Date of Graduation

1996

Document Type

Thesis

Abstract

The sodium pump activity declines rapidly after denucleation of erythroblastic cells. To investigate whether this decrease in the rat erythroblastic cells is due to changes in isoforms of the {dollar}\\alpha{dollar} and {dollar}\\beta{dollar} subunits of the Na,K-ATPase during maturation, techniques of cryoultramicrotomy and immunoelectron microscopy with protein-A-gold were used. Specific antisera against various isoforms of the {dollar}\\alpha{dollar} and {dollar}\\beta{dollar} subunits of rat Na,K-ATPase were used to label the bone marrow and red blood cells of rats. Three types of cells of the erythroid series at different stages of maturation were studied: erythroblasts, reticulocytes and mature red blood cells. It was found that all the tested isoforms of the {dollar}\\alpha{dollar} subunit ({dollar}\\alpha1,\\ \\alpha2{dollar} and {dollar}\\alpha{dollar}3) and the {dollar}\\beta{dollar} subunit ({dollar}\\beta1{dollar}, and {dollar}\\beta{dollar}2) are present in these cells. The {dollar}\\alpha{dollar}3 isoform is the major isozyme in the cytoplasm, whereas {dollar}\\alpha{dollar}1 is the most abundant isoform on the cell membrane. The {dollar}\\beta{dollar}1 and {dollar}\\beta{dollar}2 isoforms are much less abundant than the {dollar}\\alpha{dollar}1 or {dollar}\\alpha{dollar}3 isoforms, indicating that there is no coordinate synthesis of the {dollar}\\alpha{dollar} and {dollar}\\beta{dollar} subunits. During maturation of the cells, there is a transient increase in the cytoplasmic subunits, particularly the {dollar}\\alpha{dollar}1 and {dollar}\\alpha{dollar}3 isoforms, in reticulocytes. The reason for this increase is not clear. This increase could be a transient response to the increase in intracellular sodium during denucleation. The numbers of these isoforms decrease drastically in the mature red blood cells. Changes of the {dollar}\\alpha\\beta{dollar} complexes, which may represent the fully assembled pump units, were studied by labeling the two different subunits with two different sized protein-A-gold particles (5 and 10 nm). Although there are some {dollar}\\alpha1\\beta{dollar}1 complexes in the cytoplasm of reticulocytes, their number on the membrane decreases continuously from erythroblast to mature red blood cells. The {dollar}\\alpha{dollar}3 isoforms are trapped in the endoplasmic reticulum, probably due to not having enough {dollar}\\beta{dollar} subunits to complex with them. Therefore the reduction in sodium pump activity during maturation of erythroblastic cells is referable to a decrease in the {dollar}\\alpha1\\beta{dollar}1 complex on the cell membrane.

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