Properties of the chloride-ATPase fromLimonium salt glands: Activation by, and binding to, specific sugars

Summary

Attempts to separate membrane fractions enriched in Cl⁻-ATPase activity fromLimonium 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 0.1m 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.