PROPERTIES OF THE CHLORIDE-ATPASE FROM LIMONIUM SALT-GLANDS - ACTIVATION BY, AND BINDING TO, SPECIFIC SUGARS

Attempts to separate membrane fractions enriched in Cl--ATPase activity from Limonium leaf microsomes were hampered because, it seemed, the microsomal membranes were aggregated in clumps. We found hemagglutination activity, specific for N-acetylgalactosamine and to a lesser extent galactose, in the soluble phase of the homogenate, and we were able to prevent membrane aggregation by adding galactose to the microsomes. We discovered that the Cl--ATPase activity of the microsomes was increased by galactose and to an even greater extent by N-acetylgalactosamine. We report that the Cl--ATPase binds to galactosamine-sepharose, from which it can be eluted in 0.1 m galactose, i.e., the enzyme is associated with a saccharide-binding site similar to that of the hemagglutinins. This procedure results in a 100-fold enrichment of the Cl--ATPase activity and represents a new way of purifying a membrane-bound enzyme from a heterogeneous membrane preparation in one step. Enzyme isolated by affinity chromatography of Triton-solubilized membranes was essentially free of other ATPase and accounted for a substantial proportion (sometimes all) of the Cl--ATPase of the microsomes. This purified preparation of the enzyme shows N-acetylgalactosamine-specific hemagglutination activity. However, we can show that the Cl--ATPase and the hemagglutinins are different entities. Thus, material isolated in the same way from salt-free plants showed hemagglutination but not Cl--ATPase activity. Also, the hemagglutinins, but not the Cl--ATPase, will bind to galactosaminesepharose in the absence of ATP. This is the first report of enzyme activity associated with a carbohydrate receptorspecific protein. Possible roles for saccharide-binding in the control, assembly, and orientation of the chloride-pump are discussed. © 1979 Springer-Verlag New York Inc.