Comparative kinetic properties of tissue-specific Na,K-pumps

The catalytic $ alpha$ subunit of the heterodimeric Na,K-ATPase comprises three distinct isoforms which are expressed in a tissue-specific manner. For example, the $ alpha sb1$ isoform can be detected in virtually all mammalian tissues, whereas the appearance of the $ alpha sb2$ and $ alpha sb3$ isoforms is more restricted to particular tissues such as muscle and nervous tissue. Previous comparative functional studies of Na,K-ATPases isolated from various tissues indicated that there are differences, including apparent cation affinities, among these enzymes. While these differences often appear to correlate with the presence of distinct isozymes, their precise molecular bases remain to be determined. Moreover, certain studies suggest that the behavior of the same isoform can vary from tissue to tissue (e.g., the erythrocyte versus the kidney, both of which contain only the $ alpha sb1$ isoform). An hypothesis that may explain these observations is that the cell-specific membrane environment influences Na,K-ATPase activity. To investigate this possibility, polyethylene glycol-mediated membrane fusion was used to deliver pumps from high-specific-activity microsomes derived from various tissues into mammalian erythrocyte membranes. The success of this methodology was verified using two distinct experimental systems. In the first system, rabbit sarcoplasmic reticulum Ca-ATPase was delivered into human erythrocyte membranes. Cellular Ca$ sp{2+}$ uptake fueled by extracellular ATP was used as a measure of the functional delivery of the Ca-ATPase into these membranes. In the second system, ATP- and cardiac glycoside-dependent rubidium fluxes verified the functional delivery of axolemma or kidney Na,K-ATPases into mammalian erythrocytes. Among these studies was a series of experiments demonstrating that the L$ rm sb{p}$-antigen of sheep erythrocyte membranes is a distinct membrane component that interacts with and alters the behavior of rat kidney pumps fused into LK sh