alpha-chymotrypsin and pimagedine

Topics: Animals; Calcimycin; Chymotrypsin; Cromolyn Sodium; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanidines; Guinea Pigs; Ileum; In Vitro Techniques; Ionophores; Male; Muscle Contraction; Muscle, Smooth; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Receptors, Proteinase-Activated; Serine Proteinase Inhibitors; Tosylphenylalanyl Chloromethyl Ketone

Glycation is a process whereby sugar molecules form a covalent adduct with protein amino groups. In this study, we used ascorbic acid (AsA) as a glycating agent and purified cucumber (Cucumis sativus L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a model protein in chloroplast tissues, and examined effects of glycation on the activity and susceptibility of Rubisco to proteases. Glycation proceeded via two phases during incubation with AsA and Rubisco in vitro at physiological conditions (10 mM AsA, pH 7.5, 25 degrees C in the presence of atmospheric oxygen). At the early stage of glycation (phase 1), the amount of AsA attaching to Rubisco increased at an almost linear rate (0.5-0.7 mol AsA incorporated (mol Rubisco)(-1) d(-1)). By Western blotting using monoclonal antibodies recognizing glycation adducts, a major glycation adduct, N( epsilon )-(carboxymethyl)lysine was detected. At the late stage of glycation (phase 2), incorporation of AsA reached saturation, and a glycation adduct, pentosidine mediating intramolecular cross-linking, was detected corresponding to formation of high molecular weight aggregates cross-linked between subunits. Glycation led to a decrease in Rubisco activity (half-life about 7-8 d). Furthermore, glycated Rubisco of phase 2 drastically increased protease susceptibility in contrast to unchanged susceptibility of glycated Rubisco of phase 1 compared to that of native Rubisco. Results obtained here suggest that AsA is possibly an important factor in the loss of activity and turnover of Rubisco.

Topics: Ascorbic Acid; Blotting, Western; Carbon Radioisotopes; Chymotrypsin; Endopeptidases; Enzyme Stability; Glutathione; Glycation End Products, Advanced; Guanidines; Immunochemistry; Lysine; Ribulose-Bisphosphate Carboxylase

Recent reports show a pro-oxidant activity of aminoguanidine. Aminoguanidine is able to generate hydrogen peroxide in the presence of Cu (II). These observations have been confirmed by the present studies in that aminoguanidine is, indeed, able to generate oxidants similar in reactivity to the hydroxyl radical and is also able to fragment BSA in a Cu (II)-dependent manner. Studies on glycated bovine serum albumin show that aminoguanidine can affect a number of parameters associated with the nonenzymatic glycation of protein. This includes an ability to decrease glucose attachment and levels of protein fluorescence termed glycophore, resulting from protein glycation. Aminoguanidine also increases the generation of dicarbonyl compounds by glycated protein. All of these effects on parameters of glycation appear to be Cu (II) dependent. Further studies show that one effect of protein glycation is to decrease its susceptibility to proteolysis. The reverse is true of protein oxidation, which has previously been shown to increase the susceptibility of proteins to proteolytic digestion. Evidence is presented suggesting that aminoguanidine is able to enhance the proteolytic digestion of glycated BSA, a protein shown to be protease resistant. Our observations are discussed within the context of current concepts of protein glycation in the development of diabetic complications and aminoguanidine's potential use as a prophylactic agent in diabetes mellitus.

Topics: Chymotrypsin; Diabetes Mellitus; Endopeptidases; Glycated Serum Albumin; Glycation End Products, Advanced; Guanidines; Hydrogen Peroxide; Oxidants; Pepsin A; Serum Albumin; Serum Albumin, Bovine; Trypsin