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Biomedical subjects

J C Ellory

Publications and source records attributed to J C Ellory.

At least 145 records · Page 8Linked to original sources
PubMed1982

The substructure of phosphodiesterase as established by radiation inactivation. A reinterpretation of results.

A model for the activation of phosphodiesterase by calmodulin based on a conversion of inactive dimers to active monomers, derived from radiation inactivation studies J. Biol. Chem. (1981) 256, 11351-11355 has been re-examined using a simple probability argument. We conclude that the original model is not supported by the radiation inactivation studies, since our analysis of this model would predict that the rate of radiation inactivation of calmodulin-dependent phosphodiesterase activity be exactly twice that for the decay in total activity in marked contrast with the results obtained.

Calcium-Binding Proteins↗
PubMed1982

Mode of transport and possible mechanism of action of L-phenylalanine benzyl ester as an anti-sickling agent.

L-Phenylalanine benzyl ester (Phe-Bz) and a number of ester analogues prevent sickling of erythrocytes from sickle cell disease patients. The compounds tested exhibit anti-sickling activity in the concentration range 0.5-3.0 mM. A general feature of these compounds is the presence of two aromatic rings in their molecular structure. The anti-sickling agents rapidly enter the erythrocyte and are hydrolysed to their component molecules. Incubation of human erythrocytes with 3.0 mM L-phenylalanine for 30 min at 37 degrees C results in accumulation of 2.0 mmol L-phenylanine/l cells, while incubation of erythrocytes with 3.0 mM Phe-Bz under similar conditions results in the production of 4.0 mmol L-phenylalanine/l cells and an equivalent amount of benzyl alcohol. Both L-phenylalanine and benzyl alcohol are inhibitors of the gelation of deoxyhaemoglobin S (deoxy-HbS) in vitro. Moreover, Phe-Bz and related anti-sickling agents fluidize the lipid bilayer of the erythrocyte membrane, inhibiting several transport systems, including those for L-phenylalanine, uridine and sulphate ions, as well as the Na+ pump and the Na+/K+ cotransporter, but increasing the passive influx and efflux of both cations and anions. The accumulation of Phe-Bz hydrolysis products within the erythrocyte together with the effects of Phe-Bz on cation permeability result in the influx of water causing the cell to swell. Thus, treatment of erythrocytes with 3.0 mM Phe-Bz at 37 degrees C for 30 min causes an increase in mean cell volume of 14.8%, decreasing the mean intracellular haemoglobin concentration from 34 to 29.6 g%. The increase in cell volume caused by Phe-Bz and its analogues together with the direct effects of their hydrolysis products on HbS probably act in concert to bring about the anti-sickling effect.

Anemia, Sickle Cell↗
PubMed1982

Anion-dependent cation transport in erythrocytes.

A selective survey of the literature reveals at least three major anion-dependent cation transport systems, defined as Na+ + Cl-, K+ + Cl- and Na+ + K+ + Cl- respectively. In human red cells, kinetic data on the fraction of K+ and Na+ influx inhibitable by bumetanide are presented to indicate an Na+:K+ stoichiometry of 1:2. For LK sheep red cells the large Cl- -dependent K+ leak induced by swelling is shown to share many characteristics with that induced by N-ethylmaleimide (NEM) treatment. NEM has complex effects, both inhibiting and then activating Cl- -dependent K+ fluxes dependent on NEM concentration. The alloantibody anti-L can prevent the action of NEM. In human red cells NEM induces a large Cl- -dependent specific K+ flux, which shows saturation kinetics. Its anion preference is Cl- greater than Br- greater than SCN- greater than I- greater than NO3- greater than MeSO4-. This transport pathway is not inhibited by oligomycin or SITS, although phloretin and high concentrations of furosemide and bumetanide (over 0.3 mM) do inhibit. Quinine (0.5 mM) is also an inhibitor. It is concluded that at least two distinct Cl- -dependent transport pathways for K+ are inducible in mammalian red cells, although the evidence for their separation is not absolute.

Animals↗
PubMed1982

Thiocyanate transport across fish intestine (Pleuronectes platessa).

When bathed on both sides with identical chloride-containing salines the in vitro preparation of the plaice intestine maintains a negative (serosa to mucosa) short-circuit current of 107 +/- 11 muA/cm2, a transepithelial potential difference of 5.5 +/- 0.6 mV (serosa negative), and a mean mucosal membrane potential of -45.4 +/- 0.6 mV. Under these conditions the intracellular chloride activity is 32 mM. If chloride in the bathing media is partially, or completely substituted by thiocyanate the measured electrical parameters do not change but transepithelial flux determinations show a reduction in chloride fluxes and the presence of a significant thiocyanate flux. The addition of piretanide (10-4M) reduced the short-circuit current and the mucosa-to-serosa fluxes of chloride and thiocyanate; this inhibition is similar to the effect of piretanide on chloride transport in this tissue. The results indicate that thiocyanate is transported in this tissue via the piretanide-sensitive "chloride" pathway and are compared with the effects of thiocyanate on other tissues reported in the literature.

Animals↗
PubMed1982

Interaction of L antibody with low potassium-type sheep red cells: resolution of two separate functional antibodies.

Antibodies of two specificities in alloimmune sheep anti-L sera, anti-Lp and anti-L1, were separated by a new technique and characterized. Absorption of anti-L serum with trypsinized LK(LL) sheep red cells left anti-Lp antibodies; the absorbed anti-L1 antibodies were then eluted. Anti-Lp was only weakly lytic in the presence of complement; it had no effect on passive K influx, but stimulated active K influx. The stimulation could be reversed by eluting the antibody in glycine buffer at low pH. Stimulatory activity in the eluted cells could be restored by resensitization with anti-Lp. Anti-L1 was more strongly lytic than anti-Lp in the presence of complement; it had no effect on active K influx, but inhibited passive K influx. Pig anti-ruminant IgG conjugated to hemocyanin was used to visualize by electron microscopy the number of Lp and L1 antigen sites on LL sheep red cells sensitized with anti-Lp an L1 antigen sites on LL sheep red cells sensitized with anti-Lp and anti-L1. The values obtained were 590 Lp sites/cell and 847 L1 sites/cell.

Animals↗
PubMed1982

Pressure and temperature effects on human red cell cation transport.

The effects of hydrostatic pressure and temperature on the three components of K+ uptake in human red cells have been investigated, using ouabain and bumetanide to distinguish between the pump, passive diffusion and cotransport. The pressure sensitivity for passive diffusion has been shown to depend on the counter-ion present. The order of this effect, Cl- greater than Br- greater than NO3- greater than I-, is the same as for the ionic partial molal volumes and the Hofmeister series. We have analyzed our experimental results thermodynamically, and propose a model for the activated transition-state complex of the potassium ion which involves the loss of water molecules from the secondary hydration shell, cosphere II.

Anions↗
PubMed1982

The transition from HK to LK phenotype in the red cells of newborn genetically LK lambs.

Red cells from newborn lambs were separated into different age populations by centrifugation, and cells with fetal hemoglobin (Hb) were distinguished from those with adult Hb by an acid elution technique. Changes were followed during development in rates of K+ transport (active and passive), numbers of Na+/K+ pump sites per cell, cell volumes, and numbers of Lp and L1 antigen sites per cell. These changes were correlated with the percentage of cells with adult hemoglobin. (The Lp and L1 antigens are associated with K+ transport in that specific alloantibody against Lp, anti-Lp, stimulates active transport, and anti-L1 inhibits passive transport.) Active K+ transport decreased during development because of a decline in number of Na+/K+ pumps (from measurements of ouabain binding) and because of an alteration in the affinity of the pumps for intracellular K+ (from kinetic studies in which the intracellular K+ concentration was varied). Cells with fetal Hb had fewer Lp sites and were larger than cells with adult Hb. As transport properties changed, the number of Lp sites increased and continued to increase after all the cells had adult Hb Cells with fetal Hb had as many L1 sites as lamb cells with adult Hb, but the number of L1 sites was less than those found previously for adult sheep. A population of small cells with intermediate K+ concentration and intermediate numbers of Lp sites appeared soon after birth. The various points of evidence suggested that the developmental process leading to cells with adult transport properties was a gradual one and did not coincide precisely with the switch from fetal to adult Hb.

Animals↗
PubMed1982

Kinetics of the sodium pump in red cells of different temperature sensitivity.

Ouabain-sensitive K influx into ground squirrel and guinea pig red cells was measured at 5 and 37 degrees C as a function of external K and internal Na. In both species the external K affinity increases on cooling, being three- and fivefold higher in guinea pig and ground squirrel, respectively, at 5 than at 37 degrees C. Internal Na affinity also increased on cooling, by about the same extent. The effect of internal Na on ouabain-sensitive K influx in guinea pig cells fits a cubic Michaelis-Menten-type equation, but in ground squirrel cells this was true only at high [Na]i. There was still significant ouabain-sensitive K influx at low [Na]i. Ouabain-binding experiments indicated around 800 sites/cell for guinea pig and Columbian ground squirrel erythrocytes, and 280 sites/cell for thirteen-lined ground squirrel cells. There was no significant difference in ouabain bound per cell at 37 and 5 degrees C. Calculated turnover numbers for Columbian and thirteen-lined ground squirrel and guinea pig red cell sodium pumps at 37 degrees C were about equal, being 77-100 and 100-129 s-1, respectively. At 5 degrees C red cells from ground squirrels performed significantly better, the turnover numbers being 1.0-2.3 s-1 compared with 0.42-0.47 s-1 for erythrocytes of guinea pig. The results do not accord with a hypothesis that cold-sensitive Na pumps are blocked in one predominant form.

Animals↗
PubMed1982

The human erythrocyte Cl-dependent Na-K cotransport system as a possible model for studying the action of loop diuretics.

1 The recent demonstration of the chloride-dependence of the red cell Na-K cotransport system suggests an analogy between this process and the active Cl- absorption in the ascending loop of Henle, which is the target transport system for loop diuretics. 2 Using red cell K influx, four known loop diuretics, six experimental frusemide analogues, two thiazides, two K-retaining diuretics and one organomercurial were compared for inhibitory potency on the red cell Na-K cotransport system. 3 Except for mersalyl, whose exact mode of action in the kidney is still in doubt, the inhibition of the red cell system by various loop diuretics was consistent with both published whole body diuretic data and isolated perfused tubule studies, while the system did not respond to the thiazides or the K-retaining diuretics. 4 It is concluded that the human red cell Na-K cotransport system is a possible valid model process on which to study the activity of loop diuretics.

Biological Transport, Active↗
PubMed1982

Protein and glycoprotein antifreezes in the intestinal fluid of polar fishes.

Measurements of ion concentrations, freezing points and melting points of intestinal fluid were made for several Antarctic fishes and two North Atlantic species. These measurements indicated that plasma and intestinal fluid are nearly isosmotic. Freezing points of intestinal fluid were approximately 0.9 degrees C below the melting points, suggesting the presence of glyco-protein antifreeze within the intestinal fluid of the Antarctic fishes. Polyacrylamide gel electrophoresis and specific immunoprecipitation with glyco-protein antifreeze antibody confirmed the presence of appreciable quantities of antifreeze and showed that the major antifreeze fractions present in the intestinal fluid are low molecular weight glycopeptides.

Adaptation, Physiological↗
PubMed1981

Radiation inactivation of (Na+ + K+)-ATPase. A small target size for the K+-occluding mechanism.

Radiation inactivation of partially purified (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pig kidney outer medulla shows that the target size for Rb+ occlusion by the enzyme (in the absence of phosphorylation) is much smaller than the target size for p-nitrophenyl phosphatase activity, which is itself smaller than the reported target size for (Na+ + K+)-ATPase activity.

Animals↗
PubMed1981

Amino acid transport via the red cell anion transport system.

Evidence is presented that the red cell anion-exchange transporter (Band 3) can selectively transport small neutral amino acids, including glycine, serine and cysteine, but not alanine, proline, valine and threonine. This transport is inhibited by micromolar concentrations of SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate), and increased by raising the pH from 6.5 to 8.5.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1981

Passive potassium transport in low potassium sheep red cells: dependence upon cell volume and chloride.

The major pathway of passive K influx (ouabain-insensitive) was characterized in low-K type (LK) red cells of sheep. 1. Passive K transport in these cells was highly sensitive to variations in cell volume; it increased threefold or more in cells swollen osmotically by 10%, and decreased up to twofold in cells shrunken 5-10%. Active K influx was insensitive to changes in cell volume. Three different methods for varying cell volume osmotically all gave similar results. 2. The volume-sensitive pathway was specific for K in that Na influx did not vary with changes in cell volume. 3. The volume-sensitive K influx was a saturable function of external K concentration. It was slightly inhibited by Na, whereas K influx in shrunken cells was unaffected by Na. 4. Passive K influx was dependent on the major anion in the medium in that replacement of Cl with any of six other anions resulted in a reduction of K influx by 50-80% (replacement of Cl by Br caused an increase in K influx). The activation of K influx by Cl followed sigmoid kinetics. 5. Passive K influx is inhibited by anti-L antibody. The antibody affected only that portion of influx which was Cl-dependent and volume-sensitve. Of the subfractions of the antibody, it is anti-L1 which inhibits passive K transport. 6. Pretreatment of cells with iodoacetamide reduced the sensitivity of K influx to cell volume in that the influx was reduced in swollen IAA-treated cells and increased in shrunken IAA-cells. 7. Intracellular Ca has no role in altering passive K transport in LK sheep cells. Therefore, the major pathway of passive K transport in LK sheep red cells is sensitive to changes in cell volume, specific for K, dependent on Cl, and inhibited by anti-L1 antibody, The minor pathway, observed in shrunken cells, has none of these properties.

Animals↗
PubMed1981

Glycine transport in human erythrocytes.

1. Glycine transport in human erythrocytes was resolved into five separate components of uptake. The first and major component of uptake was transport by a high-affinity (apparent Km 25 microM) Na+- and Cl- -dependent system. This system was specific for glycine, sarcosine and proline; Br- but not I- was able to substitute for Cl-. Uptake by this route was inhibited less than 20% by the loop diuretics, bumetanide and furosemide (10(-4) M), suggesting that it was distinct from the Cl- -dependent system responsible for Na+/K+ transport. Its properties closely resembled those of the gly transport system described previously in avian erythrocytes. 2. The second uptake route was transport by the Na+-dependent, Cl- -independent small neutral amino acid transport system (designated ASC). Neither NA+-dependent uptake route was present in sheep erythrocytes. 3. Two Na+-independent uptake mechanisms were also identified; first, uptake by the amino acid transport system (designated L), and secondly, SITS-sensitive uptake by the anion-exchange (band 3) transport mechanism (SITS is 4-acetamido-4'-iso-thiocyanatostilbene-2,2'-disulphonic acid, an effective inhibitor of anion transport by this route). Uptake by the latter route was increased markedly when fluxes were measured in isotonic SO42- medium or when the pH was increased. 4. At 0.2 mM extracellular glycine, the relative contributions of each of these uptake routes to the total glycine flux were 42, 11, 15 and 16% for the gly, ASC, L and band 3 systems, respectively. 5. Finally, there was a residual Na+-independent component of glycine uptake which contributed 16% of the total flux. With the exception of the gly system, all uptake routes showed a linear concentration dependence up to 2 mM-glycine.

Anions↗
PubMed1980

Amino acid transport in human and in sheep erythrocytes.

Amino acid transport was compared in human and in sheep erythrocytes. Kinetic studies established that human cells have three discrete amino acid transport systems, designated L, Ly+ and ASC. The L system is partially stereospecific, with a preference for large neutral amino acids. L-leucine has a threefold lower apparent Km and a twofold smaller Vmax than D-leucine. Alanine, cysteine and possibly dibasic amino acids are transported by this route, but with a low affinity. The Ly+ system is highly stereoselective, and specific for dibasic amino acids, including arginine. The ASC system is Na-dependent and selective for neutral amino acids of intermediate size. It has a particularly low apparent Km for cysteine and is stereospecific. Sheep erythrocytes lack these systems. Instead they possess an additional system (C system) responsible for the transport both of neutral and of dibasic amino acids, with cysteine as the optimal substrate. Although the substrate specificities of the human ASC and sheep C systems are similar, the sheep system does not require Na and has considerably higher apparent Km values. Dibasic amino acid transport (of lysine, but not of arginine) by the C system occurs with a low affinity.

Amino Acids↗
PubMed1980

Nucleoside transport in human erythrocytes. Apparent molecular weight of the nitrobenzylthioinosine-binding complex estimated by radiation-inactivation analysis.

Nitrobenzylthioninosine, a potent nucleoside-transport inhibitor, binds specifically to functional nucleoside transport sites. Irradiation of freeze-dried human erythrocyte membranes with high-energy electrons was used to estimate the apparent molecular weight of the nitrobenzylthioninosine-binding complex in situ. The nitrobenzylthioinosine-binding complex had an apparent mol.wt. of 122000.

Binding Sites↗