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 ketoamine adduct (Amadori product), which leads to the formation of advanced glycation end-products. These compounds are involved in the development of tissue modifications such as cross-linking and fluorescence in diabetes and aging. Searching for an enzyme to reverse protein glycation, we isolated a Pseudomonas sp. soil strain growing selectively on the Amadori product epsilon-fructosyl-aminocaproate. An Amadori product binding protein (ABP) was purified from the bacterial extract by single-step affinity chromatography on glycated lysine-Sepharose. The protein, a monomer of 45 kDa, did not bind to unglycated or NaBH4-reduced glycated lysine-Sepharose suggesting specificity for the Amadori compound. The concentration-dependent binding of glycated aminocaproate showed saturation with Kd = 1.49 microM and Bmax = 17.63 nmol/mg of protein corresponding to 0.8 mol/mol of protein. The binding of epsilon-1-[14C]fructosyl-aminocaproate to the protein was inhibited by other glucose-derived Amadori products, but not by free sugars, unglycated amines, 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 synthesis was inducible by growth of the organism in Amadori product. Immunoblotting studies showed that ABP is not found in cell extracts from other prokaryotes, yeast, or liver homogenate and does not bind Amadori products in glycated proteins. ABP has no enzymatic activity toward glycated substrates and may thus have transport or permease function for glycated amino acids.
Gerhardinger
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C
Institute of Pathology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Taneda
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S
Marion
M S
MS
Monnier
V M
VM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000737:chemistry*; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D011549:Pseudomonas / Q000737:chemistry* / Q000378:metabolism
C090149:Amadori product-binding protein, Pseudomonas sp.; D001426:Bacterial Proteins; D002352:Carrier Proteins; D017127:Glycation End Products, Advanced; D047908:Intracellular Signaling Peptides and Proteins