PubMed HealthSearch

Biomedical subjects

T J Simons

Publications and source records attributed to T J Simons.

25 records · Page 2Linked to original sources

The preparation of human red cell ghosts containing calcium buffers.

1. Ca buffers may be introduced into human red cells by reversible haemolysis. The resealed ghosts retain Ca and chelating anions in the same ratio as in the haemolysing solution, enabling the intracellular Ca2+ concentration to be calculated simply. 2. The passive permeability of the ghosts to Na and Cl is unaffected by intracellular Ca2+ concentrations in the 10(-8)-10(-4) M range, whereas the K permeability is greatly increased at concentrations above 10(-7) M. 3. These preparations enable Ca-dependent K movements to be studied under stable conditions. When the ghosts contain about 5 X 10(-6) M-Ca2+, over 96% of K transport occurs via the Ca-sensitive route.

Buffers

Calcium-dependent potassium exchange in human red cell ghosts.

1. The properties of the Ca-dependent K transport system of human red cell ghosts have been examined under equilibrium exchange conditions. 2. K transport is stimulated half-maximally by about 0-4muM-Ca2i+ or 5muM-Sr2i+, but much higher concentrations of Ba2i+ give only slight stimulation. Mg is a weak antagonist to Ca. 3. The free Ca2+ concentration in human red cells is estimated to be below 0-25muM. 4. The curve relating the rate of K transport to the intracellular Ca2+ concentration is complicated and suggests that internal Ca acts at three or more sites. 5. K, Rb and possibly Cs ions are transported by the Ca-dependent system. Under comparable conditions the relative rates are 1(K):1-5(Rb): less than 0-05(Cs). 6. No Ca-dependent transport of Na, Li or choline could be detected. If Na is transported, it must be at less than 1/40 of the rate of K. 7. The rate of K transport is almost linearly related to the K concentration in the 0-200 mM range, but the curve is sigmoid close to the origin. 8. Intracellular, but not extracellular Na inhibits K transport, in a way that suggests competition with K at more than one site. 9. These results suggest that the transport system has a complex mechanism.

Barium

The interaction of ATP-analogues possessing a blocked gamma-phosphate group with the sodium pump in human red cells.

1. The (Na++K+)-ATPase of red cell membranes is unable to hydrolyse ATP-analogues in which the oxygen atom linking the beta- and gamma-phosphate groups is replaced by a minusCH2minus or minusNH-bridge. 2. In resealed ghosts both these ATP-analogues support K:K exchange but not Na:K exchange. ATP supports both modes of operation of the sodium pump, whereas neither occurs without any nucleotide. 3. These results support the hypothesis that ATP is needed as a cofactor for K:K exchange to occur, and make it extremely unlikely that phosphorylation from ATP is involved.

Adenine Nucleotides

Potassium: potassium exchange catalysed by the sodium pump in human red cells.

1. When red cells were so depleted of Na that Na:K exchange had almost ceased, the ouabain-sensitive K efflux seen in K-containing media was accompanied by an almost equal ouabain-sensitive K influx.2. This suggests that the Na pump in these cells was carrying out a one-for-one K:K exchange across the erythrocyte membrane.3. 30-40% of the (42)K efflux from resealed ghosts was sensitive to ouabain when the ghosts contained 1 mM-ATP, 2 mM orthophosphate, 10 mM-K and less than 1 mM-Na, and the suspending medium contained 10 mM-K and 0-Na, choline being the predominant cation.4. In resealed ghosts, the rate of K:K exchange saturated as internal K was increased, and was half-maximal at about 10 mM-K.5. When internal ATP was maintained with a phosphocreatine:creatine phosphokinase regenerating system, K:K exchange saturated as internal ATP was increased, and was half-maximal at about 100 muM-ATP.6. The rate of K:K exchange did not depend on whether the ADP concentration was roughly the same as the ATP concentration or very much less, suggesting that ADP did not affect the rate of K:K exchange.7. GTP, ITP and UTP were unable to substitute for ATP in supporting K:K exchange. CTP was a poor substitute.8. There was no evidence to support the hypothesis that K:K exchange is accompanied by a ouabain-sensitive hydrolysis of ATP.9. Internal Na was a strong inhibitor of ouabain-sensitive K efflux from ghosts containing 9 mM-K. 4 mM-Na was sufficient to produce 90% inhibition.10. The rate of K:K exchange depended on the orthophosphate concentration inside the ghosts (confirming Glynn, Lew & Lüthi, 1970). The curve obtained suggested that the rate was half-maximal at about 1.7 mM orthophosphate.11. These experiments suggested that inhibition by internal K is an important factor affecting the Na efflux from intact red cells. Experiments measuring Na:K exchange as a function of internal Na in low-K ghosts supported this hypothesis.12. The significance of these findings is discussed.

Adenosine Triphosphate