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

25. GONZÁLEZ FLECHA, F.L., CASTELLO, P.R., CARIDE, A.J., GAGLIARDINO, J.J. AND ROSSI, J.P.F.C.

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Lugar: Londres; Año: 1993 vol. 293 p. 369 - 375

0264-6021

In a previous paper we demonstrated that incubation of eitherintact erythrocytes or erythrocytes membranes with glucosedecreases the activity of the membrane Ca2+-ATPase [GonzailezFlecha, Bermiudez, Cedola, Gagliardino and Rossi (1990)Diabetes 39, 707-711]. The aim of the present work was to obtaininformation about the mechanism of this inhibition. For thispurpose, experiments were carried out with purified Ca2+-ATPase, inside-out vesicles and membranes from humanerythrocytes. Incubation of the purified Ca2+-ATPase with glucoseled to a decay in the enzyme activity of up to 50 % of thecontrol activity under the conditions used. The decrease inATPase activity was concomitant with labelling by [6-3H]glucoseofthe purified Ca2+ pump; the kinetic properties ofboth processeswere almost identical, suggesting that inhibition is a consequenceof the incorporation of glucose into the Ca2+-ATPase molecule.In inside-out vesicles, glucose also promoted inhibition of Ca2+-INTRODUCTIONWe have previously reported [1] that erythrocytes from diabeticpatients show lower Ca2+-ATPase activity than comparable cellsfrom healthy volunteers. In a subsequent paper we found thatpreincubation of normal erythrocytes with glucose decreased theCa2+-ATPase activity, and that this effect was correlated withincreased glycosylation of the erythrocyte membrane proteins[2]. In that study we also observed that preincubation of eitherintact erythrocytes or isolated erythrocyte membranes withglucose caused inactivation of the Ca2+-ATPase, which wasenhanced by an increase of the pH of the preincubation medium,a typical characteristic of the non-enzymic glycosylation process.The reaction of reducing sugars with proteins, which results innon-enzymic 'browning' have been extensively studied [3]. Theformation of adducts between sugars and proteins has beenreported for several proteins such as haemoglobin [4], RNAase[5] and, more recently, Na+/K+-ATPase [6]. Non-enzymicglycosylation of proteins is a reaction which proceeds throughseveral steps; the early ones are reversible and develop inrelatively short periods, while the later ones take longer timesand become irreversible [7]. Both the initial and end products ofthe reaction modify the physicochemical properties of theglycosylated protein [8]. Based on such changes, and the permanenthigh blood glucose levels which characterize diabetesmellitus, glycosylation of proteins has been postulated as thedeveloping mechanism of the chronic complications of thisdisease.The experiments in the present paper were designed to: (1) testATPase activity as well as of active Ca2l transport. Arabinose,xylose, mannose, ribose, fructose and glucose 6-phosphate (butnot mannitol) were also able to inactive the ATPase. Theactivation energy for both the decrease in ATPase activity byglucose and the labelling of the pump with [6-3H]glucose wasabout 65 kJ/mol. Furthermore, inorganic phosphate enhancedthe inactivation of the Ca2+-ATPase by glucose. This evidencestrongly suggests that inhibition is a non-enzymically catalysedprocess. Inactivation of the Ca2+-ATPase by glucose was enhancedby reductive alkylation with sodium borohydride.Aminoguanidine, an inhibitor of the formation of the advancedend products of glycosylation, did not prevent the deleteriouseffect of glucose on the enzyme activity. Therefore it is concludedthat inactivation of the Ca2+ pump is a consequence of theglycation of this protein.whether