The Na+-K+-ATPase consists of α and β subunits proposed to function as an α-β heterodimer. Skeletal muscle is characterized by expression of α1, α2, β1, and β2 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 [3H]ouabain binding. The results indicate that in kidney microsomes, which express predominantly α1 and β1 isoforms, the α:β subunit molar ratio is approximately 1:1. In brain microsomes, the sum of all α (α1, α2, and α3) and all β (β1 and β2) subunits also yielded a molar ratio of approximately 1:1. In contrast, in red (oxidative) skeletal muscles, the all α:β subunit ratio was 0.2 in plasma membranes and 0.4 in intracellular membranes. The ratio of α2 subunits to α1 subunits ranged from 1.6 in surface membranes to up to 7 in internal membranes, while the β1 subunits exceded the β2 subunits by ≃4-fold in all membrane fractions. Thus, intracellular membranes of red skeletal muscles contain primarily α2 and β1 subunits. When these intracellular membranes were further subfractionated by velocity gradient centrifugation, the α2:β1 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 β1 subunits without a change in the concentration of α2 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 β to α subunits may serve to regulate the catalytic activity of the Na+-K+-ATPase in skeletal muscle.
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