Properties of the chloride-ATPase from Limonium salt glands: activation by, and binding to, specific sugars

Hill, B.S.; Hanke, D.E.

Journal of Membrane Biology 51(2): 185-194

1979


ISSN/ISBN: 0022-2631
PMID: 160948
DOI: 10.1007/bf01869168
Accession: 068522889

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Abstract
Attempts to separate membrane fractions enriched in Cl--ATPase activity from L. vulgare leaf microsomes were hampered because, it seemed, the microsomal membranes were aggregated in clumps. Hemagglutination activity, specific for N-acetylgalactosamine and to a lesser extent galactose, was found in the soluble phase of the homogenate, and membrane aggregation was prevented by adding galactose to the microsomes. The Cl--ATPase activity of the microsomes was increased by galactose and to an even greater extent by N-acetylgalactosamine. 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 1 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. The Cl--ATPase and the hemagglutinins are different entities. 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 galactosamine-sepharose in the absence of ATP. This is the 1st report of enzyme activity associated with a carbohydrate receptor-specific protein. Possible roles for saccharide-binding in the control, assembly, and orientation of the chloride-pump are discussed.