ISOLATION, PURIFICATION, AND CHARACTERIZATION OF AN AMADORI PRODUCT BINDING-PROTEIN FROM A PSEUDOMONAS SP SOIL STRAIN

Sugars react nonenzymatically with protein amino groups to form a keto amine adduct (Amadori product), which leads to the formation of advanc ed glycation endproducts. These compounds are involved in the developm ent of tissue modifications such as cross-linking and fluorescence in diabetes and aging. Searching for an en zyme to reverse protein glycat ion, we isolated a Pseudomonas sp. soil strain growing selectively on the Amadori product epsilon-fructosyl-aminocaproate. An Amadori produc t binding protein (ABP) was purified from the bacterial extract by sin gle-step affinity chromatography on glycated lysine-Sepharose. The pro tein, a monomer of 45 kDa, did not bind to unglycated or NaBH4-reduced glycated lysine-Sepharose suggesting specificity for the Amadori comp ound. The concentration dependent binding of glycated aminocaproate sh owed saturation with K-d = 1.49 mu M and B-max = 17.63 nnol/mg of prot ein corresponding to 0.8 mol/mol of protein. The binding of epsilon-[C -14]fructosyl-aminocaproate to the protein was inhibited by other gluc ose-derived Amadori products, but not by free sugars, unglycated amine s, or ribated lysine. The sequence of the first 16 NH2-terminal amino acids and a GenBank search revealed that ABP is a novel protein. Its s ynthesis was inducible by growth of the organism in Amadori product. I mmunoblotting studies showed that ABP is not found in cell extracts fr om other prokaryotes, yeast, or liver homogenate and does not bind Ama dori products in glycated proteins. ABP has no enzymatic activity towa rd glycated substrates and may thus have transport or permease functio n for glycated amino acids.