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R M Johnstone

Publications and source records attributed to R M Johnstone.

At least 55 records · Page 3Linked to original sources
PubMed1986

Asymmetric reconstitution of the glucose transporter from Ehrlich ascites cell plasma membrane: role of alkali cations.

Gel chromatography of solubilized Ehrlich cell plasma membranes and preformed asolectin vesicles coupled to a freeze-thaw cycle results in the reconstitution of 3-O-methyl-D-glucose transport. The transport activity of the liposomes formed is critically dependent on the cation present during reconstitution. Liposomes formed in K+ show high levels of carrier-mediated 3-O-methyl-D-glucose uptake (495 pmol/min/mg protein) while those formed in Na+ do not (33 pmol/min/mg protein). The inactivity in Na+ is not due to a diminished incorporation of glucose transporter nor is it due to carrier molecules reconstituted with a different orientation from those in K+ liposomes. Instead, the low glucose transport level in Na+ liposomes is related to the small size of vesicles formed with Na+. A second freeze-thaw cycle in K+ causes a two- to threefold increase in the available intravesicular volume of Na+ liposomes and results in an eightfold increase in carrier-mediated 3-O-methyl-D-glucose uptake. K+ liposomes, treated in an identical manner, show only a twofold increase in uptake. The glucose transporter was identified as a protein with a molecular mass range of 44.7 to 66.8 kDa, by the D-glucose-inhibitable photoincorporation of [3H]cytochalasin B. The carrier protein is inserted in reconstituted vesicles in a nonrandom manner with at least 80% of the molecules oriented with the cytoplasmic domain accessible to the external medium. In contrast, the neutral Na+-dependent amino acid transport system appears to be randomly reconstituted.

3-O-Methylglucose↗
PubMed1985

Effect of alkali cations on freeze-thaw-dependent reconstitution of amino acid transport from Ehrlich ascites cell plasma membrane.

Na+-dependent amino acid transport can be reconstituted by gel filtration of disaggregated plasma membrane and asolectin vesicles coupled to a freeze-thaw cycle. The resultant transport activity is markedly affected by the nature of the reconstitution medium. Reconstitution in K+ permits the formation of active liposomes, whereas reconstitution in Na+, Li+, or choline does not. Electron micrographs of K+ liposomes show a wide variation in liposome sizes. Ficoll density gradient fractionation of K+ liposomes shows that the largest vesicles are lipid rich, have the lowest density, and have the highest level of Na+-dependent amino acid transport. Liposomes formed in Na+ have a 34% smaller trapped volume than K+ liposomes and lack a population of large vesicles. A second freeze-thaw in K+ restores activity to Na+ liposomes which now contain large low density active vesicles. Fluorescence measurements of freeze-thaw-induced mixing of vesicle lipids indicates that the absence of large vesicles in Na+ liposomes is due to inhibition by Na+ of lipid vesicle fusion events during freezing and thawing. The large vesicle fraction is enriched in a 125-kDa peptide. It has not yet been established whether this peptide is part of the transport system for neutral amino acids.

Amino Acids↗
PubMed1985

Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes.

Using ferritin-labeled protein A and colloidal gold-labeled anti-rabbit IgG, the fate of the sheep transferrin receptor has been followed microscopically during reticulocyte maturation in vitro. After a few minutes of incubation at 37 degrees C, the receptor is found on the cell surface or in simple vesicles of 100-200 nm, in which the receptor appears to line the limiting membrane of the vesicles. With time (60 min or longer), large multivesicular elements (MVEs) appear whose diameter may reach 1-1.5 micron. Inside these large MVEs are round bodies of approximately 50-nm diam that bear the receptor at their external surfaces. The limiting membrane of the large MVEs is relatively free from receptor. When the large MVEs fuse with the plasma membrane, their contents, the 50-nm bodies, are released into the medium. The 50-nm bodies appear to arise by budding from the limiting membrane of the intracellular vesicles. Removal of surface receptor with pronase does not prevent exocytosis of internalized receptor. It is proposed that the exocytosis of the approximately 50-nm bodies represents the mechanism by which the transferrin receptor is shed during reticulocyte maturation.

Animals↗
PubMed1984

In vitro acylation of the transferrin receptor.

In vitro fatty acylation of the transferrin receptor with [3H]tetradecanoate or [3H]tetradecanoyl-CoA has been demonstrated for isolated sheep reticulocyte plasma membranes. Although less than 5% of the receptor was labeled in vitro, the acylated protein could be readily observed after sodium dodecyl sulfate-gel electrophoresis. The acylated transferrin receptor in the reticulocyte membrane was specifically precipitated with a monoclonal antibody and was absent from mature red cell membranes. Incorporation of fatty acid was dependent on ATP, and fatty acid was 5-10 times less effective as an acyl donor than the acyl-CoA derivative, pointing out the strong potential of this reagent for in vitro acylation of membrane proteins. During in vitro maturation of reticulocytes, the receptor is released in vesicles into the incubation medium. Using reticulocytes labeled with [3H]tetradecanoate, it can be shown that the 3H-labeled receptor is transferred from the cells to the vesicles without loss of acyl groups, suggesting that the vesiculation process does not involve deacylation.

Acyl Coenzyme A↗
PubMed1984

A simple and efficient method for reconstitution of amino acid and glucose transport systems from Ehrlich ascites cells.

Solubilized Ehrlich cell plasma membrane proteins were incorporated into lipid vesicles in the presence of added phospholipid, using Sephadex G-50 chromatography combined with a freeze-thaw step. Liposomes formed in K+ exhibited high levels of Na+-dependent, alpha-aminoisobutyric acid uptake which was electrogenic and inhibited by other amino acids. The transport activity reconstituted was similar to that observed in native plasma membrane vesicles. In addition to transport by system A, leucine exchange activity (system L), Na+-dependent serine exchange activity (system ASC), and stereospecific glucose transport activity were also reconstituted. The latter was inhibited by D-glucose, D-galactose, cytochalasin B, and mercuric chloride. The medium used for reconstitution was critical for the recovery of Na+-dependent amino acid transport. The use of Na+ in the reconstitution procedure led to formation of liposomes which displayed little Na+-dependent and gradient-stimulated amino acid uptake. In contrast, all transport activities studied were efficiently reconstituted in K+ medium.

3-O-Methylglucose↗
PubMed1984

Density gradient localization of plasma membrane and tonoplast from storage tissue of growing and dormant red beet : characterization of proton-transport and ATPase in tonoplast vesicles.

Membranes from homogenates of growing and of dormant storage roots of red beet (Beta vulgaris L.) were centrifuged on linear sucrose gradients. Vanadate-sensitive ATPase activity, a marker for plasma membrane, peaked at 38% to 40% sucrose (1.165-1.175 grams per cubic centimeter) in the case of growing material but moved to as low as 30% sucrose (1.127 grams per cubic centimeter) during dormancy.A band of nitrate-sensitive ATPase was found at sucrose concentrations of 25% to 28% or less (around 1.10 grams per cubic centimeter) for both growing and dormant material. This band showed proton transport into membrane vesicles, as measured by the quenching of fluorescence of acridine orange in the presence of ATP and Mg(2+). The vesicles were collected on a 10/23% sucrose step gradient. The phosphate hydrolyzing activity was Mg dependent, relatively substrate specific for ATP (ATP > GTP > UTP > CTP = 0) and increased up to 4-fold by ionophores. The ATPase activity showed a high but variable pH optimum, was stimulated by Cl(-), but was unaffected by monovalent cations. It was inhibited about 50% by 10 nanomolar mersalyl, 20 micromolar N,N'-dicyclohexylcarbodiimide, 80 micromolar diethylstilbestrol, or 20 millimolar NO(3) (-); but was insensitive to molybdate, vanadate, oligomycin, and azide. Proton transport into vesicles from the 10/23% sucrose interface was stimulated by Cl(-), inhibited by NO(3) (-), and showed a high pH optimum and a substrate specificity similar to the ATPase, including some proton transport driven by GTP and UTP.The low density of the vesicles (1.10 grams per cubic centimeter) plus the properties of H(+) transport and ATPase activity are similar to the reported properties of intact vacuoles of red beet and other materials. We conclude that the low density, H(+)-pumping ATPase of red beets originated from the tonoplast. Tonoplast H(+)-ATPases with similar properties appear to be widely distributed in higher plants and fungi.

Journal Article↗
PubMed1984

Phosphorylation of the transferrin receptor in isolated sheep reticulocyte plasma membranes.

The transferrin receptor of sheep reticulocyte plasma membranes undergoes phosphorylation with [gamma-32P]ATP in isolated plasma membranes. The phosphorylation is stimulated by Mn2+, Co2+, and Mg2+, but not by Ca2+, Ba2+, Zn2+, Fe2+, or Cu2+. There is no detectable effect of cyclic nucleotides on the phosphorylation of the receptor. Transferrin and a monoclonal antibody against the transferrin receptor have no apparent effect on receptor phosphorylation in intact cells or isolated membranes. Immunoprecipitates of the receptor retain ability to phosphorylate the receptor. The phosphorylation appears to be at a serine residue which turns over with a half time of 20-30 min. ATP appears to be the best, but not the only substrate for receptor phosphorylation.

Amino Acids↗
PubMed1984

The fate of the transferrin receptor during maturation of sheep reticulocytes in vitro.

The transferrin receptor of sheep reticulocytes is excised from the cell during in vitro maturation to erythrocytes. The excised receptor may be recovered from the medium by centrifugation at 100 000 X g. Loss of transferrin-binding activity parallels the loss of binding of anti-receptor antibody, as well as RNA content. The released receptor retains the molecular size of the receptor isolated from the plasma membranes (93 000 monomer, 186 000 dimer), has an identical iodotyrosyl peptide map, and is still capable of binding transferrin, as well as an antibody directed against the receptor. The receptor is released in a vesicular form. The major peptides of the vesicles are the receptor and an unidentified peptide of 70 000 whose iodotyrosyl peptide map is distinct from that of the receptor. Although the transferrin receptor has been shown to undergo posttranslational modification (phosphorylation, acylation, and glycosylation) in cultured cells, it has not been established whether any of the transformations are retained in nongrowing cells. In the present communication, it is shown that isolated reticulocyte plasma membranes are capable of receptor phosphorylation, a process previously shown only with intact, cultured cells. The phosphorylating activity is retained in immunoprecipitates of the receptor, but is absent in the vesicles released during maturation. No evidence has been obtained for an effect of either transferrin or an anti-receptor antibody on receptor phosphorylation in intact cells or isolated membranes.

Animals↗
PubMed1983

Loss of the transferrin receptor during the maturation of sheep reticulocytes in vitro. An immunological approach.

Sheep reticulocyte-specific antiserum absorbed with mature sheep red cells has been used to isolate and identify reticulocyte-specific plasma-membrane proteins and to monitor their loss during incubation in vitro. Specific precipitation of labelled plasma-membrane proteins is obtained when detergent-solubilized extracts of 125I-labelled reticulocyte plasma membranes are incubated with this antiserum and Staphyloccus aureus, but not when mature-cell plasma membranes are treated similarly. During maturation of reticulocytes in vitro (up to 4 days at 37 degrees C), there is a marked decrease in the immunoprecipitable material. The anti-reticulocyte-specific antibodies have been identified as anti-(transferrin receptor) antibodies. By using these antibodies as a probe, the transferrin receptor has been shown to have a subunit molecular weight of 93 000. The data are consistent with reported molecular weights of this receptor and with the proposal that the receptor may exist as a dimer, since [125I]iodotyrosyl-peptide maps of the 93 000- and 186 000-mol.wt. components isolated are shown to be identical. Evidence is presented for the transmembrane nature of the receptor and for the presence of different binding sites for transferrin and these antibodies on the receptor.

Animals↗
PubMed1983

Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor.

The fate of the transferrin receptor during in vitro maturation of sheep reticulocytes has been followed using FITC- and 125I-labeled anti-transferrin-receptor antibodies. Vesicles containing peptides that comigrate with the transferrin receptor on polyacrylamide gels are released during incubation of sheep reticulocytes, tagged with anti-transferrin-receptor antibodies. Vesicle formation does not require the presence of the anti-transferrin-receptor antibodies. Using 125I-surface-labeled reticulocytes, it can be shown that the 125I-labeled material which is released is retained by an immunoaffinity column of the anti-transferrin-receptor antibody. Using reticulocytes tagged with 125I-labeled anti-transferrin-receptor antibodies to follow the formation of vesicles, it can be shown that at 0 degree C or in phosphate-buffered saline the rate of vesicle release is less than that at 37 degrees C in culture medium. There is selective externalization of the antibody-receptor complex since few other membrane proteins are found in the externalized vesicles. The anti-transferrin-receptor antibodies cause redistribution of the receptor into patches that do not appear to be required for vesicle formation.

Animals↗
PubMed1983

Na+-independent exchange of amino acids in Ehrlich cells.

Many hydrophilic amino acids are transported via exchange by the Na+-independent L system in the Ehrlich cells. However, this exchange is measurable only at relative high concentrations of the small hydrophilic amino acids. Whereas leucine saturated this transport system at 0.2 mM, with threonine, serine, or alanine the system was below saturation at 30 mM. All amino acids capable of exchanging showed similar EA values (congruent to 20 X 10(3) cal X mol-1, 1 cal = 4.1868 J) and maximum velocities. Depending on the temperature, the apparent Km values for different amino acids differed by one or two orders of magnitude. These data suggest that for the small hydrophilic amino acids, the relatively slow rate of transport is due to the unfavorable Km values.

Amino Acids↗
PubMed1982

The use of fluorescent dyes to measure membrane potentials: a critique.

Under controlled conditions, fluorescent cyanine dyes can be used to measure membrane potentials of cell suspensions. Similar changes in membrane potential can be followed both with fluorescent dyes and electrophysiological probes in response to changes in the ion composition of the medium. Recent reports that attempt to abrogate the use of the cyanine dyes in measurements of the membrane potential are misleading.

Adenosine Triphosphate↗
PubMed1982

Reversal of Na+-dependent glycine transport in sheep reticulocyte membrane vesicles.

Inside-out membrane vesicles have been prepared from sheep reticulocytes. With these vesicles, Na+-dependent glycine uptake and net accumulation have been demonstrated to occur in reverse, i.e., from extravesicular (normal cytoplasmic) to intravesicular (normal extravesicular) surface. Uptake and accumulation are inhibited by energization of the sodium pump by ATP whereby the Na+ electrochemical gradient is dissipated. Glycine-dependent Na+ uptake was also observed, providing evidence that Na+-dependent glycine influx into these vesicles, equivalent to normal efflux, is characterized by Na+-glycine co-transport.

Adenosine Triphosphate↗
PubMed1982

Partial purification of amino acid transport systems in Ehrlich ascites tumor cell plasma membranes.

Ehrlich ascites tumor cell plasma membranes were subjected to sequential selective protein extraction to identify protein components associated with amino acid transport. These membranes were extracted with Triton X-100 followed by 2,3-dimethylmaleic anhydride. Approximately 80% of the membrane proteins were extracted by these procedures while the original lipids were largely retained (approximately 70%). The quantity of carbohydrate per milligram protein in the residue increased on extraction, consistent with an enrichment of glycoprotein in the residue. The residual vesicles display the characteristic properties of Na+-coupled amino acid transport. These properties include Na+-stimulated uptake and Na+-gradient-stimulated uptake leading to an accumulation of the solute against its chemical gradient as well as inhibition of uptake by a competitive amino acid, L-methionine. The extracted vesicles exhibit a peak level of alpha-aminoisobutyrate uptake six times greater than that expected from equilibration of alpha-aminoisobutyrate. This accumulation is greater than that obtained with native vesicles, albeit slower. The accelerated exchange diffusion of L-leucine is not measurable in the residual vesicles after dimethylmaleic acid anhydride treatment, although it can be measured after Triton extraction. These results are consistent with the conclusion that the amino acid transport systems "A" (Na+-coupled) and "L" (Na+-independent) in Ehrlich cells, though having overlapping specificities for amino acids, and distinct physical entities.

Amino Acids↗
PubMed1982

[Disappearance of specific membrane components during reticulocyte maturation in sheep].

Using antibodies prepared against whole reticulocyte suspensions, it is possible to identify specific membrane proteins from sheep reticulocytes. These membrane proteins can be isolated using the antibodies and Staphylococcus A protein to precipitate the immune complex. With fluorescein-labelled antireticulocyte antibody it may be shown that only reticulocytes and not red cells become fluorescent on exposure to the antibody, confirming that the antibody is directed against reticulocytes. Using either immobilized antibody columns or immobilized transferring columns, the same peptide bands from the membrane are retained by either column. The data suggest that the antibody is directed against the transferrin receptor. When the antibody is incubated for several hours with the reticulocytes, the cells release vesicles containing the transferrin receptor. This release is energy dependent since the process is inhibited by agents which interfere with metabolic activity.

Animals↗
PubMed1981

Alterations in membrane permeability with trypsin treatment.

Trypsin treatment of Ehrlich cells reduced sodium-coupled amino acid transport, lowered ATP levels, and abolished cation gradients. The data suggest that the decrease in active amino acid transport results from an alteration in membrane permeability caused by DNA released from a fraction of the cells upon trypsin treatment. The reduced amino acid transport appears to be the result of an abolition of ion gradients required for transport since vesicles prepared from these cells show near normal transport activity. The altered permeability of the membrane can be restored by incubation of the cells with serum. Protein synthesis does not appear to play a role in the restoration of Na+-dependent amino acid transport since restoration is unaffected by the presence of cycloheximide. The recovery of activity does depend partly on the presence of Ca2+ in the incubation medium since reversal of the trypsin inhibition of glycine transport can be obtained on incubation with Ca2+ and glucose.

Adenosine Triphosphate↗
PubMed1980

Bimodal effects of cellular amino acids on Na+-dependent amino acid transport in Ehrlich cells.

Cells depleted of amino acids show lower rates of glycine or aminoisobutyric acid uptake than do freshly isolated cells. In the amino acid-depleted cells, addition of valinomycin stimulates amino acid influx at least to the level observed in freshly isolated cells. In cells containing high levels of cellular amino acids, valinomycin has little effect on influx of amino acids. It is concluded that the transport of amino acids in freshly isolated cells is elevated compared to depleted cells because the cells are hyperpolarized by the continuous loss of cellular amino acids during the transport assay. During this hyperpolarization by amino acid loss, transport of amino acids is not further stimulated by valinomycin at low external [K+] (10 mM +/- 5 mM). With the exception of preloading with glycine, cells preloaded with a single amino acid to a concentration greater than 20 mM show reduced rates of glycine and aminoisobutyric acid influx at early times (less than 15 min) compared to amino acid-depleted cells. The reduction of influx is transient and by 30 min, influx is greater in preloaded than in amino acid-depleted cells. Knowing that increases and decreases in the membrane potential are achieved by using varying external [K+] in the presence of valinomycin and propranolol, and using amino acid-depleted cells, it can be shown that an increased membrane potential increases the V for glycine and aminoisobutyric acid influx. A decrease in the potential difference results in a decreased V. Changes in Km also occur when the membrane potential is varied.

Amino Acids↗