The molar ratios of alpha and beta subunits of the Na+-K+-ATPase differ in distinct subcellular membranes from rat skeletal muscle

The Na+-K+-ATPase consists of alpha and beta subunits proposed to function as an alpha-beta heterodimer. Skeletal muscle is characterized by expression of alpha 1, alpha 2, beta 1, and beta 2 subunit isoforms. The relative molar proportions of each subunit or each protein isoform are not known, yet their subcellular distribution and expression in muscles of different fiber types are markedly different. In this study, the molar ratio of each pump subunit isoform was measured in purified membranes from skeletal muscle and compared with those in kidney and brain microsomes. Recombinant proteins were used as standards to quantitate each isoform by immunoblotting in combination with measurements of [H-3]ouabain binding. The results indicate that in kidney microsomes, which express predominantly alpha 1 and beta 1 isoforms, the alpha:beta subunit molar ratio is approximately 1:1. In brain microsomes, the sum of all alpha (alpha 1, alpha 2, and alpha 3) and all beta (beta 1 and beta 2) subunits also yielded a molar ratio of aproximately 1:1. In contrast, in red (oxidative) skeletal muscles, the all alpha:beta subunit ratio was 0.2 in plasma membranes and 0.4 in intracellular membranes. The ratio of alpha 2 subunits to alpha 1 subunits ranged from 1.6 in surface membranes to up to 7 in internal membranes, while the beta 1 subunits exceded the beta 2 subunits by approximate to 4-fold in all membrane fractions. Thus, intracellular membranes of red skeletal muscles contain primarily alpha 2 and beta 1 subunits. When these intracellular membranes were further subfractionated by velocity gradient centrifugation, the alpha 2:beta 1 subunit ratio was 0.5 in the faster migrating (larger) membranes and 1.0 in the slower migrating (smaller) ones. This was due to a progressive decrease in abundance of the beta 1 subunits without a change in the concentration of alpha 2 subunits per unit protein. The Na+-K+-ATPase hydrolytic activity was higher in the larger vesicles than in the smaller ones along the sucrose gradient. These results suggest that the ratio of beta to alpha subunits may serve to regulate the catalytic activity of the Na+-K+-ATPase in skeletal muscle.