Non-enzymatic glycosylation of alkaline phosphatase alters its biological properties.
Hyperglycaemia in poorly controlled diabetic patients induces non-enzymatic glycosylation (glycation) of proteins, altering their structure and physiological bioactivity. Alkaline phosphatase (ALP) is a membrane-bound exoenzyme which faces the extracellular compartment. We have investigated the glycation of intestinal alkaline phosphatase in vitro and the consequences of such molecular modifications on certain structural and functional characteristics. The effect of glycation on alkaline phosphatase specific activity was determined after incubation of the enzyme with different sugars for various periods of time. The formation of early reversible glycation products was determined by the measurement of fructosamine levels, while the appearance of advanced glycation end products was estimated by spectrofluorometric analysis. A decrease in the specific activity of ALP was associated both with an increase in fructosamine levels and with the appearance of AGE-characteristic fluorescence. Changes in these parameters were found to depend on the incubation time, and on the concentration and glycating capability of the sugar employed. Co-incubation with aminoguanidine slowed down the appearance of protein-linked fluorescence, and additionally curbed the decrease in enzymatic specific activity. A significant correlation between the levels of ALP-fructosamine and ALP-advanced glycation end product was observed. Patterns of protein bands fractionated by SDS-PAGE were essentially identical for the nonglycated controls and the glycated samples. The electrophoretic mobility of the band of alkaline phosphatase on cellulose acetate gels increased as a function of the incubation time and the glycosylating power of the carbohydrate used. The present study provides evidence for the in vitro glycation of alkaline phosphatase, and for the consecutive alteration of its activity and structure.
McCarthy
A D
AD
Cátedra de Bioquímica Patológica, Facultad de Ciencias Exactas, Universidad Nacional de la Plata, Argentina.
Cortizo
A M
AM
Giménez Segura
G
G
Bruzzone
L
L
Etcheverry
S B
SB
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D050260:Carbohydrate Metabolism; D002417:Cattle; D019270:Fructosamine / Q000032:analysis; D006031:Glycosylation; D007422:Intestines / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D019270:Fructosamine; D000469:Alkaline Phosphatase