Biomedical subjects

E Schweinzer

Publications and source records attributed to E Schweinzer.

7 recordsLinked to original sources

PubMed1996

Reduction of extracellular dehydroascorbic acid by K562 cells.

K562 erythroleukaemic cells produced ascorbate when incubated with dehydroascorbic acid. The reduction depended on the number of cells and on the concentration of dehydroascorbic acid. The observed rate consists of a high affinity (apparent Km 7 mu M, Vmax 3 center dot 25 pmol min-1 (10(6) cells)-1 and a low affinity component, which was non-saturable up to 1 mM of DHA (rate increase of 0 center dot 1 pmol min-1 (10(6) cells)-1 (1 mu M of DHA-1). The rate was dependent on temperature and was stimulated by glucose and inhibited by phloretin, N-ethylmaleimide, parachloro-mercuribenzoate and the noyltrifluoroacetone. Although uptake of DHA proceeded at a higher rate than its extracellular reduction, the generation of extracellular ascorbate from DHA cannot be accounted for by intracellular reduction and the release of ascorbate, since the latter was not linear with time and had an initial rate of approximately 3 pmol min-1 (10(6) cells-1). At a concentration of DHA of 100 mu M this is 25 per cent of the observed reduction.

Ascorbic Acid↗

PubMed1994

Transport of vitamin C in animal and human cells.

The transport systems of animal and human tissues for vitamin C are reviewed with respect to their properties. It emerges that pure diffusion plays only a very minor role while a variety of more or less specific transporters is found on cellular membranes. Although most tissues prefer the reduced ascorbate over the oxidized dehydroascorbic acid and have high-affinity transporters for it, there are several examples for the reversed situation. Special attention is given to similarity or identity with glucose transporters, especially the GLUT-1 and the sodium-dependent intestinal and renal transporters, and to the very widespread dependence of ascorbate transport on sodium ions. The significance of ascorbate transport for vitamin C-requiring and nonrequiring species as well as alterations in states of disease can be seen from ample experimental evidence.

Animals↗

PubMed1993

Monodehydroascorbate reductase activity in the surface membrane of leukemic cells. Characterization by a ferricyanide-driven redox cycle.

A transmembrane monodehydroascorbate reductase activity with a high affinity in the subpicomolar concentration range of the free radical can be measured at the surface of erythroleukemic cells using a ferricyanide-driven redox cycle. The activity is dependent on the membrane potential and can therefore only be found in intact cells. It is independent of the glutathione content of the cells. Thenoyltrifluoroacetone is an efficient inhibitor of the activity, whereas ouabain, monensin and tetraethylammonium show no effect. Cells are able to generate ascorbate from dehydroascorbic acid. This explains why both forms of vitamin C show practically the same affinity for the redox cycle but why it does not drive the redox cycle by itself because it is much slower and is not inhibited by thenoyltrifluoroacetone. The reductase activity is independent of the degree of differentiation of the leukemic cells.

Animals↗

PubMed1993

No enzymatic activities are necessary for the stabilization of ascorbic acid by K-562 cells.

We disprove that living cells stabilize ascorbate by the activity of a trans plasma membrane semidehydroascorbate reductase. The two processes show different specificities for both substrate and inhibitor. Not only cells but also cell-conditioned buffers stabilize ascorbate as long as compounds with molecular weights above 10 kDa are not removed. The effect is most probably due to chelation of traces of transition metals.

Ascorbic Acid↗

PubMed1993

Influence of conjugation of doxorubicin to transferrin on the iron uptake by K562 cells via receptor-mediated endocytosis.

The influence of conjugation of doxorubicin to holotransferrin on the receptor-mediated endocytosis of and on the iron uptake from transferrin was studied using K562 cells. 125I-labelled transferrin and doxorubicin-transferrin conjugates were used in the binding, dissociation, and ligand-exchange experiments at 0 degree C, and 59Fe,125I-labelled (double-labelled) ligands were used in the endocytosis, iron uptake, and recycling experiments at 37 degrees C. The binding affinity of conjugates was about half of that of transferrin. Binding of 125I-labelled ligands was blocked by both unlabelled ligands to the same degree, however, it was not blocked at all by an 8000-fold excess of doxorubicin. After saturation bindings, slightly more 125I-labelled conjugates dissociated from the surface of cells than transferrin. Exchange of 125I-labelled ligands for unlabelled ligands resulted in different EC50 values (defined as the concentration of unlabelled ligand at which half as much radioligand is exchanged for unlabelled ligand as would be exchanged at infinitely high concentration of unlabelled ligand under similar assay conditions). While transferrin exchanged transferrin with an EC50 value close to the binding affinity, conjugates exchanged conjugates with much lower efficiency. The heterolog exchange experiments yielded EC50 values inbetween the two extrema. For studying iron uptake, K562 cells were loaded with the double-labelled ligands either at 37 degrees C (endosome-loading only) or at 0 degree C (surface-loading only). Results obtained for the endocytosis of, the iron uptake from, and the recycling of double-labelled ligands indicate that (a) the rate of iron uptake is smaller from conjugates than from transferrin, (b) there are at least two parallel recycling processes for both ligand.receptor complexes, and (c) each time constant characterizing the different steps of iron uptake via receptor-mediated endocytosis is smaller for conjugates than for transferrin (or, the half times characterizing the different steps are higher for conjugates than for transferrin). Endocytosis and iron uptake were unaffected by free doxorubicin (12.5 microM) or colchicine (1 mM). Benzyl alcohol (30 mM) slowed down the rate of both endocytosis and iron uptake, while dithiothreitol (5 mM) decreased the rate of iron uptake and increased the rate of endocytosis. N-Ethylmaleimide (1 mM) completely stopped both endocytosis and iron uptake. The results suggest that the binding of conjugates to the surface of cells is governed by the binding of the transferrin part of conjugates to the transferrin receptor. However, conjugation of doxorubicin to transferrin seems to influence all properties of transferrin.(ABSTRACT TRUNCATED AT 400 WORDS)

Doxorubicin↗

PubMed1993

Binding of doxorubicin-conjugated transferrin to U937 cells.

Binding of transferrin (Trf) and its doxorubicin-conjugated forms (Conj) to U937 cells at 0 degrees C were compared using 125I-labelled Trf or Conj. The apparent binding affinity (Ka) of Conj to the surface of U937 cells was (1.9 +/- 0.4).10(8) l/mol; it is about 40% of that of Trf [(5.0 +/- 1.2).10(8) l/mol]. Binding of 125I-labelled ligands was blocked by the unlabelled ligands to the same degree, however, it was not blocked by a great excess of doxorubicin (Dox). N-ethylmaleimide caused about 10% inhibition while dithiothreitol was without effect. Dissociation of 125I-labelled ligands in the presence of different concentrations of unlabelled ligands (Trf and Conj in the all 4 variations) resulted in different R50 values (the concentration of the unlabelled ligand where 50% of the radiolabelled ligand was released). While Trf displaced Trf with an R50 value close to the binding affinity, Conj displacement by Conj occurred with much lower efficiency. The heterolog displacement experiments yielded R50 values in between the two extrema. These results suggest that 1) binding of Conj to the surface of cells is governed by the binding of the Trf part of Conj to the transferrin receptor, 2) -SH groups are not involved in the binding, and 3) a second interaction between the Conj and some constituent(s) of the plasma membrane may modify the binding of Conj in comparison to that of Trf.

Doxorubicin↗

PubMed1992

Ascorbate-mediated transmembrane electron transport and ascorbate uptake in leukemic cell lines are two different processes.

Transmembrane reduction of extracellular oxidants by K562 and U937 leukemic cells was stimulated by catalytic amounts of ascorbate or dehydroascorbate. This stimulation was not due to transport of ascorbate in different redox states in and out of the cells. The membrane redox cycle was strictly dependent on the presence of the cells at every stage, and showed high affinity for ascorbate with simple linear kinetics. Metabolic inhibitors and sulfhydryl reagents inhibited this stimulation. Ascorbate uptake was also dependent on oxidation, but in a very different manner and with much lower affinity for ascorbate. The uptake was non-saturable in the concentration range used. There was some release of ascorbate from the cells, which cannot account for an appreciable part of the reduction of extracellular electron acceptors.

Ascorbic Acid↗