THE ERYTHROCYTE CALCIUM-PUMP IS INHIBITED BY NONENZYMATIC GLYCATION -STUDIES IN-SITU AND WITH THE PURIFIED ENZYME

In a previous paper we demonstrated that incubation of either intact e rythrocytes or erythrocytes membranes with glucose decreases the activ ity of the membrane Ca2+-ATPase Gonzalez Flecha, Bermudez, Cedola, Ga gliardino and Rossi (1990) Diabetes 39, 707 -71 1 !. The aim of the pr esent work was to obtain information about the mechanism of this inhib ition. For this purpose, experiments were carried out with purified Ca 2+-ATPase, inside-out vesicles and membranes from human erythrocytes. Incubation of the purified Ca2+-ATPase with glucose led to a decay in the enzyme activity of up to 50% of the control activity under the con ditions used. The decrease in ATPase activity was concomitant with lab elling by 6-H-3!glucose of the purified Ca2+ pump, the kinetic proper ties of both processes were almost identical, suggesting that inhibiti on is a consequence of the incorporation of glucose into the Ca2+-ATPa se molecule. In inside-out vesicles, glucose also promoted inhibition of Ca2+-ATPase activity as well as of active Ca2+ transport. Arabinose , xylose, mannose, ribose, fructose and glucose 6-phosphate (but not m annitol) were also able to inactive the ATPase. The activation energy for both the decrease in ATPase activity by glucose and the labelling of the pump with 6-H-3!glucose was about 65 kJ/mol. Furthermore, inor ganic phosphate enhanced the inactivation of the Ca2+-ATPase by glucos e. This evidence strongly suggests that inhibition is a non-enzymicall y catalysed process. Inactivation of the Ca2+-ATPase by glucose was en hanced by reductive alkylation with sodium borohydride. Aminoguanidine , an inhibitor of the formation of the advanced end products of glycos ylation, did not prevent the deleterious effect of glucose on the enzy me activity. Therefore it is concluded that inactivation of the Ca2+ p ump is a consequence of the glycation of this protein.