Biomedical subjects
Publications and source records attributed to J C Ellory.
1. There is substantial evidence for a volume-sensitive KCl cotransport system in young human RBC. 2. The KCl cotransport system becomes latent on cell maturation. 3. There is a correlation between the activation of the KCl cotransporter by either pressure, NEM, ghosting or in certain anemias with disc/cup cell shape change. 4. The stretch-activated KCl transporter may be coupled to some component of the cell cytoskeleton.
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Isolated trout hepatocytes exposed to hypotonic Hank's medium (isotonicity x 0.70) swelled to 1.17 times the control volume after 3 min; by 15 min the cell volume had returned to normal. The ouabain-insensitive K+ uptake increased, indicating an immediate rise in K+ membrane permeability. As indicated by analysis of cellular contents, the regulatory volume decrease (RVD) was ensured by a release of intracellular K+. Na+ was not implicated in this mechanism. This potassium permeability induced by hypotonic shock was transient (maximum at 6 min), insensitive to blocking agents of voltage- and Ca2+-dependent K+ channels, and chloride-dependent. This result, together with a time-course of Cl- uptake similar to that of K+, suggests a K+/Cl- cotransport mechanism. This cotransport is inhibited by high furosemide concentrations (10(-3) mol l-1) but not by bumetanide (10(-4) mol l-1) or piretanide (10(-4) mol l-1).
1. The action of sodium periodate and neuraminidase on active and passive K+ transport in low-potassium type (LK) sheep red cells was investigated in relation to the contribution of the Lp and Ll antigens. 2. Active K+ transport in LK sheep red cells was not affected by treatment with sodium periodate (2 mM), or with neuraminidase. 3. Passive K+ transport in LK sheep red cells was increased by sodium periodate treatment in a concentration-dependent manner. The increase was not Cl- dependent, and so differed from the increased passive K+ uptake resulting from N-ethylmaleimide treatment. 4. HK sheep red cells treated with sodium periodate showed small increases in passive K+ uptake, and N-ethylmaleimide treatment used sequentially with sodium periodate resulted in further small increases in passive K+ uptake. 5. In LK sheep red cells the stimulation of active K+ transport by anti-L was impaired by 50% in cells treated with sodium periodate (2 mM) and was slightly lowered in cells treated with neuraminidase. 6. In LK sheep red cells inhibition of passive K+ transport by anti-L was not impaired by sodium periodate treatment (2 mM), or by neuraminidase treatment.
The quaternary structure of the membrane-bound mannitol permease (EIIMtl) of the bacterial phosphotransferase system in Escherichia coli has been investigated in the membrane by using the radiation inactivation method. The experiments reveal two distinct but interconvertible forms of the permease. The first state is a dimer, and the second state consists of a less active higher molecular weight complex involving the dimer. The equilibrium between these two forms in the membrane can be shifted by changing the pH. At pH 8.1 the dimer is the dominant form. Decreasing the pH results in increased binding of a regulatory protein to the dimer, thus increasing the amount of the higher molecular weight form involving the dimer. Cross-linking EIIMtl in situ, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, resulted in the formation of two cross-linked forms. One is the dimer, and the other has a higher molecular weight. Two-dimensional electrophoresis using a reversible cross-linker revealed no other protein except EIIMtl in these complexes.
The monoamine transporter of chromaffin granule membrane has two distinct high-affinity binding sites for tetrabenazine and reserpine, which can be assayed by [3H]dihydrotetrabenazine and [3H]reserpine binding, respectively. The functional molecular mass of the components bearing these sites has been investigated by the radiation inactivation technique. The decline of [3H]dihydrotetrabenazine binding activity with increasing radiation doses followed a single exponential, from which a functional molecular mass of 68 kDa was derived for tetrabenazine binding sites. [3H]Reserpine binding activity declined in a more complex way; however, under conditions where high-affinity reserpine binding sites were specifically assayed, the decline was also exponential, corresponding to a functional molecular mass of 37 kDa for these sites. The figures obtained for high-affinity tetrabenazine and reserpine binding sites are consistent with previous values obtained by photoaffinity of tetrabenazine and serotonin binding sites, respectively. It is thus concluded that the monoamine transporter has an oligomeric structure. By the radiation inactivation technique, cytochrome b561 and dopamine beta-hydroxylase have functional molecular masses of 25 and 123 kDa, respectively. The latter value might be attributed to the dimeric form of the enzyme.
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1. The potassium concentration in red cells of 21 Barbary sheep showed a bimodal distribution, with five animals of LK type (K+ conc. 30-45 mM) and 16 of HK type (K+ conc. 80-95 mM). 2. Evidence is presented that both Lp and Ll antigens are present on LK Barbary sheep red cells. 3. Active K+ transport in LK Barbary sheep red cells was stimulated 3-5 fold by sheep and goat anti-L. 4. Active K+ transport in HK Barbary sheep red cells was higher than in LK red cells. Five out of six HK animals tested showed no stimulation of active K+ transport with anti-L. One HK animal (2BA2) showed some stimulation of active K+ transport, and also absorbed some anti-L from antisera, suggesting that Lp antigen is present on these red cells. 5. Ouabain-sensitive ATPase in membranes from HK and LK Barbary sheep red cells showed kinetics characteristic of HK and LK membranes of domestic goats and sheep; the ATPase of LK Barbary sheep membranes sensitized with anti-L was stimulated 2-fold due to an alteration in the internal sodium and potassium affinities in favour of sodium.
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The effect of procedures known to stimulate the volume-sensitive KC1 pathway has been studied in human red cells. This system is functional in young, but not mature cells and may mediate large fluxes in individuals with large fractions of reticulocytes. The pathway is apparently latent in mature cells, but may be unmasked by certain procedures suggesting that the transporter is not lost from the membrane during cell maturation.
'Young' human red cells are shown to possess a specific K+ pathway which is dependent on Cl- and sensitive to cell volume. This system was latent in 'mature' cells but was revealed by high hydrostatic pressure. This suggests the pathway is functionally active in 'young' cells but becomes masked with cell maturation.
The human erythrocyte nucleoside and glucose transporters, identified previously as band 4.5 peptides (apparent Mr 66 000-45 000) on SDS-polyacrylamide gels, have been characterized in situ by radiation inactivation analysis. Target size analysis of lyophilized membranes indicates an apparent Mr of 110 000 +/- 12 000 and 124 000 +/- 11 000 for the nucleoside and glucose carriers, respectively. These data suggest that both transporters exist in the membrane as dimers.
The effects of high hydrostatic pressure (up to 400 ATA) on the 'passive' (defined as ouabain + bumetanide + EGTA-insensitive) influx and efflux of radiotracer cations (K+ Rb+, Na+, Cs+) has been studied in human red cells suspended at different medium tonicities giving altered cell volumes. Under all conditions studied, cation permeability was raised at pressure, and at least two distinct components were found to comprise this flux. Thus, increasing pressure caused a generalized increase in cation permeability which was unaffected by the anion present, demonstrated linear concentration dependence, and was reduced with cell swelling, and stimulated a specific KCl pathway which was Cl- dependent, demonstrated saturation kinetics with raised [K]0 and was increased with cell swelling. High hydrostatic pressure caused a significant alteration to red cell morphology from the normal biconcave disc to cup-shaped forms and it is proposed that this is associated with the unmasking of the volume-sensitive KCl system.
Electron inactivation analysis with 16 MeV electrons was used to determine the functional target size of a number of commonly studied lysosomal hydrolases. Observed values ranged from a low of 62 000 +/- 4000 Da for beta-galactosidase to a high of 200 000 +/- 17 500 Da (mouse beta-glucuronidase). One group of lysosomal hydrolases (N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, alpha-galactosidase, beta-mannosidase, beta-glucosidase, arylsulphatase A and sphingomyelinase) had target sizes in the range 100 000-120 000 Da, whereas alpha-glucosidase and alpha-fucosidase exist as complex multimers in the 150 000-160 000 Da range. Analysis of freeze-dried cell material showed little evidence of species (mouse versus human) variation in the functional size of most lysosomal hydrolases with the exception of beta-glucuronidase. Our findings suggest the potential usefulness of lysosomal hydrolases as endogenous marker enzymes in studies where the target size of proteins of unknown molecular mass is to be determined.
The bumetanide-sensitive uptake of Na+, K+(Rb+) and Cl- has been measured at 21 degrees C in ferret red cells treated with (SITS + DIDS) to minimize anion flux via capnophorin (Band 3). During the time course of the influx experiments tracer uptake was a first-order rate process. At normal levels of external Na+ (150 mM) the bumetanide-sensitive uptake of K+ was dependent on Cl- and represented almost all of the K+ uptake, the residual flux demonstrating linear concentration dependence. The uptake of Na+ and Cl- was only partially inhibited by bumetanide indicating that pathways other than (Na + K + Cl) cotransport participate in these fluxes. The diuretic-sensitive uptake of Na+ or Cl- was, however, abolished by the removal of K+ or the complementary ion indicating that bumetanide-sensitive fluxes of Na+, K+ and Cl- are closely coupled. At very low levels of [Na]o (less than 5 mM) K+ influx demonstrated complex kinetics, and there was evidence of the unmasking of a bumetanide-sensitive Na+-independent K+ transport pathway. The stoichiometry of bumetanide-sensitive tracer uptake was 2Na:1K:3Cl both in cells suspended in a low and a high K+-containing medium. The bumetanide-sensitive flux was markedly reduced by ATP depletion. We conclude that a bumetanide-sensitive cotransport of (2Na:1K:3Cl) occurs as an electroneutral complex across the ferret red cell membrane.
Radioisotopic cation transport studies are described in a family whose erythrocytes had previously been found to show an abnormal net efflux of potassium when cooled to room temperature. This net efflux effect, which was inherited as an autosomal dominant trait, was associated with a few target cells on the blood film and a mild compensated haemolytic state. Measurements of intracellular electrolyte concentrations, cell water and of Na+ and K+ transport rates across the membrane at 37 degrees C were consistent with a diagnosis of mild hereditary xerocytosis. Studies of cation transport in the temperature range 20-37 degrees C revealed that the fluxes attributable to the Na+-K+ pump showed a temperature dependence comparable with that in normal cells, but that the ouabain plus loop-diuretic insensitive fluxes of K+, which probably represent the 'passive diffusional leak' to K+, were less sensitive to temperature than normal over the range 20-37 degrees C. These findings were held to account for the net efflux effect previously reported.