 and 1.13 g/cm3 (20% and 30% sucrose). Lanosterol was in a peak of density 1.13 g/cm3. Pulse-chase experiments showed that lanosterol synthesized in the membranes at 20% sucrose moved rapidly to the membranes at 30% sucrose where it was converted to cholesterol. The density gradient profiles of the following organelle markers also were monitored: plasma membrane, cholesterol mass; Golgi apparatus, galactosyltransferase; endoplasmic reticulum, RNA, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cytochrome c reductase; peroxisomes, catalase. None of these markers appeared at the buoyant density of 1.08 g/cm3. We conclude that 1) cholesterol biosynthesis may be topographically heterogeneous and 2) newly synthesized squalene 2,3-oxide resides in a buoyant membrane fraction distinct from markers for the major organelles.

Cells, Cultured↗
PubMed1988

Plasma membrane of cultured oligodendrocytes: II. Possible structural and functional domains.

An oligodendrocyte plasma membrane-rich fraction, F2.2, was resolved by equilibrium density centrifugation on a linear sucrose gradient from 0.5 M to 1.3 M into three fractions, F2.2a, F.2.2b, F2.2c, and a pellet F2.2p. F2.2a and F.2.2b were enriched 1.5-fold relative to F2.2 in plasma membrane markers at the expense of F2.2c and F2.2p, which became correspondingly impoverished. This gave F2.2a and F2.2b a 42-fold and 37-fold enrichment, respectively, in plasma membrane markers relative to the initial cell homogenate. F2.2c had a sevenfold enrichment in a Golgi marker; together with F2.2p, they contained all the Golgi marker initially present in F2.2. Preliminary data indicated that the F2.2-subfractions differed from one another in their molar ratios of cholesterol to phospholipids and protein to lipids but had similar protein profiles when examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Their content of fucosylated glycoproteins appeared also to be different. Morphologically, F2.2a and F2.2b were very similar: they contained large membrane vesicles, membrane sheets, and vesicles entrapped within other vesicles. Membrane-membrane interaction was apparent in these fractions. F2.2c had many of the same elements, but most of the membrane structures contained amorphous material. F2.2p differed morphologically from the other fractions in that it had principally electron-dense structures. It is postulated that F2.2a, F2.2b, and perhaps F2.2c represent different domains of oligodendrocyte plasma membrane. Alternatively, these fractions might correspond to the plasma membrane of oligodendrocyte subtypes.

Animals↗
PubMed1988

Localization and movement of newly synthesized cholesterol in rat ovarian granulosa cells.

The distribution and movement of cholesterol were studied in granulosa cells from the ovaries of estrogen-stimulated hypophysectomized immature rats cultured in serum-free medium. Plasma membrane cholesterol was distinguished from intracellular cholesterol with cholesterol oxidase, an enzyme that converts cell surface cholesterol to cholestenone, leaving intracellular cholesterol untouched. Using this approach we showed that 82% of unesterified cholesterol was associated with the plasma membrane in granulosa cells cultured for 48 h in serum-free medium in both the presence and absence of added androstenedione and FSH. FSH and androstenedione stimulated a marked increase in steroid hormone (progestin) production. The movement of newly synthesized cholesterol to the plasma membrane also was followed using cholesterol oxidase. Newly synthesized cholesterol reached the plasma membrane too rapidly to be measured in unstimulated cells (t1/2 less than 20 min); however, in cells stimulated by FSH and androstenedione, this rate was considerably slower (t1/2 approximately 2h). Therefore, cholesterol movement to the plasma membrane appears to be regulated by gonadotropins in these cells. We tested whether steroid biosynthesis used all cell cholesterol pools equally. To this end we administered [3H]acetate and [14C]acetate at different times and determined their relative specific contents in various steroids after defined intervals. The relative ages of the steroids (youngest to oldest) were: lanosterol, progestins, intracellular cholesterol, and plasma membrane cholesterol. This finding suggests that progestins use newly synthesized intracellular cholesterol in preference to preexisting intracellular or cell surface cholesterol. A measure of this effect is that the specific activity of secreted hormone was 15- to 30-fold greater than that of intracellular cholesterol. We conclude that the various cholesterol compartments in granulosa cells are discrete. While the major fraction of cholesterol in these steroidogenic cells resides in the plasma membrane, it is not in rapid equilibrium with intracellular cholesterol. Furthermore, steroidogenesis appears to use newly synthesized over preexisting cholesterol, suggesting a shunt pathway.

Acetates↗
PubMed1987

Cholesterol is not synthesized in membranes bearing 3-hydroxy-3-methylglutaryl coenzyme A reductase.

We have shown previously that newly synthesized lanosterol and cholesterol in homogenates of cultured human fibroblasts do not have the same equilibrium buoyant density as the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) in the smooth endoplasmic reticulum (SER) (Lange, Y., and Steck, T. L. (1985) J. Biol. Chem. 260, 15592-15597). This finding suggested two alternative and novel hypotheses: (a) that lanosterol and cholesterol might be transported rapidly from the SER to other internal membranes or (b) that synthesis of the sterols is not associated with the SER, or at least not with that portion of this organelle bearing HMG-CoA reductase. We therefore compared the subcellular distribution of HMG-CoA reductase with that of enzymes which convert lanosterol to cholesterol. The two activities studied were the consumption of exogenous [3H]lanosterol and the conversion of exogenous radiolanosterol to radiocholesterol. Differential centrifugation, rate zonal centrifugation, and equilibrium sucrose gradient centrifugation of rat liver homogenates all showed that these enzyme activities did not comigrate with HMG-CoA reductase. The subcellular distribution of newly synthesized sterols also was examined in cultured human fibroblasts. Cells were incubated with radioactive acetate to label endogenous sterols biosynthetically, homogenized, and spun to equilibrium on sucrose gradients. The buoyant density profiles of radioactive cholesterol and lanosterol both had a peak at 1.12 g/cm3. Digitonin treatment shifted both sterols to higher densities, strong evidence that they resided in cholesterol-rich membranes. Pretreatment of intact cells with cholesterol oxidase, which selectively oxidizes plasma membrane cholesterol, abolished the digitonin shift of lanosterol but not of intracellular cholesterol. These findings provide support for the hypothesis that newly synthesized cholesterol and lanosterol are not in the same membrane.

Animals↗
PubMed1985

Cholesterol-rich intracellular membranes: a precursor to the plasma membrane.

The disposition of newly synthesized sterols in cultured human fibroblasts has been examined in this study. We began by demonstrating that cholesterol mass and exogenously added [3H]cholesterol both are markers for the plasma membrane, perhaps better than 5'-nucleotidase. Cells were incubated with radioactive acetate to label their endogenous sterols biosynthetically, treated with cholesterol oxidase to convert plasma membrane cholesterol to cholestenone, and then homogenized and spun to equilibrium on sucrose gradients. The density gradient profiles of the various organelles were monitored using these markers: plasma membrane, radioactive cholestenone; smooth endoplasmic reticulum, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase); and Golgi apparatus, galactosyltransferase. The buoyant density profiles of radioactive intracellular cholesterol and lanosterol both had a peak at 1.12 g/cm3, similar to 5'-nucleotidase and galactosyltransferase but not to HMG-CoA reductase. This result suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. Digitonin treatment shifted the profiles of both plasma membrane and intracellular cholesterol to higher densities. Pretreatment of intact cells with cholesterol oxidase abolished the digitonin shift of plasma membranes but not the intracellular cholesterol, indicating that these two membrane pools are not entirely physically associated. Because intracellular cholesterol was shifted more than any of the organelle markers, it must reside in a separate membrane. Since digitonin selectively shifts the density of membranes rich in cholesterol, we infer that newly synthesized cholesterol accumulates in such membranes prior to its delivery to the plasma membrane. Taken together, these results suggest that cholesterol may be concentrated for delivery to the plasma membrane by being synthesized from a sterol precursor such as lanosterol in a discrete but undefined intracellular membrane.

5'-Nucleotidase↗
PubMed1985

Kinetics of proton exchange of phosphatidylethanolamine in phospholipid vesicles.

The rate of proton exchange of the amino protons of phosphatidylethanolamine (PE) in sonicated mixed phospholipid vesicles has been determined by NMR spectroscopy. The rate of exchange increases with increasing pH and phosphate concentration. In the absence of buffer the dominant exchange process is an intrasurface reaction in which NH2 groups react via water with NH3+ groups on the outer surface. Addition of cholesterol reduces the rate constant for intrasurface exchange. The experiments are evidence that such reactions could be dominant in proton transport in and to membrane surfaces.

Hydrogen-Ion Concentration↗
PubMed1984

Transfer of cholesterol from its site of synthesis to the plasma membrane.

We have followed the transfer of newly synthesized cholesterol to the plasma membrane in cultured fibroblasts using cholesterol oxidase as a probe. Since the enzyme has access only to the plasma membrane in intact cells, it permits the discrimination of cell surface and endogenous cholesterol. Cholesterol synthesized from radiolabeled acetate was transferred to the plasma membrane in a strictly first order fashion with a half-time of 1-2 h at 37 degrees C. The rate of transfer was similar in rapidly growing and confluent cells and was not affected by preincubating the cells in lipoprotein-deficient serum which greatly stimulated cholesterol synthesis. We used equilibrium density gradient centrifugation of homogenates from cholesterol oxidase-treated cells to examine further the distribution of newly synthesized cholesterol between cellular pools. We identified membrane fractions enriched in newly synthesized cholesterol yet inaccessible to cholesterol oxidase. The cholesterol in these membranes eventually moved to the plasma membrane. The movement of exogenous radiocholesterol from the plasma membrane to the cell interior also was examined by this method. No detectable transfer was observed over several hours, during which time endogenous cholesterol moved to the plasma membrane. We conclude that the transfer of newly synthesized cholesterol to the plasma membrane is a vectorial process and is not mediated by a simple diffusional equilibrium.

Acetates↗
PubMed1984

Interaction of chlorpromazine with the human erythrocyte membrane.

The interaction of the amphipath chlorpromazine (CPZ) with the human erythrocyte membrane was evaluated. The partition coefficient of CPZ between the membrane bilayer and the aqueous compartment, measured spectrophotometrically, ranged between 1 and 3 X 10(3). An independent estimate, 4.6 X 10(3), was obtained by a novel method which avoided the measurement of binding and determined instead the variation of the hemolytic potency of the amphipath with the ratio of buffer volume to membrane volume. The maximal uptake of CPZ exceeded 2 X 10(9) molecules/red cell, corresponding to a volume greater than that of the bilayer itself. Such heavily loaded membranes were increased in thickness more than 2-fold, suggesting the formation of a CPZ-rich zone at the center of the bilayer. Ghosts loaded with massive levels of CPZ condensed approximately 20-fold in surface area and increased proportionately in thickness, suggesting the formation of a novel CPZ-lipid solution. CPZ caused hemolysis by a colloid-osmotic mechanism. By measuring the simultaneous uptake of mannitol and sucrose, we determined that CPZ induced holes of constant size but variable number. If circular, the holes would have had a diameter of approximately 14 A. The time-averaged number of holes ranged from 0.09 per cell (signifying intermittency) to 16. Freeze-fracture electron microscopy of CPZ-treated red cells revealed multiple round patches of nearly particle-free bilayer up to 0.3 micron in diameter with crowding of the intramembrane particles into the surrounding membrane. We interpret these images to signify lateral phase separation within the CPZ-treated bilayer. Hemolysis could, therefore, result from the intermittent opening of weak seams at phase boundaries; these could then be fluctuating slits approximately 14 A in width and of variable length, rather than simple circular holes.

Biological Transport↗
PubMed1984

Acyl chain organization and protein secondary structure in cholesterol-modified erythrocyte membranes.

Fourier transform infrared and Raman spectroscopies are used to study the effects of cholesterol on human erythrocyte membrane acyl chain organization (mobility and conformation) and protein secondary structure. Compared to normal red cell membranes (approximately equal to 0.8 mol of cholesterol/mol of phospholipid) (C/P), acyl chain mobility is greater for the depleted (C/P approximately equal to 0.6) and less for the enriched (C/P approximately equal to 1.2) membranes as monitored by shifts of the IR symmetric methylene C-H stretching band (2852 wave numbers, cm-1) over the temperature range 5 to 40 degrees C. There is a continuous first order trend to the IR shifts, but no evidence of a phase change for any of the three cholesterol contents. Raman scattering of C-C stretching vibrations (1065-1130 cm-1) revealed that acyl chain conformation in the three membrane preparations is in a similar state of high disorder; however, compared to depleted and control membranes, the enriched membrane acyl chains display higher order lattice packing. The alpha-helical content of membrane proteins is correlated with the relative intensity of the Raman peptide backbone C-C stretching band (940 cm-1). Spectra of cholesterol-enriched erythrocyte membranes indicate a substantial increase in protein helical structure compared to those of the cholesterol-depleted membranes.

Acylation↗
PubMed1984

Cholesterol oxidase susceptibility of the red cell membrane.

We have used the highly variable and conditional susceptibility of cholesterol oxidase to probe molecular rearrangements in the human red cell membrane. Cholesterol in the intact erythrocyte normally is not a substrate for this enzyme. Susceptibility was induced however, by these pretreatments: mild enrichment in membrane cholesterol, exposure to greater than or equal to 0.03% (3 mM) glutaraldehyde and warming in dilute salt solutions (mu approx. 0.001). Cholesterol reactivity in dilute salt solutions emerged only following a lag of 30 min or more. The lag time was shortened by raising the temperature, by reducing the salt concentration or by treating with glutaraldehyde. The induced sensitivity to the enzyme was inhibited by restoring physiologic ionic strength or by introducing 0.1 mol lysophosphatidylcholine per mol cholesterol into the membrane. (In striking contrast, lysophosphatidylethanolamine and lysophosphatidylserine did not inhibit oxidation). The various effectors of cholesterol oxidase sensitivity strongly influenced the impact of the others, suggesting that each shifted cholesterol toward or away from an enzyme-sensitive disposition. None of these effects was observed in pure cholesterol or red cell membrane lipids dissolved in detergent, which were uniformly highly reactive with the enzyme. We conclude that the observed variation in cholesterol oxidase sensitivity reflects changes in the organization of the bilayer, perhaps a lateral redistribution of lipids which creates cholesterol-rich phases or domains in which cholesterol is more or less accessible to the enzyme. If so, the time-dependent increase in cholesterol susceptibility during warming at low ionic strength might be a novel indicator of the kinetics of phase changes in the bilayer of the red cell.

3-Hydroxysteroid Dehydrogenases↗
PubMed1984

Mechanism of red blood cell acanthocytosis and echinocytosis in vivo.

Patients with abetalipoproteinemia have an inborn absence of the major apoprotein of low density plasma lipoproteins, an abnormal serum and red cell lipid profile, and spiny erythrocytes, called acanthocytes. We now show that these deformed cells are reversibly converted to a normal shape, that of a biconcave disk, by incubation with 3 to 10 X 10(-5) M chlorpromazine. We suppose that chlorpromazine acts by expanding the cytoplasmic leaflet of the bilayer, thus promoting inward curvature. Ghosts isolated from the acanthocytes are themselves spiny but are also converted to normal, concave disks by chlorpromazine or merely by a brief incubation at 37 degrees C in low ionic strength buffer. We attribute the latter to a redistribution of lipids between the two leaflets of the membrane bilayer. Similar observations were made with red cells and ghosts from a patient with benign echinocytosis. These observations suggest that the morphological abnormality in acanthocytes and echinocytes can be ascribed to the same mechanism as crenation in vitro; that is, a bilayer couple effect in which an excess of surface area in the outer leaflet over the inner leaflet of the membrane bilayer drives outward curvature. It is striking that cells which were chronically abnormal in shape in vivo contain the information to become biconcave disks immediately upon simple chemical treatment in vitro.

Abetalipoproteinemia↗
PubMed1983

Analysis of the distribution of cholesterol in the intact cell.

We have used the enzyme cholesterol oxidase, which catalyzes the oxidation of cholesterol to cholest-4-en-3-one, to examine the distribution of cholesterol in cultured fibroblasts, Chinese hamster ovary cells, and isolated rat liver hepatocytes. While the plasma membrane normally was not attacked by cholesterol oxidase, we found that treating cells with low ionic strength buffer and glutaraldehyde rendered their cholesterol highly susceptible to oxidation. Most of the cholesterol was oxidized in all three cell types: 94% in fibroblasts, 92% in Chinese hamster ovary cells, and 80% in hepatocytes. Given that the enzyme had access only to the outer surface of the cells and cholesterol can move rapidly across the fixed plasma membrane, these values are taken to reflect the fraction of cellular cholesterol present in the plasma membrane. Additional experiments confirmed this interpretation. Fibroblasts were labeled with [3H]cholesterol by brief exposure to exogenous radiolabel and incubated with [14C]mevalonic acid to label cholesterol biosynthetically. Cholesterol oxidase attacked at least 97% of the exogenous label but as little as 10% of the biosynthetically labeled cholesterol. These data suggest that the cholesterol oxidase did not reach the intracellular pool and that cholesterol in the plasma membrane is not in rapid equilibrium with internal membranes. A study of the transfer of cholesterol to plasma from cells labeled biosynthetically with [3H]cholesterol and exogenously with [14C]cholesterol confirmed the different subcellular distribution of the two labels. These studies demonstrate that an unexpectedly high proportion of cell cholesterol is associated with plasma membranes and that this cholesterol pool can be rapidly and selectively labeled and oxidized. These features make cholesterol a useful specific marker for the plasma membrane.

Animals↗
PubMed1983

On the mechanism of transfer of cholesterol between human erythrocytes and plasma.

The kinetics of transfer of [3H]cholesterol between human erythrocytes and plasma at 37 degrees C in physiological buffer had these features. 1) Cholesterol transfer was strikingly similar in both directions. 2) Transfer progressed to isotopic equilibrium in a monotonic, apparently first order fashion, except for a minor rapid component (approximately 15%) observed in the transfer of cholesterol from cells to plasma. 3) The mechanism of transfer was not via transient collisions in that the rate of the reaction was quite insensitive to the concentration of reactants over a wide range. 4) The mechanism of transfer did not involve specific, stable complex formation in that there was little difference in the behavior of erythrocytes and inside-out plasma membrane vesicles derived therefrom or between plasma and sonicated liposomes as acceptors. Furthermore, transfer was not affected by vigorous proteolysis of either the cells or the plasma. 5) The kinetics of transfer were fully compatible with diffusion of cholesterol through the aqueous compartment. This was shown by fitting our data to a rigorous model for diffusion equilibrium between three compartments. 6) The partition coefficient of [3H]cholesterol between red cells and buffer was shown to be 10(7). 7) The rate constants for cholesterol release from both red cells and plasma were approximately 1 X 10(-4) s-1 (t 1/2 approximately 2 h). The rate constant for cholesterol uptake into red cells was approximately 1 X 10(3) s-1 (t 1/2 approximately 1 ms). 8) The similarity of the corresponding kinetic constants among red cells, plasma, and liposomes suggests that phospholipids in a variety of physical forms are equivalent solvents for cholesterol. We conclude that despite its extremely low solubility in water, cholesterol moves between lipid compartments by aqueous diffusion.

Biological Transport↗
PubMed1982

Role of the bilayer in the shape of the isolated erythrocyte membrane.

The determinants of cell shape were explored in a study of the crenation (spiculation) of the isolated erythrocyte membrane. Standard ghosts prepared in 5 mM NaPi (pH 8) were plump, dimpled disks even when prepared from echinocytic (spiculated) red cells. These ghosts became crenated in the presence of isotonic saline, millimolar levels of divalent cations, 1 mM 2,4-dinitrophenol or 0.1 mM lysolecithin. Crenation was suppressed in ghosts generated under conditions of minimal osmotic stress, in ghosts from red cells partially depleted of cholesterol, and, paradoxically, in ghosts from red cells crenated by lysolecithin. The susceptibility of ghosts to crenation was lost with time; this process was potentiated by elevated temperature, low ionic strength, and traces of detergents or chlorpromazine. In that ghost shape was influenced by a variety of amphipaths, our results favor the premise that the bilayer and not the subjacent protein reticulum drives ghost crenation. The data also suggest that vigorous osmotic hemolysis induces a redistribution of lipids between the two leaflets of the bilayer which affects membrane contour through a bilayer couple mechanism. Subsequent relaxation of that metastable distribution could account for the observed loss of crenatability.

Chlorpromazine↗