Abstract
Extracts of Dictyostelium discoideum grown axenically in a chemically defined medium were evaluated for binding to discoidin I and discoidin II, endogenous lectins of this slime mold. Binding activity was measured by competitive inhibition of 125I-lactosyl-bovine serum albumin binding to the immobilized lectins. With the solubilization procedure used extracts of vegetative cells and of early aggregates had no significant inhibitory activity, but an abundant discoidin-binding substance was detected in late aggregates and fruiting bodies. This material was purified by ethanol and acid precipitation followed by precipitation with discoidin. It is a polysaccharide composed of 77% galactose, 15% N-acetylgalactosamine, 5% glucose, and 3% N-acetylglucosamine and may be a biologically functional ligand for the slime mold lectins, in particular discoidin II. Use of axenic cells was critical in these experiments, since extracts of Escherichia coli and Klebsiella aerogenes, commonly used as food for D. discoideum, were found to contain substances that react with discoidin. This would complicate isolation of endogenous discoidin ligands from cells raised on bacteria.
Highlights
MATERIALS AND METHODSFound to contain substances that react with discoidin. This would complicate isolation of endogenous discoidin ligandsfrom cells raised on bacteria
It is a polysaccharide composed of 77%galactose, 15% N-acetylgalactosamine, 5%glucose, and 3%N-acetylglucosamine and may be a biologically functional ligand for the slime mold lectins, in particular discoidin galactose and N-acetylgalactosamine andclosely resembles a spore coat polysaccharide previously identified [7,8,9]
Dictyostelium discoideum produces two isolectins, discoidin I and discoidin 11, which are distinguished by isotrient medium with streptomycin sulfate
Summary
Found to contain substances that react with discoidin. This would complicate isolation of endogenous discoidin ligandsfrom cells raised on bacteria. The dialysate, which is free of macromolecules that react with discoidin, was autoclaved and innoculated withAX3 cellsgrown in defined medium To obtain differentiatedcells they were harvested from log growth this is notestablished [1].Even less is known aboutthe phaseand initially starved for 16 h in suspension,as previously function of discoidin 11. Psychiatry, "003, University of California, San Diego, La Jolla,CA Tris buffer containing 1 mM CaC12; BSA,bovine serumalbumin; lactosyl-BSA, lactose conjugated to BSA (37 mol/mol) Each lectin prepa- tate was removed by centrifugation at lo X g for 1 h, andthe ration was mixed with the heads (1 mgof lectin/ml of heads) in a supernatant was extensively dialyzed against distilled waterand dialysis bag and dialyzed against Ca"/Na+/K'/Tris buffer containing lyophilized This material was redissolved in distilled water for anal-. Andria, VA) were incubated with 100 pl of a 40 pg/ml lectin solution in Ca'+/Na'/K'/Tris buffer containing 25% ethylene glycol and 0.3
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