Molecular characterization of the glycated plasma membrane calcium pump

We have previously demonstrated (Diabetes 39:707-711, 1990) that in vitro g lycation of the red cell Ca2+ pump diminishes the Ca2+-ATPase activity of t he enzyme up to 50%. Such effect is due to the reaction of glucose with lys ine residues of the Ca2+ pump (Biochem. J. 293:369-375, 1993). The aim of t his work was to determine whether the effect of glucose is due to a full in activation of a fraction of the total population of Ca2+ pump, or to a part ial inactivation of all the molecules. Glycation decreased the V-max for th e ATPase activity leaving unaffected the apparent affinities for Ca2+, calm odulin or ATP. The apparent turnover was identical in both, the glycated an d the native enzyme. Glycation decreased the V-max for the ATP-dependent bu t not for the calmodulin-activated phosphatase activities. Concomitantly wi th the inhibition, up to 6.5% of the lysine residues were randomly glycated . The probabilistic analysis of the relation between the enzyme activity an d the fraction of nonmodified residues indicates that only one Lys residue is responsible for the inhibition. We suggest that glucose decreases the Ca 2+-ATPase activity by reacting with one essential Lys residue probably loca ted in the vicinity of the catalytic site, which results in the full inacti vation of the enzyme. Thus, Ca2+-ATPase activity measured in erythrocyte me mbranes or purified enzyme preparations preincubated with glucose depends o n the remaining enzyme molecules in which the essential Lys residue stays u nglycated.