Modification of Proteins In Vitro by Physiological Levels of Glucose

Abstract

Hyperglycemic conditions of diabetes accelerate protein modifications by glucose leading to the accumulation of advanced glycation end-products (AGEs). We have investigated the conversion of protein-Amadori intermediate to protein-AGE and the mechanism of its inhibition by pyridoxamine (PM), a potent AGE inhibitor that has been shown to prevent diabetic complications in animal models. During incubation of proteins with physiological diabetic concentrations of glucose, PM prevented the degradation of the protein glycation intermediate identified as fructosyllysine (Amadori) by 13 C NMR using [2-13 C]-enriched glucose. Subsequent removal of glucose and PM led to conversion of proteinAmadori to AGE N ⑀ -carboxymethyllysine (CML). We utilized this inhibition of post-Amadori reactions by PM to isolate protein-Amadori intermediate and to study the inhibitory effect of PM on its degradation to protein-CML. We first tested the hypothesis that PM blocks Amadori-to-CML conversion by interfering with the catalytic role of redox metal ions that are required for this glycoxidative reaction. Support for this hypothesis was obtained by examining structural analogs of PM in which its known bidentate metal ion binding sites were modified and by determining the effect of endogenous metal ions on PM inhibition. We also tested the alternative hypothesis that the inhibitory mechanism involves formation of covalent adducts between PM and proteinAmadori. However, our 13 C NMR studies demonstrated that PM does not react with the Amadori. Because the mechanism of interference with redox metal catalysis is operative under the conditions closely mimicking the diabetic state, it may contribute significantly to PM efficacy in preventing diabetic complications in vivo. Inhibition of protein-Amadori degradation by PM also provides a simple procedure for the isolation of proteinAmadori intermediate, prepared at physiological levels of glucose for relevancy, to study both the biological effects and the chemistry of post-Amadori pathways of AGE formation.Chemical modifications of circulating, cellular, and matrix proteins by glucose are thought to be a major factor in pathogenesis of diabetes, atherosclerosis, and neurodegenerative diseases, as well as in the process of aging (1-4). These modifications derive, in part, from glycation reactions, i.e. the reversible condensation of the aldehyde group of glucose with a protein amino group, forming a Schiff base followed by an essentially irreversible rearrangement to an Amadori intermediate (Fig.

The Role Formation of Methylglyoxal, Carbonyl Compound, Hydrogen Peroxide and Advance Oxidation Protein Product Induced Cadmium in Ovarian Rat

“…The linear aldehyde glucose reactive structure that can bind to the amine group protein become glycated protein. This is consistent with the mechanism proposed by Voziyan et al [6] can accelerate the formation of compounds dicarbonyl [e.g., glyoxal (GO), methylglyoxal (MG) and 3 deoxyglucosone].…”

“…Massanyi et al in their research showed that the number of atretic follicles was significantly higher in all groups administered cadmium and the diameter of the follicles was significanlty smaller in the primary follicles of group C compared with control group [4], [5]. The mechanism cadmium damage the ovarium might through the formation of advance glycation end products (AGEs) by nonenzymatic reaction and the formation of advance oxidation protein products (AOPP) [6].…”

Modulation of renal-specific oxidoreductase/ myo -inositol oxygenase by high-glucose ambience

“…These changes are related to the increased cellular flux of glucose intermediaries (2), de novo synthesis of intra-and extracellular advanced glycation end products (3,4), activation of protein kinase C (5, 6), increased expression of transforming growth factor-␤ (7), increased activity of GTP-binding and cell-cycle proteins (6,8), and generation of reactive oxygen species (9,10), with consequential compromise in renal functions (11,12). Such complex interrelated cellular signaling events, also involving various forms of MAP͞ERK kinases and Smad proteins, have been defined mainly in glomerular cells (13,14); information relevant to the tubulointerstitial cells, although notably affected, is limited (15,16).…”

Oxidative Stress and Kidney Glycation in Rats Exposed Cadmium

“…Glycation is nonenzymatic reaction between reducing sugars and proteins. Voziyan et al proposed that heavy metal can accelerate the formation of compounds dicarbonyl [e.g., glyoxal (GO), methylglyoxal (MG) and 3 deoxyglucosone] [13], [14].…”

“…The mechanism Cd damage the kidney might through the formation of Reactive Oxygen Species (ROS) [12] and Advance Glycation End Products (AGEs) by nonenzymatic reaction [13].…”