Characterization of a microsomal fraction from rabbit parotid gland: Partial separation of alkaline phosphatase from Na,K-ATPase

Subfractionation of the 140,000 g_max pellet from the rabbit parotid gland revealed 3 fractions. Both linear and discontinuous sucrose-density gradients were used to separate a region of high-density particles with considerable mitochondria, a middle region rich in Na⁺,K⁺-ATPase (sodium-potassium activated adenosine triphosphatase) and a low-density (top) region with elevated alkaline phosphatase. The top and middle fractions appeared to represent subfractions of plasma-membrane enriched membranes. Treatment of these 2 gradient fractions with low concentrations of sodium dodecyl sulphate confirmed that the middle fraction was preferentially enriched in Na⁺,K⁺-ATPase. Thus, alkaline phosphatase-enriched membranes are at least partially separable from Na⁺,K⁺-ATPase-enriched membranes. Separation of alkaline phosphatase from Na⁺,K⁺-ATPase may be an important purification step in future work involving isolation of plasma membranes of secretory cells, as both alkaline phosphatase and Na⁺,K⁺-ATPase have been used as plasma-membrane markers in the parotid gland. The present findings demonstrate that considerable caution should be exercised in defining the purity of plasma-membrane fractions from the parotid gland in view of the heterogeneity of the plasma-membrane enriched fraction.