Macromolecular characterization of muscle membranes. I. Proteins and sialic acid of normal and denervated muscle

Two major membrane fractions have been isolated from denervated and contralateral limb control rat muscle by a subcellular fractionation technique involving lithium bromide and potassium chloride extraction and differential and density gradient centrifugation. Membranes isolated from the initial nuclear pellet (Fraction I) contained Na⁺K⁺(Mg⁺⁺)ATPase (20 μmoles of P(i) per hour per mg), sialic acid (24 nmoles per mg), and Ca⁺⁺/Mg⁺⁺ATPase (19 μmoles of P(i) per hour per mg). With subfractionation of Fraction I the membranes enriched in Na⁺K⁺(Mg⁺⁺)ATPase and sialic acid (IM(L)) also had the highest specific activity of membrane protein iodination using extracellular lactoperoxidase. These membrane subfractions were separated from ones containing Ca⁺⁺/Mg⁺⁺ATPase activity (IM(H)). Membranes isolated from the post mitochondrial supernatant (Fraction II) contained Ca⁺⁺/Mg⁺⁺ATPase (22 μmoles of P(i) per hour per mg), Na⁺K⁺(Mg⁺⁺)ATPase (5 μmoles of P(i) per hour per mg), and sialic acid (10 nmoles per mg). With further subfractionation the Ca⁺⁺/Mg⁺⁺ATPase containing membranes [IIM(M) and IIM(H)] were separated from those most accessible to iodination by extracellular lactoperoxidase and containing Na⁺K⁺(Mg⁺⁺)ATPase and sialic acid (IIM(L) and IIL). Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed different protein profiles for Fractions I and II with polypeptide molecular weights ranging from 20,000 to 200,000. Both subfractions IM(L) and IIM(L) contained a single major glycoprotein species with an apparent molecular weight of approximately 110,000. These data would suggest that both lighter subfractions [IM(I) and IIM(L)D derived from the nuclear pellet (Fraction I) and the postmitochondrial supernatant (Fraction II), respectively, were enriched in surface membranes. The heavier subfraction (IIM(H)) from the postmitochondrial supernatant was enriched in membranes derived from sarcoplasmic reticulum. Following 1 week of denervation no change was observed in either electron micrographs or sodium dodecyl sulfate polyacrylamide gels of the membrane fractions. The ATPase and sialic acid of Fraction I remained unaltered while a 2 fold increase was noted in the Ca⁺⁺/Mg⁺⁺ATPase (51 μmoles of P(i) per hour per mg) of heavier membranes of Fraction II and sialic acid (20 nmoles per mg) of ligher membranes of Fraction II.