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T L Steck

Publications and source records attributed to T L Steck.

At least 55 records · Page 3Linked to original sources
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

Mode of interaction of phosphofructokinase with the erythrocyte membrane.

Phosphofructokinase is known to associate with the human erythrocyte membrane both in vitro and in vivo. Such association activates the enzyme in vitro by relieving the allosteric inhibition imposed by ATP (Karadsheh, N.S., and Uyeda, K. (1977) J. Biol. Chem. 252, 7418-7420). We now demonstrate that ADP, ATP, and NADH, all of which are known to bind to the enzyme's adenine nucleotide activation site, are particularly potent in eluting the enzyme from the membrane. In addition, both inside-out red cell membrane vesicles and a 23-kDa fragment containing the amino terminus of the membrane protein, band 3, cause a slow, partial, and reversible inactivation of phosphofructokinase. The dependence of the residual phosphofructokinase activity on phosphofructokinase concentration demonstrates that inactivation occurs through the dissociation of active tetramers to inactive dimers. Dimers of phosphofructokinase associate with the membrane more avidly than tetramers. The kinetics of phosphofructokinase inactivation are consistent with the dissociation of tetramers in solution followed by the binding of dimers to the membrane. There is no indication of an association equilibrium between tetramers and dimers of phosphofructokinase bound to the membrane. Taken together, these results suggest that the amino-terminal segment of band 3 binds to the adenine nucleotide activation site, which is thought to be located in a cleft between the dimeric subunits of phosphofructokinase. As a result, band 3 not only rapidly activates the phosphofructokinase tetramer but also slowly inactivates the enzyme by preferentially binding its dissociated subunits.

Acetylcholinesterase↗
PubMed1984

The interaction of hemoglobin with the cytoplasmic domain of band 3 of the human erythrocyte membrane.

Previous studies point to the acidic amino-terminal segment of band 3, the anion transport protein of the red cell, as the common binding site for hemoglobin and several of the glycolytic enzymes to the erythrocyte membrane. We now report on the interaction of hemoglobin with the synthetic peptide AcM-E-E-L-Q-D-D-Y-E-D-E, corresponding to the first 11 residues of band 3, and with the entire 43,000-Da cytoplasmic domain of the protein. In the presence of increasing concentrations of the peptide, the oxygen binding curve for hemoglobin is shifted progressively to the right, indicating that the peptide binds preferentially to deoxyhemoglobin. The dissociation constant for the deoxyhemoglobin-peptide complex at pH 7.2 in the presence of 100 mM NaCl is 0.31 mM. X-ray crystallographic studies were carried out to determine the exact mode of binding of the peptide to deoxyhemoglobin. The difference electron density map of the deoxyhemoglobin-peptide complex at 5 A resolution showed that the binding site extends deep (approximately 18 A) into the central cavity between the beta chains, along the dyad symmetry axis, and includes Arg 104 beta 1 and Arg 104 beta 2 as well as most of the basic residues within the 2,3-diphosphoglycerate binding site. The peptide appears to have an extended conformation with only 5 to 7 of the 11 residues in contact with hemoglobin. In agreement with the crystallographic studies, binding of the peptide to deoxyhemoglobin was blocked by cross-linking the beta chains at the entrance to the central cavity. Oxygen equilibrium studies showed that the isolated cytoplasmic fragment of band 3 also binds preferentially to deoxyhemoglobin. The binding of the 43,000-Da fragment to hemoglobin was inhibited in the cross-linked derivative indicating that the acidic amino-terminal residues in the intact cytoplasmic domain also bind within the central cavity of the hemoglobin tetramer.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1984

Association of phosphofructokinase and aldolase with the membrane of the intact erythrocyte.

The binding of phosphofructokinase and aldolase to the membrane of the intact human erythrocyte was assessed by the rapid hemolysis/filtration method of Kliman and Steck (Kliman, H. J., and Steck, T. L. (1980) J. Biol. Chem. 255, 6314-6321). We found that about 50% of the phosphofructokinase was membrane-bound in fresh red cells prior to hemolysis. Binding was not significantly altered by deoxygenation. Approximately 40% of aldolase was membrane-associated in fresh red cells. In outdated, blood-banked red cells, aldolase was 73% membrane-bound while, following metabolic repletion, 40% of the enzyme was membrane-associated. These results support the hypothesis that certain glycolytic enzymes in the red cell are membrane-bound in a rapidly reversible and metabolically sensitive fashion.

Erythrocyte Membrane↗
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

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↗
PubMed1984

Ultrastructure of unit fragments of the skeleton of the human erythrocyte membrane.

We have examined fragments of the filamentous network underlying the human erythrocyte membrane by high-resolution electron microscopy. Networks were released from ghosts by extraction with Triton X-100, freed of extraneous proteins in 1.5 M NaCl, and collected by centrifugation onto a sucrose cushion. These preparations contained primarily protein bands 1 + 2 (spectrin), band 4.1 and band 5 (actin). The networks were partially disassembled by incubation at 37 degrees C in 2 mM NaPi (pH 7), which caused the preferential dissociation of spectrin tetramers to dimers. The fragments so generated were fractionated by gel filtration chromatography and visualized by negative staining with uranyl acetate on fenestrated carbon films. Unit complexes, which sedimented at approximately 40S, contained linear filaments approximately 7-8 nm diam from which several slender and convoluted filaments projected. The linear filaments had a mean length of 52 +/- 17 nm and a serrated profile reminiscent of F-actin. They could be decorated in an arrowhead pattern with S1 fragments of muscle heavy meromyosin which, incidentally, displaced the convoluted filaments. Furthermore, the linear filaments nucleated the polymerization of rabbit muscle G-actin, predominantly but not exclusively from the fast-growing ends. On this basis, we have identified the linear filaments as F-actin; we infer that the convoluted filaments are spectrin. Spectrin molecules were usually attached to actin filaments in clusters that showed a preference for the ends of the F-actin. We also observed free globules up to 15 nm diam, usually associated with three spectrin molecules, which also nucleated actin polymerization; these may be simple junctional complexes of spectrin, actin, and band 4.1. In larger ensembles, spectrin tetramers linked actin filaments and/or globules into irregular arrays. Intact networks were an elaboration of the basic pattern manifested by the fragments. Thus, we have provided ultrastructural evidence that the submembrane skeleton is organized, as widely inferred from less direct information, into short actin filaments linked by multiple tetramers of spectrin clustered at sites of association with band 4.1.

Cell Fractionation↗
PubMed1984

Maturation of the reticulocyte in vitro.

The maturation of reticulocytes into erythrocytes was demonstrated in vitro. Reticulocytosis was induced in rats by repeated bleeding or by phenylhydrazine injections. Whole blood samples were then incubated for 2 days at 37 degrees C. Reticulocytes in culture changed from polylobulated, monoconcave or triconcave forms to biconcave disks. During the first 12 h in vitro, the average reticulocyte count decreased from 39% to 12%, and the membrane-bound organelles, ribosomes and exocytic figures in the remaining reticulocytes were markedly diminished. In contrast, the number of red cells containing inclusions of denatured haemoglobin (Heinz bodies) in phenylhydrazine-treated blood did not decline. The reduction in reticulocyte count was not the result of differential cell destruction, since little haemolysis occurred in vitro. During red cell maturation three modes of organelle removal were observed particularly well when mitochondria were followed by cytochrome oxidase cytochemistry. First, some mitochondria degenerated, presumably through autolysis, by swelling, losing cristae and forming small single membrane-bound vesicles. Second, individual mitochondria became enclosed in vacuoles that fused with the plasma membrane and expelled their mitochondria by exocytosis. Third, autophagic vacuoles containing mitochondria, cytosol and membrane fragments fused with existing lysosomes. We conclude that all aspects of normal reticulocyte maturation occur in vitro, independent of the spleen, including the removal of organelles and the assumption of the mature biconcave disk shape.

Acid Phosphatase↗
PubMed1983

Amino acid sequence of the N alpha-terminal 201 residues of human erythrocyte membrane band 3.

We have determined the amino acid sequence of the N alpha-terminal portion of band 3, the anion transport protein of the human erythrocyte membrane. The material analyzed was a 201-residue, 23,053-Da fragment cleaved from the cytoplasmic end of band 3 by S-cyanylation. The sequence had these notable features. 1) The N alpha-terminal region was extraordinarily acidic, second only to a segment of similar size from the sigma factor of Escherichia coli RNA polymerase. The first 33 residues contained 6 aspartic acid and 12 glutamic acid residues, no basic residue, and a blocked N alpha-amino group. 2) The first 11 residues of the protein had a striking resemblance to the following 11 residues. 3) In contrast to the acidic N alpha-terminal third, the COOH-terminal two-thirds of the 23,053-Da fragment had a predominantly basic character. The highly acidic character of the N alpha-terminal portion of band 3 accounts for the capacity of this part of the protein to bind glycolytic enzymes in a highly electrostatic fashion, presumably through interaction with their cationic substrate-binding sites.

Amino Acid Sequence↗
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

Effect of red cell membrane binding on the catalytic activity of glyceraldehyde-3-phosphate dehydrogenase.

Band 3, the anion transport protein of the human erythrocyte, provides the site of association of certain glycolytic enzymes with the membrane. We have now demonstrated that glyceraldehyde-3-P dehydrogenase is inhibited, reversibly and completely, when membrane bound. The inhibition was competitive with respect to NAD+ and arsenate, but was noncompetitive with glyceraldehyde-3-P. Peptide fragments containing the NH2-terminal 23 residues of band 3 also inhibited the enzyme and displaced it from ghosts. Thus, the red cell membrane binding site for glyceraldehyde-3-P dehydrogenase is the same as that for aldolase, the polyanionic NH2-terminal region of the band 3 polypeptide.

Anion Exchange Protein 1, Erythrocyte↗
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↗
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↗
PubMed1981

The aldolase-binding site of the human erythrocyte membrane is at the NH2 terminus of band 3.

Band 3 is the predominant membrane-spanning polypeptide and the mediator of anion transport in the human erythrocyte. In addition, it provides the sites of association for fructose 1,6-bisphosphate aldolase and other cytoplasmic proteins with the membrane. The aldolase-binding activity of water-soluble fragments of band 3 was measured by their inhibition of aldolase catalytic activity and by their displacement of aldolase from ghosts. At saturation, the binding of one band 3 or certain of its fragments per aldolase molecule partially inhibited the catalytic activity and band 3 binding of the unliganded subunits of the tetramer through an apparently cooperative mechanism. An NH2-terminal 23,000-dalton fragment generated by S-cyanylation of the cytoplasmic pole of band 3 was approximately 20% as avid in binding aldolase as was native band 3. Several fragments cleaved from the NH2-terminal portion of the 23,000-dalton peptide by trypsin, mild acid hydrolysis, and cyanogen bromide digestion all bound aldolase, while fragments from the rest of the polypeptide were essentially inactive. The first 31 residues of band 3 contained 16 Asp plus Glu, no basic residues, and a blocked alpha-amino terminus. The highly acidic composition of this region is consistent with the strongly electrostatic character of the interaction between band 3 and aldolase, presumably at the strongly basic catalytic center of the enzyme. We conclude that the NH2-terminal region of band 3 bears the membrane-binding site for aldolase.

Amino Acids↗
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↗