Acta Pharm. 55 (2005) 107-114

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Enzyme activity and AGE formation in a model of AST glycoxidation by D-fructose in vitro


1Department of Biochemical Sciences, Charles University in Prague, Faculty of Pharmacy, Hradec Králové, Czech Republic
2Department of Medical Biochemistry and Haematology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia
3Medical Faculty Hospital, Charles University in Prague, Medical Faculty, Hradec Králové, Czech Republic
Received October 5, 2004      Accepted February 11, 2005

Non-enzymatic glycation as the reaction chain between reducing sugars and free amino groups of proteins has been shown to correlate with physiological ageing and severity of diabetes. The process involves oxidative steps (glycoxidation). In this paper, the effect of D-fructose as a reactive sugar on aspartate aminotransferase (AST) as a model protein was monitored by measurements of the enzyme activity and formation of fluorescent advanced glycation end products (AGEs). Change in the AST activity was considered as a measure of the overall protein damage caused by glycation, and total AGEs and pentosidine represent, at least partly, the formation of glycoxidation products. Catalytic activity of AST in an incubation mixture containing D-fructose (50 mmol L-1), decreased compared to control values to 42% (p < 0.05) and to 11% (p < 0.05) on 5th and on 21st day of incubation, respectively. In the presence of fructose, total fluorescent AGEs concentrations were significant higher since 5th day of incubation (110 %, p < 0.05) and the fluorescent pentosidine concentrations from 15th day of incubation (117 %, p < 0.05) compared to control values, respectively. Catalytic activity of AST clearly and quantitatively demonstrated functional changes in the enzyme molecule caused by structural modifications initiated by fructose, while the evaluation of AGE formation and especially that of pentosidine by fluorescence measurement was less reliable.

Keywords: non-enzymatic glycation, glycoxidation, aspartate aminotransferase, AGE, pentosidine