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Biomedical subjects

J C Ellory

Publications and source records attributed to J C Ellory.

At least 127 records · Page 7Linked to original sources
PubMed1985

Changes in the cation composition and active K+ transport in the red cells of fetal sheep prepartum.

The red blood cells of lambs, genotypically low potassium type, undergo a transition from high potassium to low potassium cell type from parturition onwards. This involves gradual changes in cell ion content, sodium pump activity, and ouabain binding. In the present study we investigated the properties of fetal red blood cells from 30 days prepartum using the chronically cannulated pregnant ewe preparation. We demonstrate that intracellular sodium increases and potassium decreases from -30 days onwards. Sodium pump activity monitored either by tracer potassium influx or ouabain binding is markedly higher in the early fetal samples examined and declines fourfold during the final month in utero. Unlike the maternal low potassium cells the early fetal red cells are refractory in terms of sodium pump stimulation by anti-L, the antibody in fact consistently inhibiting the pump. Finally, we have investigated the volume sensitivity and development of the ouabain-insensitive potassium fluxes in these cells and found that both fetal and maternal cells show a marked chloride-dependent, volume-sensitive passive potassium flux. We conclude that the decrease in active sodium transport between fetal red cells and adult low potassium cells is achieved partly by a reduction in the density of sodium pumps per cell, and then later by the introduction into the circulation of cells with Lp-antigen-modified sodium pumps.

Animals↗
PubMed1984

Nucleoside transport in human erythrocytes. Nitrobenzylthioinosine binding and uridine transport activities have similar radiation target sizes.

Intact human erythrocytes were irradiated in the frozen state with a high-energy electron beam. Nitrobenzylthioinosine-sensitive uridine influx, equilibrium exchange uridine influx and high-affinity nitrobenzylthioinosine binding were inactivated as a simple exponential function of the radiation dose, indicating an in situ target size of 122 000. The results suggest that the nitrobenzylthioinosine-binding site(s) and the permeation site(s) of the transporter are present on the same transporter element.

Biological Transport↗
PubMed1984

Molecular weight estimates of insect cholinergic receptors by radiation inactivation.

Molecular weight (MW) estimates for sites to which the radiolabelled cholinergic receptor probes alpha-bungarotoxin and N-methylscopolamine bind in CNS extracts of the cockroach Periplaneta americana have been made by radiation inactivation analysis. The MW of 77,600 determined for [3H]N-methylscopolamine binding sites agrees well with published values for vertebrate muscarinic acetylcholine receptor sites. In contrast, N-[propionyl-3H]propionylated alpha-bungarotoxin binds to a separate membrane component of lower MW (108,000) than previously reported values for vertebrate and insect nicotinic acetylcholine receptors. This appears to represent one or more subunits of the receptor complex, containing the recognition site.

Animals↗
PubMed1984

Threonine uptake in Trypanosoma brucei.

Threonine uptake by the parasitic protozoan Trypanosoma brucei has been assessed using an oil-phase separation technique for measuring rapid amino acid fluxes. It was shown that the storage conditions for the organism were critical for the production of reproducible data. Using this method it has been shown that threonine uptake occurs rapidly, being linear for less than 30 s at 37 degrees C in contrast with previous reported results. Kinetic analysis of threonine uptake at 25 degrees C and at physiological plasma threonine levels (20-700 microM) gave a Km of 250 microM and a Vmax of 8 nmol mg-1 cell protein min-1. At these concentrations threonine uptake takes place against a concentration gradient.

Alanine↗
PubMed1984

Effect of amphotericin B and gestational age on sodium transport across the rat visceral yolk sac placenta in vitro.

The transport properties of the rat visceral yolk sac placenta from Days 14.5 to 18.5 of gestation were studied in vitro. All tissues had a positive potential difference, fetal side relative to maternal side, and showed net Na transport towards the fetus. Basal short-circuit current and net Na flux increased rapidly with gestational age over the period studied. Amphotericin B applied to the maternal surface of the yolk sac stimulated current and net Na flux, indicating that the apical membrane Na permeability limited transport and revealing a reserve capacity for transport. Contrary to their basal values, current and Na flux following treatment with amphotericin were independent of gestational age.

Amphotericin B↗
PubMed1983

Radiation inactivation of (Na,K)-ATPase, an enzyme showing multiple radiation-sensitive domains.

The radiation inactivation of membrane-bound (Na,K)-ATPase from pig kidney was studied by irradiating lyophilized enzyme preparations in vacuo. Various results were obtained depending upon the function assayed. The apparent target size of (Na,K)-ATPase activity was found to be 264 kDa an the high affinity binding sites for ATP, vanadate, and ouabain (bound in the presence of Na+, Mg2+, and ATP) had an apparent target size of 145 kDa. The binding of ouabain in the presence of Mg2+ alone seemed to depend upon the integrity of a domain with a target size of approximately 100 kDa. Na-ATPase and p-nitrophenyl phosphatase, assayed under a variety of conditions, gave inactivation kinetics that did not conform to classical target theory. All the results have been assembled into a model according to which 1) the membrane-bound enzyme is an (alpha beta)2-dimer; 2) there is radiation energy coupling between all four peptides in a molecule, and 3) each alpha-peptide consists of five discrete and independent radiation-sensitive domains with specific functions in the enzymatic reactions catalyzed by the sodium pump.

Adenosine Triphosphate↗
PubMed1983

Red-cell amino acid transport. Evidence for the presence of system ASC in mature human red blood cells.

The properties of Na+-dependent L-alanine transport in human erythrocytes were investigated using K+ as the Na+ substitute. Initial rates of Na+-dependent L-alanine uptake (0.2 mM extracellular amino acid) for erythrocytes from 22 donors ranged from 40 to 180 mumol/litre of cells per h at 37 degrees C. Amino acid uptake over the concentration range 0.1-8 mM was consistent with a single saturable component of Na+-dependent L-alanine transport. Apparent Km and Vmax. values at 37 and 5 degrees C measured in erythrocytes from the same donor were 0.27 and 0.085 mM respectively, and 270 and 8.5 mumol/litre of cells per h respectively. The transporter responsible for this uptake was identified as system ASC on the basis of cross-inhibition studies with a series of 42 amino acids and amino acid analogues. Apparent Ki values for glycine, L-alpha-amino-n-butyrate, L-serine and L-leucine as inhibitors of Na+-dependent L-alanine uptake at 37 degrees C were 4.2, 0.12, 0.16 and 0.70 mM respectively. Reticulocytes from a patient with inherited pyruvate kinase deficiency were found to have a 10-fold elevated activity of Na+-dependent L-alanine uptake compared with erythrocytes from normal donors. Separation of erythrocytes according to cell density (cell age) established that even the oldest mature erythrocytes retained significant Na+-dependent L-alanine transport activity. Amino acid transport was, however, a more sensitive indicator of cell age than acetylcholinesterase activity. Erythrocytes were found to accumulate L-alanine against its concentration gradient (distribution ratio approx. 1.5 after 4 h incubation), an effect that was abolished in Na+-free media. Na+-dependent L-alanine uptake was shown to be associated with L-alanine-dependent Na+ influx, the measured coupling ratio being 1:1.

Alanine↗
PubMed1983

The solubilization of the L and M antigens from sheep red cell membranes.

The Lp, L1 and M antigens from sheep red cells were solubilized using the non-ionic detergent Triton X-100 in the presence of dithiothreitol. Recovery rates were improved when membranes were sonicated at 4 degrees C in the presence of the detergent; values in the range 16-25% (M) and 9-17% (Lp and L1) were achieved for recovery.

Animals↗
PubMed1983

Determination of the molecular size of the nitrendipine-sensitive Ca2+ channel by radiation inactivation.

The molecular size of the [3H] nitrendipine receptor of transverse tubules prepared from rabbit skeletal muscle and from rat cortex synaptic membranes have been investigated. Radiation inactivation of the specific binding of [3H] nitrendipine was consistent with Mr equals 210 000 +/- 20,000 for the receptor in each membrane preparation indicating a common size for the [3H] nitrendipine receptor.

Animals↗
PubMed1983

Molecular size of opiate (enkephalin) receptors in neuroblastoma-glioma hybrid cells as determined by radiation inactivation analysis.

Opiate receptor binding decayed exponentially in mouse neuroblastoma-rat glioma (NG108-15) hybrid cell preparations following exposure to increasing doses of ionizing radiation (0.2 to 7.0 Mrads; 2.0 Mrads/min). Target size analysis revealed that [3H][D-Ala2, D-Leu5]enkephalin (agonist) and [3H]naloxone (antagonist) bound specifically to a component with an apparent molecular size of 200,000 +/- 20,000. Lyophilization of cells for the irradiation procedure did not significantly alter receptor affinity or binding capacity for these ligands. Furthermore, the loss of opiate receptor binding in irradiated cell samples could not be attributed to reduced receptor affinity since increasing concentrations of radiolabeled ligand failed to reverse the inhibition; nonspecific binding decreased only slightly under identical experimental conditions. The value of determining molecular size by radiation inactivation analysis was confirmed by showing that apparent target sizes for two representative lysosomal enzymes (beta-galactosidase and alpha-mannosidase) were consistent with results obtained previously using conventional methods. Thus, the data suggest that the ligand binding component of delta-opiate (enkephalin) receptors in NG108-15 cells has a minimum functional size of approximately 200,000.

Animals↗
PubMed1983

Molecular size of different neurotoxin receptors on the voltage-sensitive Na+ channel.

Measurements were made of the molecular sizes of two distinct receptors on the Na+ channel in rat brain synaptosomes that are specific for different neurotoxins. Radiation inactivation of the binding of radiolabeled derivatives of the toxins was consistent with Mr = 260,000 for the tetrodotoxin receptor and Mr = 266,000 for the receptor specific for two scorpion toxins, toxin II from Centruroides suffusus suffusus and toxin gamma from Tityus serrulatus serrulatus. Covalent cross-linking of the latter to its receptor similarly indicated Mr = 270,000. It seems most likely that these two distinct receptors reside on the same molecule.

Amphibian Proteins↗
PubMed1983

Glycosylation-dependent regulation of opiate (enkephalin) receptors in neurotumor cells.

Electron inactivation analysis revealed that the opiate (enkephalin) binding site in neurotumor cell lines NG108-15 and NCB-20 had an apparent target size of 200,000 daltons. Expression of functional opiate receptors in neurotumor cells appeared to require glycosylation, as treatment of such cells with tunicamycin (TM; under conditions where de novo glycosylation of asparagine residues in protein was reduced by 80%, but overall protein and DNA synthesis were inhibited by less than 10%) resulted in the loss of 50% of the opiate binding sites. The loss of binding sites could not be prevented by addition of protease inhibitors to cell cultures, but binding sites were partially restored 48-60 h after removal of the TM. In addition, the number of enkephalin binding sites in TM-treated cells was also restored to near-normal levels by addition of physiological concentrations (1-10 mM) of manganese ions to the in vitro receptor binding incubation mixture. TM treatment resulted in receptor supersensitivity to manganese ions for both opiate agonists and antagonists, no change in the sodium effect for either agonists or antagonists, and subsensitivity to GTP for both agonists and antagonists. However, opiate binding to cell membranes was not substantially inhibited by either neuraminidase treatment or short-term incubation with lectins such as wheat germ agglutinin, ricin, or concanavalin A. Thus, the data suggest that oligosaccharide units are not directly involved in opiate receptor-ligand interactions, but protein glycosylation is required for functional expression of receptors.

Animals↗
PubMed1983

Inhibition of human red cell sodium and potassium transport by divalent cations.

The influx and efflux of Na and K across the human red cell membrane by the bumetanide-sensitive (Na-K co-transport) and residual (ouabain- and bumetanide-insensitive) routes were inhibited by increasing concentrations of external Mg. Ca, Sr, Ba, Mn and Co also inhibited bumetanide-sensitive and residual K transport. External Mg inhibited choline uptake and the Na-dependent fractions of L-alanine and L-serine uptakes. External Mg reduced the maximal rate (app. Vmax) but not the affinity (app. Km) of the bumetanide-sensitive K and Na influxes when they were measured as functions of external K and Na respectively. The inhibitory effect of Mg was not due to a small reduction in zeta potential since much larger reductions in zeta potential produced by neuraminidase did not affect transport. Internal Mg stimulated the ouabain-sensitive K influx but inhibited the co-transport and residual components of K influx. Bumetanide was a poor co-transport inhibitor in red cells pre-treated with A23187 and EDTA. It was concluded that the inhibitory effects of external Mg were probably not due to changes in the ionic composition of the diffuse double layer adjacent to the cell membrane. Mg and other divalent cations should not be used as 'inert' ionic substitutes in human red cell Na and K transport studies.

Amino Acids↗
PubMed1983

Transport of amino acids for glutathione biosynthesis in human and dog red cells.

Cysteine and glycine for glutathione biosynthesis in human red cells enter via specific amino acid transport systems, principally system ASC and gly respectively. In contrast, human red cells are impermeable to glutamate, but glutamine enters via a Na-dependent, saturable pathway, which apparently obeys Michaelis-Menten kinetics with appKm and Vmax values of 90-160 microM and 140-240 mumol/1.cells/h respectively. Dog red cells however, do show a Na-dependent glutamate uptake which apparently obeys Michaelis-Menten kinetics with appKm and Vmax values of 4-6 microM and 70-110 mumol/1.cells/h respectively.

Animals↗