CARBAMYLATION OF ERYTHROCYTE-MEMBRANE PROTEINS - AN IN-VITRO AND IN-VIVO STUDY

Objectives: To establish the degree of erythrocyte membrane protein ca rbamylation in uremic and nonuremic patients, and to characterize the in vitro binding of cyanate to the individual proteins of the cytoskel etal matrix. Design and Methods: For in vivo studies, erythrocyte ghos ts were digested with proteinase K and the released peptides colorimet rically assayed for carbamylation, using the diacetyl monoxime reagent , and quantitated using homocitrulline. For in vitro studies, erythroc yte ghosts were incubated with [C-14] cyanate, and the membrane protei ns separated by SDS-PAGE. Cyanate incorporation was quantitated by liq uid scintillation counting and imaging densitometry of the excised ban ds. Results: Erythrocytes from uremic patients were found to have a gr eater level of carbamylation than those from nonuremic patients (47.09 +/- 7.80 and 25.89 +/- 6.92 nmol homocitrulline/mg proteolyzed protei n released, respectively). in vitro incorporation of [C-14] cyanate in to membrane protein followed the sequence: spectrin > ankyrin > Band 4 .1 > Band 3 > actin > Band 7. Conclusions: The increased level of eryt hrocyte membrane protein carbamylation in uremic compared to nonuremic patients may lead to membrane destabilization and contribute to the d ecreased erythrocyte survival time observed in uremia.