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. 1993 Jul 15;293(Pt 2):369–375. doi: 10.1042/bj2930369

The erythrocyte calcium pump is inhibited by non-enzymic glycation: studies in situ and with the purified enzyme.

F L González Flecha 1, P R Castello 1, A J Caride 1, J J Gagliardino 1, J P Rossi 1
PMCID: PMC1134369  PMID: 8393658

Abstract

In a previous paper we demonstrated that incubation of either intact erythrocytes or erythrocytes membranes with glucose decreases the activity of the membrane Ca(2+)-ATPase [González Flecha, Bermúdez, Cédola, Gagliardino and Rossi (1990) Diabetes 39, 707-711]. The aim of the present work was to obtain information about the mechanism of this inhibition. For this purpose, experiments were carried out with purified Ca(2+)-ATPase, inside-out vesicles and membranes from human erythrocytes. Incubation of the purified Ca(2+)-ATPase with glucose led to a decay in the enzyme activity of up to 50% of the control activity under the conditions used. The decrease in ATPase activity was concomitant with labelling by [6-3H]glucose of the purified Ca2+ pump; the kinetic properties of both processes were almost identical, suggesting that inhibition is a consequence of the incorporation of glucose into the Ca(2+)-ATPase molecule. In inside-out vesicles, glucose also promoted inhibition of Ca(2+)-ATPase activity as well as of active Ca2+ transport. Arabinose, xylose, mannose, ribose, fructose and glucose 6-phosphate (but not mannitol) 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 [6-3H]glucose was about 65 kJ/mol. Furthermore, inorganic phosphate enhanced the inactivation of the Ca(2+)-ATPase by glucose. This evidence strongly suggests that inhibition is a non-enzymically catalysed process. Inactivation of the Ca(2+)-ATPase by glucose was enhanced by reductive alkylation with sodium borohydride. Aminoguanidine, an inhibitor of the formation of the advanced end products of glycosylation, did not prevent the deleterious effect of glucose on the enzyme activity. Therefore it is concluded that inactivation of the Ca2+ pump is a consequence of the glycation of this protein.

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Selected References

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