Molecular characterization of the glycated plasma membrane calcium pump.
We have previously demonstrated (Diabetes 39:707-711, 1990) that in vitro glycation of the red cell Ca(2+) pump diminishes the Ca(2+)-ATPase activity of the enzyme up to 50%. Such effect is due to the reaction of glucose with lysine residues of the Ca(2+) pump (Biochem. J. 293:369-375, 1993). The aim of this work was to determine whether the effect of glucose is due to a full inactivation of a fraction of the total population of Ca(2+) pump, or to a partial inactivation of all the molecules. Glycation decreased the V(max) for the ATPase activity leaving unaffected the apparent affinities for Ca(2+), calmodulin or ATP. The apparent turnover was identical in both, the glycated and the native enzyme. Glycation decreased the V(max) for the ATP-dependent but not for the calmodulin-activated phosphatase activities. Concomitantly with the inhibition, up to 6.5% of the lysine residues were randomly glycated. The probabilistic analysis of the relation between the enzyme activity and 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 located in the vicinity of the catalytic site, which results in the full inactivation of the enzyme. Thus, Ca(2+)-ATPase activity measured in erythrocyte membranes or purified enzyme preparations preincubated with glucose depends on the remaining enzyme molecules in which the essential Lys residue stays unglycated.
González Flecha
F L
FL
Departamento de Química Biológica-IQUIFIB, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires-CONICET, Junín 956, 1113-Buenos Aires, Argentina.
Castello
P R
PR
Gagliardino
J J
JJ
Rossi
J P
JP
D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology*; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D014316:Tritium
D014316:Tritium; D000252:Calcium-Transporting ATPases; D005947:Glucose; D008239:Lysine