muramidase and pentosidine

Bovine lens alpha-crystallin was immobilized on EAH-Sepharose gel and glycated using d-ribose. Incubation with 500 and 100 mm d-ribose for 2 and 15 days produced short-term glycated (STGP gel) and long-term glycated proteins (LTGP gel). Both STGP and LTGP gels produced oxygen free radicals. Hydroxyl radical production was twice that in STGP gel compared with the LTGP gel. Incubation with the glycated gels produced pentosidine in a mixture of N-alpha-acetylarginine + N-alpha-acetyllysine, bovine lens proteins (BLP), and lysozyme; the amounts measured with STGP gel were higher than those with LTGP gel. Reactive oxygen species scavengers decreased the formation of pentosidine. Pentosidine was also formed in BLP when incubated with water-insoluble proteins extracted from aged or brunescent human lenses. Early glycated proteins from aged or diabetic lenses were bound to a boronate affinity column, the protein-containing gel was incubated with BLP, and pentosidine was measured in the incubation mixtures. With this method we found that diabetic lens proteins produced more pentosidine on BLP than did aged lens proteins. Further investigation indicates that two and three carbon carbohydrates possibly formed from oxidative cleavage of early glycation products are involved in pentosidine formation. Based on our findings, we propose a novel pathway for pentosidine formation on native proteins from glycated proteins.

Topics: Animals; Arginine; Carbohydrates; Cattle; Chelating Agents; Crystallins; Glucose; Humans; Lysine; Muramidase; Oxygen; Proteins; Reactive Oxygen Species

Cross-linking of proteins by sugars is thought to be involved in the pathogenesis of a variety of disorders associated with diabetes and aging; however, the molecular mechanisms involved in this process are not well understood. A new, general method for cross-link analysis, using precolumn derivatization with the reagent diphenylborinic acid (DPBA), was applied to the problem of detection and characterization of Maillard (sugar-derived) cross-links. DPBA analysis of reaction mixtures containing ribose, lysine, and arginine showed the presence of the Maillard cross-link pentosidine, along with a novel cross-link which was also found in acid hydrolyzates of ribose-treated proteins. Evidence is presented that this cross-link and pentosidine are the major acid-stable fluorescent cross-links formed by ribose with basic amino acids. Since it was determined that the new cross-link is derived from two lysine residues and at least one pentose per molecule, it was named penK2. PenK2 was found to be identical, by three different methods of chromatographic analysis, to the Maillard product "LM1', which was isolated from cataractous lens by Nagaraj and Monnier, but not yet structurally characterized (Biochim. Biophys. Acta 1116, (1992) 34-42).

Topics: Amino Acids; Arginine; Boron Compounds; Cross-Linking Reagents; Histidine; Hydrochloric Acid; Hydrogen-Ion Concentration; Hydrolysis; Lysine; Maillard Reaction; Muramidase; Ribose; Serum Albumin, Bovine; Spectrometry, Fluorescence