ISSN:
1432-1424
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01869168
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