AJP - Cell Physiology, Vol 259, Issue 2 C241-C250, Copyright © 1990 by American Physiological Society
Decrease in Na(+)-K(+)-ATPase associated with maturation of sheep reticulocytes
R. Blostein and E. Grafova
Department of Medicine, McGill University, Montreal, Quebec, Canada.
Na(+)-K(+)-ATPase of immature and mature sheep red blood cells of both the
high-K+ and low-K+ genotype and of immature cells matured in vitro was
detected using polyclonal antiserum to purified sheep kidney
Na(+)-K(+)-ATPase. This antiserum detects both alpha (alpha 1)- and alpha +
(alpha 2 and/or alpha 3)-isoforms of the catalytic subunit as well as the
beta-subunit of brain and kidney Na(+)-K(+)-ATPase. After sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and immunoblotting, a single
major immunologically reactive component corresponding to the alpha-subunit
was detected in membranes of immature and mature cells from sheep of both
genotypes. Cells separated according to increasing density showed a
corresponding decrease in ouabain binding sites on the cells and
Na(+)-activated ATP hydrolysis of membranes isolated from the cells. A
progressive decrease in immunologically reactive alpha-subunit was also
observed. After in vitro culture of reticulocytes, reduction in ouabain
binding to the cells was also associated with loss in alpha-subunit. As
well, appearance of immunologically reactive alpha-subunit was detected in
membranous material shed into the incubation medium, accounting for a
fraction (less than or equal to 30%) of the material lost from the cells.
Proteolytic sensitivity of the alpha-subunit indicates that, in this
material, the cytoplasmic surface of the enzyme is exposed to the medium.
The shed material was largely devoid of function as evidenced in little, if
any, Na(+)-dependent phosphorylation of Na(+)-K(+)-ATPase. The existence in
reticulocytes of an intracellular pool of ouabain binding sites was
indicated by the transient appearance on the cell surface of ouabain
binding sites after rapid ATP depletion and also after addition of
chloroquine to cells during culture. Taken together, these findings
indicate that the maturation-associated loss of sodium pump protein
involves, at least partly, energy-dependent endocytosis and, presumably,
processing whereby inactivation of function occurs as well as release of
pump protein into the extracellular milieu.
