Transport systems of sheep reticulocytes and their changes during cell maturation

Maturation and aging-associated changes in membrane transport of sheep reticulocytes of the high-K('+) and low-K('+) genotypes were studied. The results indicate that during short term (less than 10 days) in vitro maturation, there is a progressive decrease in ouabain-sensitive Na('+),-K('+)-pump activity (('86)Rb('+) uptake in low-K('+) cells) and Na('+)-ATPase activity (high-K('+) cells). However, during long term (several weeks) in vivo maturation in which HbC was used as a marker of newly formed reticulocytes, kinetic changes, as well as a decline in total activity, were observed.The effect of metabolic depletion on the maturation-associated loss of two membrane transport functions was also studied using in vitro incubation. Both Na('+)-dependent glycine transport and the Na('+),K('+)-pump, estimated from measurements of the number of ('3)H-ouabain binding sites per cell, were decreased during maturation. ATP also enhanced the decrease in both activities when a 'reconstituted' vesicle system comprised of inside-out vesicles plus cell lysates was incubated at 37(DEGREES)C. Associated with this ATP-dependent loss of activity was an increase in the amount of concomitantly measured ninhydrin-positive material. It is concluded that the loss of certain functions during reticulocyte maturation is retarded by metabolic depletion.Membrane vesicles of distinct sidedness were prepared from sheep reticulocytes. Using these vesicles the Na('+)-dependent glycine transport system was found to be symmetrical with respect to: (a) the Na('+) dependency of glycine transport, (b) the ability to accumulate glycine against a concentration gradient, (c) the Na('+): glycine stoichiometry, i.e. two Na('+) ions are transported per molecule of glycine, and (d) the apparent Michaelis-Menten constants for Na('+) and glycine