Studies on the glycoprotein component of (Na+ +K+)-ATPase from dog fish salt gland. Binding to concanavalin A and removal of sialic acid by neuraminidase.

1. The presence of concanavalin A binding sugars in the glycoprotein component of a partially purified (Na++K+) ATPase preparation from dog fish salt gland was demonstrated by binding of a Triton X-100 extract of the enzyme and isolated glycoprotein to concanavalin A-Sepharose, and by binding of membrane-associated enzyme to free concanavalin A. 2. The binding of concanavalin A to the glycoprotein in both membrane-associated enzyme and a Lubrol extract of the enzyme had no effect on (Na++K+)-ATPase activity. Binding was completely inhibited by methyl-alpha-mannoside. Also, enzyme activity was not affected by removal of 50% of glycoprotein sialic acid by neuraminidase. These results suggest that the carbohydrate moiety of the glycoprotein does not play a catalytic role in the (Na++K+)-ATPase. 3. When a Triton X-100 extract of (Na++K+)-ATPase was chromatographed on concanavalin A-Sepharose, 37% of total protein was bound to the column and eluted by methyl-alpha-mannoside. The bound fraction was free of lipid, and contained not only the glycoprotein but also the large protein which is the catalytic subunit of the enzyme, and small amounts of other membrane derived proteins. The ratio of large protein to glycoprotein, as measured by the relative Coomassie blue absorbance of the two proteins separated by gel electrophoresis, was the same in the bound fraction as in the membrane. These results suggest that the glycoprotein and lareg protein are either associated together in the membrane or become associated during lipid replacement by Triton.