PubMed Health⌕ Search

Biomedical subjects

R M Johnstone

Publications and source records attributed to R M Johnstone.

At least 19 recordsLinked to original sources
PubMed1992

Evidence for an internal pool of nucleoside transporters in mammalian reticulocytes.

Comparisons of the site specific binding of nitrobenzylthioinosine (NBMPR) to intact and lysed red cells from various mammalian and avian species suggest the presence of a cytoplasmic pool of nucleoside transporters. In some species the cytoplasmic pool is about 50% of the total (mouse). On the average, the cytoplasmic pool is approx. 20% of the surface pool of NBMPR-binding sites. In sheep reticulocytes, both pools disappear in an energy-dependent manner during the maturation of the reticulocyte in vitro.

4-Chloromercuribenzenesulfonate↗
PubMed1991

Intracellular localization of newly synthesized transferrin receptors in the peripheral sheep reticulocyte.

In this paper, we provide evidence for an incompletely glycosylated transferrin receptor (TfR) which is not transported to the plasma membrane in the sheep reticulocyte. Cleveland peptide maps of the native (preexisting) TfR and [35S]methionine-labeled TfR were different. If the receptors were deglycosylated before mapping, the peptides were identical. There was preferential binding of the [35S]TfR to Con A-Sepharose, indicating the existence of a higher density of high mannose chains on the 35S-labeled TfR. Moreover, when total [3H]mannose-labeled glycopeptides from reticulocytes were separated on a column of Bio-Gel P6, the [3H]mannose was associated with endoglycosidase H-sensitive high mannose or hybrid oligosaccharides, but not with complex sugars. After Percoll density gradient centrifugation, the [35S]TfR peaked in a fraction which separated from the bulk of the native TfR. The transmembrane glycoproteins, Band 3 and mature glycophorins, are not synthesized in the sheep reticulocyte. It appears that the reticulocyte, at this stage of red cell development, has lost the vesicles and/or proteins which are required to transport proteins from the site of translation to the cell surface.

Animals↗
PubMed1991

Molecular size of a Na(+)-dependent amino acid transporter in Ehrlich ascites cell plasma membranes estimated by radiation inactivation.

Radiation inactivation was used to estimate the molecular size of a Na(+)-dependent amino acid transport system in Ehrlich ascites cell plasma membrane vesicles. Na(+)-dependent alpha-aminoisobutyric acid uptake was measured after membranes were irradiated at -78.5 degrees C in a cryoprotective medium. Twenty-five percent of the transport activity was lost at low radiation doses (less than 0.5 Mrad), suggesting the presence of a high molecular weight transport complex. The remaining activity (approximately 75% of total) decreased exponentially with increasing radiation dose, and a molecular size of 347 kDa was calculated for the latter carrier system. Vesicle permeability and intravesicular volume were measured to verify that losses in transport activity were due to a direct effect of radiation on the transporter and not through indirect effects on the structural integrity of membrane vesicles. Radiation doses 2-3-fold higher than those required to inactivate amino acid transport were needed to cause significant volume changes (greater than 15%). Vesicle permeability was unchanged by the irradiation. The structural integrity of plasma membrane vesicles was therefore maintained at radiation doses where there was a dramatic decrease in amino acid transport. The relationship between the fragmentation of a 120-130-kDa peptide, a putative component of the Na(+)-dependent amino acid carrier [McCormick, J. I., & Johnstone, R. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881], and loss of transport activity in irradiated membranes was also examined. Peptide loss was quantitated by Western blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Transport Systems↗
PubMed1991

Exosome formation during maturation of mammalian and avian reticulocytes: evidence that exosome release is a major route for externalization of obsolete membrane proteins.

We have assessed whether exosome formation is a significant route for loss of plasma membrane functions during sheep reticulocyte maturation in vitro. Although the recovery of transferrin binding activity in exosomes is at best approximately 25-30% of the lost activity, recoveries of over 50% of the lost receptor can be obtained if 125I-labelled transferrin receptor is measured using an that receptor instability may contribute to the less than quantitative recovery of the transferrin receptor. Significantly higher (75-80%) levels of the nucleoside transporter can be recovered in exosomes during red cell maturation using 3H-nitrobenzylthioinosine binding to measure the nucleoside transporter. These data suggest that exosome formation is a major route for removal of plasma membrane proteins during reticulocyte maturation and plasma membrane remodelling. We have also shown that both in vivo and in vitro, embryonic chicken reticulocytes form exosomes which contain the transferrin receptor. Thus, exosome formation is not restricted to mammalian red cells, but also occurs in red cells, which retain organelles, such as nuclei and mitochondria, into the mature red cell stage.

Animals↗
PubMed1990

Role of specific acidic lipids on the reconstitution of Na(+)-dependent amino acid transport in proteoliposomes derived from Ehrlich cell plasma membranes.

The effect of acidic phospholipids on the activity of a Na(+)-dependent amino acid transporter (A system) from Ehrlich ascites cell plasma membranes was examined. Plasma membranes were solubilized in cholate/urea and reconstituted with Ba2(+)-precipitated asolectin (soybean phospholipid free of anionic phospholipids) replenished with different acidic phospholipids. In the absence of added acidic phospholipids, transport activity was very low. However, three acidic lipids [cardiolipin greater than phosphatidic acid (PA) greater than phosphatidylinositol] were capable of restoring transport activity (in the order given) to proteoliposomes made from Ba2(+)-precipitated asolectin, while other acidic phospholipids (phosphatidylserine and phosphatidylglycerol) were much less active in this respect. For restoration of optimal activity, PA containing at least one unsaturated fatty acyl moiety, particularly in the beta position, was required. PA containing only saturated fatty acids in the beta and gamma positions was largely inactive. No difference in restoration of function was observed on varying the saturated fatty acyl chain length in PA from 10 carbons to 18 carbons. The specific effects of PA on the A-system transporter were not shared by the Na(+)-independent amino acid exchange system (L system) or the glucose transport system. Treatment with poly(ethylene glycol) 8000 was shown to reduce the nonspecific permeability of the reconstituted proteoliposomes and to enhance Na(+)-dependent amino acid transport.

Amino Acids↗
PubMed1990

Evidence for an essential sulfhydryl group at the substrate binding site of the A-system transporter of Ehrlich cell plasma membranes.

Plasma membrane suspensions of Ehrlich ascites cells solubilized with cholic acid were used to study the effects of sulfhydryl reagents on Na(+)-dependent amino acid transport. These suspensions were treated with the sulfhydryl binding agents p-chloromercuribenzenesulfonic acid or N-ethylmaleimide prior to reconstitution for the assay of transport activity. The proteoliposomes formed from dissolved membranes treated with p-chloromercuribenzenesulfonic acid showed no Na(+)-dependent alpha-aminoisobutyric acid transport, while N-ethylmaleimide pretreated membranes retained approximately 90% of the original activity. To avoid interference by the N-ethylmaleimide component, further studies were carried out with membranes pretreated with 200 microM N-ethylmaleimide prior to p-chloromercuribenzenesulfonic acid treatment. A concentration of 25 microM p-chloromercuribenzenesulfonic acid inhibited Na(+)-dependent alpha-aminoisobutyric acid transport by 50%. The degree of inhibition was dramatically reduced in the presence of substrates specific for the A transport system. Using an inhibition index to address the efficacy of inhibition in presence and absence of substrates, it could be shown that an index of 1.0 in presence of p-chloromercuribenzenesulfonic acid was reduced to 0.84 with (methylamino)isobutyric acid alone and 0.05 in the presence of 100 mM Na+ and 5 mM (methylamino)isobutyric acid. Na+ alone offered no protection. The results show that sulfhydryl group(s) on the amino acid carrier may be directly involved in substrate binding and that substrate binding sites are functional in the disaggregated membrane state. Furthermore, Na+ directly affects (methylamino)isobutyrate binding, since the degree of protection by the amino acid analogue against p-chloromercuribenzenesulfonic acid inhibition was influenced by the presence of Na+.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloromercuribenzenesulfonate↗
PubMed1990

A common mechanism may be involved in the selective loss of plasma membrane functions during reticulocyte maturation.

During the maturation of the mammalian reticulocyte to the erythrocyte, 50nm bodies (called exosomes) are released from the cell. These exosomes carry the transferrin receptor (TFR) as well as other plasma membrane and lysosomal activities which the cells are known to lose during maturation. Using an antibody attached to an iron core bead, we have shown that other plasma membrane activities (nucleoside transporter and acetylcholine esterase) are packaged in the same exosome as the transferrin receptor. This population of exosomes is devoid of lysosomal enzymes. Exosomes were detected in the circulation of several species of phlebotomized animals, suggesting that exosome formation is a natural phenomenon, and probably a route by which the red cell decreases in size and sheds specific plasma membrane activities during maturation.

Animals↗
PubMed1989

Solubilization and reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.

Uptake of [3H]uridine by Ehrlich cells was mediated by both nitrobenzylthioinosine (NBMPR)-sensitive (75%) and NBMPR-insensitive (25%) mechanisms. Each cell contained approx. 26,000 high-affinity (KD = 0.19 nM) recognition sites for [3H]NBMPR, and binding was inhibited by dipyridamole and adenosine at concentrations similar to those required for inhibition of [3H]uridine uptake. Calculations show that each cell contains a total of about 35,000 nucleoside transporters. Photoaffinity labelling of a partially purified preparation of plasma membranes with [3H]NBMPR resulted in a single broad 3H-labelled band on SDS/polyacrylamide gels, with an apparent molecular-mass peak of 42 kDa. This is in contrast with human erythrocyte membranes, where [3H]NBMPR photolabelled two broad bands with peaks at 55 and 80 kDa. Treatment of photoaffinity-labelled membranes with endoglycosidase F decreased the apparent molecular masses of both the Ehrlich-cell and erythrocyte [3H]NBMPR-labelled proteins to approx. 40 kDa. These results suggest that the human erythrocyte [3H]NBMPR-binding polypeptides are more extensively glycosylated than the corresponding Ehrlich-cell polypeptides. Octyl beta-D-glucopyranoside [1.0% (w/v) + asolectin] solubilized over 90% of the [3H]NBMPR-binding sites, with near-complete retention of [3H]NBMPR-binding characteristics. The only major change was a 65-fold decrease in affinity for dipyridamole, which was partly reversed upon incorporation of the solubilized proteins into asolectin membranes. Proteoliposomes, prepared by using asolectin and the octyl glucoside-solubilized plasma membranes, were capable of accumulating [3H]uridine via a protein-dependent dipyridamole/nitrobenzylthioguanosine/dilazep-sensitive mechanism. We have thus demonstrated the efficient solubilization and functional reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.

Adenosine↗
PubMed1989

Enhanced efflux of [3H]vinblastine from Chinese hamster ovary cells transfected with a full-length complementary DNA clone for the mdr1 gene.

Multidrug-resistant Chinese hamster ovary cell clones stably transfected with, and overexpressing, the mouse mdr1 complementary DNA clone along with drug-sensitive Chinese hamster ovary control cells were characterized for their capacities to accumulate and retain [3H]vinblastine. Multidrug-resistant mdr1 transfectants show a 3-4-fold decrease in [3H]vinblastine accumulation, compared to their drug-sensitive counterparts. After ATP depletion, this difference in [3H]vinblastine accumulation between mdr1 transfectants and control cells effectively disappears. This ATP-dependent decreased drug accumulation is paralleled in mdr1 transfectants by an enhanced capacity of these cells to extrude the drug in an ATP-dependent manner. In medium containing glucose and glutamine, the mdr1 transfectants release preloaded drug at a rate five times that of control, drug-sensitive cells. In ATP-depleted control and mdr1-transfected cells, there is little difference in the rate or extent of [3H]vinblastine release. The observation that the mdr1 transfectants show a decreased [3H]vinblastine accumulation and an increased vinblastine release, both of which are abolished when cellular ATP levels are reduced, provides a direct demonstration that the product of the transfected mdr1 gene is responsible for a mechanism controlling cellular drug levels in an ATP-dependent manner. However, attempts to establish competition for [3H]vinblastine transport by vincristine, daunomycin, and actinomycin D were only partly successful in mdr1 transfectants.

Adenosine Triphosphate↗
PubMed1989

Maturation-associated loss and incomplete de novo synthesis of the transferrin receptor in peripheral sheep reticulocytes: response to heme and iron.

Hemin, but not iron, in the culture medium stimulates the maturation-associated loss of the transferrin receptor from sheep reticulocytes (t1/2 for loss approximately 6 hr) and its appearance in a population of externalized vesicles. A similar pattern is seen with nucleoside binding (a measure of the nucleoside transporter), where hemin increases the loss of binding activity from the cells during culture, concomitant with an increase in nucleoside binding in the externalized vesicles. Sheep reticulocytes retain the ability to synthesize the transferrin receptor, but the 35S-labeled receptors are not detected in released vesicles. Whereas hemin stimulates the loss of 35S-labeled transferrin receptors from the cell (t1/2 for loss approximately 20 hr), nonheme iron is more effective than heme. This difference in response of native and 35S-labeled receptor to hemin and iron supplements appears to be related to the differences in the two classes of receptors. Although the 35S-labeled receptor binds transferrin and both native and 35S-labeled peptides comigrate after chemical deglycosylation, the 35S-receptor is approximately 2 kD smaller than the native receptor and fails to acquire its complete size even when chased for up to 24 hr. Moreover, the 35S-labeled receptor is not expressed at the cell surface, but is retained in a nonrecycling compartment, where it is insensitive to digestion by trypsin at both 0 degrees C and 37 degrees C.

Animals↗
PubMed1989

Reticulocyte maturation and exosome release: transferrin receptor containing exosomes shows multiple plasma membrane functions.

Vesicles (exosomes) released during sheep reticulocyte maturation contain a number of plasma membrane functions. Using an antibody coated, magnetic core bead, it has been shown unequivocally that vesicles that contain the transferrin receptor also contain other plasma membrane activities, such as the nucleoside transporter and acetylcholinesterase. Lysosomal activities, normally found in the same pellet, are excluded from the transferrin receptor-containing exosomes, suggesting that there is a common mechanism to segregate and externalize specific plasma membrane proteins. In addition to the sheep, electron micrographic studies show that exosomes can be retrieved from the circulation of anemic pigs, rats, and rabbits.

Acetylcholinesterase↗
PubMed1988

Incorporation of myristate and palmitate into the sheep reticulocyte transferrin receptor: evidence for identical sites of labeling.

The ability of sheep reticulocytes and plasma membranes isolated from them to incorporate fatty acids into the transferrin receptor has been examined using both [3H]palmitate and [3H]myristate. Both fatty acids, when incorporated into the transferrin receptor, can be released by treating the protein with 1 M hydroxylamine at pH 7.0. After treatment of the 3H-acylated receptor with borohydride, an 3H-labeled alcohol is released, suggesting that the receptor-bound fatty acid is in thioester linkage. With both [3H]myristate and [3H]palmitate, Cleveland maps from immunoprecipitates of the transferrin receptor labeled in intact cells and isolated membranes show that identical peptides are labeled. No evidence was obtained for qualitatively different labeling with the two fatty acids. In intact reticulocytes, incorporation of [3H]palmitate into the transferrin receptor is approximately 3.5 times greater than the incorporation of [3H]myristate from equivalent concentrations of the labeled fatty acids. However, in isolated reticulocyte plasma membranes, there is much less difference between palmitate and myristate incorporation (with ATP) or between their acyl-CoA derivatives. The reason for the discrepancy between cells and membranes is unknown but may be due to the presence in intact cells of more than one enzyme for activating the fatty acids. Acylation of the receptor in isolated plasma membranes is fourfold greater with the CoA derivatives than with the free fatty acids. The fatty acid activating enzyme(s) as well as the acyltransferase(s) appear to be membrane bound in reticulocytes.

Acylation↗
PubMed1988

Volume enlargement and recovery of Na+-dependent amino acid transport in proteoliposomes derived from Ehrlich ascites cell membranes.

Na+-dependent amino acid transport can be reconstituted from solubilized Ehrlich cell plasma membranes by addition of asolectin vesicles, gel filtration, and a freeze-thaw cycle. Removal of phosphatidic acid (approximately 10% of the total lipid) by Ba2+ precipitation reduces the efficiency of reconstitution of Na+-dependent amino acid transport by approximately 73% and decreases intravesicular volume of the proteoliposomes by approximately 43%. The loss of transport activity is not due to exclusion of specific proteins during reconstitution. The phosphatidic acid-free liposomes are less permeable and require more time to attain an equilibrium distribution of solute. Transport activity and intravesicular volume can be restored to Ba2+-precipitated asolectin proteoliposomes by addition of egg-phosphatidic acid during reconstitution. The extent of recovery of transport activity is proportional to the change in intravesicular volume and depends on the amount of phosphatidic acid present. Replacement of phosphatidic acid with 20% phosphatidylserine or phosphatidylglycerol leads to increases in intravesicular volume with little or no increase in amino acid transport. Generation of phosphatidic acid in situ by treatment of Ba2+-precipitated proteoliposomes with phospholipase D also restored transport. The observed increase in transport activity (9-fold) is accompanied by a 46% increase in intravesicular volume, presumably caused by vesicle fusion. Phosphatidic acid is also required for successful reconstitution of Na+-dependent amino acid transport from pure phosphatidylcholine:phosphatidylethanolamine (1:1) mixtures with only a small change (approximately 16%) in intravesicular volume. The results provide evidence for both indirect and direct effects of phosphatidic acid on reconstitution of Na+-dependent amino acid transport. The indirect effects occur through enlargement of intravesicular volume, large vesicles showing higher rates of transport. However, there is also evidence to indicate a specific effect of phosphatidic acid on the Na+-dependent amino acid transporter, since other acidic lipids may change intravesicular volume without a commensurate change in transport activity.

Amino Acids↗
PubMed1988

Simple and effective purification of a Na+-dependent amino acid transport system from Ehrlich ascites cell plasma membrane.

A reconstitution assay was used to measure transport activity during purification of a Na+-dependent amino acid transporter from Ehrlich cell plasma membrane. Cholate/urea-solubilized membranes were fractionated on a Sepharose 6B column and transport activity was recovered in the column void volume. Centrifugation of the void volume fraction at 105,000 X g and reextraction of the pellet with 1% octyl glucoside led to recovery of an extract whose specific transport activity was nearly 30-fold higher than that of the original solubilized extract with a recovery of 38% of the original activity. The properties of amino acid uptake in the purified reconstituted transporter were identical to those in native plasma membrane vesicles. The major component present in the purified fraction had a molecular mass of 120-130 kDa. Strong evidence that this 120- to 130-kDa peptide contains a component of the amino acid transporter was obtained by immunoprecipitation of transport activity from solubilized membranes with an antibody against the 120- to 130-kDa peptide. This study tentatively identifies a component of the Na+-dependent amino acid transporter as a peptide with an apparent molecular mass of 120-130 kDa.

4-Chloromercuribenzenesulfonate↗
PubMed1987

Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes).

Vesicles are released during the in vitro culture of sheep reticulocytes which can be harvested by centrifugation at 100,000 X g for 90 min. These vesicles contain a number of activities, characteristic of the reticulocyte plasma membrane, which are known to diminish or disappear upon reticulocyte maturation. The activities include acetylcholinesterase, cytochalasin B binding (glucose transporter) nucleoside binding (i.e. nucleoside transporter), Na+-independent amino acid transport, and the transferrin receptor. Enzymes of cytosolic origin are not detectable or are present at low activity in the vesicles. Cultures of whole blood, mature red cells, or white cells do not yield comparable levels of these activities, supporting the conclusion that the activities arise from the reticulocytes. In addition, the lipid composition of the vesicles shows the high sphingomyelin content characteristic of sheep red cell plasma membranes, but not white cell or platelet membranes, also consistent with the conclusion that the vesicles are of reticulocyte origin. It is suggested that vesicle externalization may be a mechanism for shedding of specific membrane functions which are known to diminish during maturation of reticulocytes to erythrocytes.

Acetylcholinesterase↗