Purified lamb kidney Na+, K+-ATPase, consisting solely of the Mτ = 95,000 catalytic subunit and the Mτ- 44,000 glycoprotein, was solubilized with Triton X-100 and incorporated into unilamellar phospholipid vesicles. Freeze-fracture electron microscopy of the vesicles showed intramembranous particles of approximately 90–100 Å in diameter, which are similar to those seen in the native Na+,K+-ATPase fraction. Digestion of the reconstituted proteins with neuraminidase indicated that the glycoprotein moiety of the Na+,K+-ATPase was asymmetrically oriented in the reconstituted vesicles, with greater than 85% of the total sialic acid directed toward the outside of the vesicles. In contrast, in the native Na+,K+-ATPase fraction, the glycoprotein was symmetrically distributed. Purified glycoprotein was also asymmetrically incorporated into phospholipid vesicles using Triton X-100 and without detergents as described by [16.], J. Biol. Chem. 250, 9206–9214). The glycoprotein-containing vesicles were 500–1000 Å in diameter, unilamellar, and, in contrast to the vesicles containing the Na+,K+-ATPase, did not contain the 90- to 100-Å intramembranous particles. These results indicate that the intramembranous particles observed in the native Na+,K+-ATPase and in the reconstituted Na+,K+-ATPase are not due to the glycoprotein alone, but represent either the catalytic subunit, or the catalytic plus the glycoprotein subunit
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