Carbamylation of erythrocyte membrane components: An in vitro and in vivo study.

Cyanate exists in equilibrium with urea and in its reactive form, isocyanic acid, forms a stable adduct with protein amino groups (carbamylation). Both carbamylated hemoglobin and carbamylated plasma proteins are elevated in patients with chronic renal failure (uremia) and are believed to be responsible for some of the pathophysiological consequences of uremia. The present study focused upon an in vitro/in vivo investigation of the carbamylation of erythrocyte membrane cytoskeletal and phospholipid components. The time course binding pattern of $\rm\lbrack \sp{14}C\rbrack $ cyanate to the individual cytoskeletal membrane proteins demonstrated that spectrin and ankyrin were the most extensively carbamylated proteins, incorporating $18.3\pm 1.6$ and $10.7\pm 3$ mol cyanate/mol protein, respectively, following a 10-h incubation. The degree of carbamylation was directly correlated with protein molecular weight, indicating the nonspecific nature of the binding. For in vivo studies, erythrocyte ghosts were digested with proteinase K and the released peptides colorimetrically assayed for carbamylation using diacetyl monoxime. Erythrocytes from uremics were found to have a greater level of carbamylation relative to nonuremics $(47.09\pm 7.90$ and $25.89\pm 6.92$ nmol homocitrulline/mg proteolyzed protein released, respectively. In addition, measurements of membrane fluidity, using the fluorescence polarization probe--fluorescein lithocholic acid, provided evidence of membrane alterations as a result of in vitro carbamylation. Solvent extraction of erythrocyte ghost membranes incubated with $\rm\lbrack \sp{14}C\rbrack $ cyanate followed by thin-layer chromatography and autoradiography clearly indicated the specific modification in vitro of the amino containing headgroups of both phosphatidylethanolamine and phosphatidylserine by cyanate. The cyanate incorporated following a 15-h incubation was $15.75\pm 0.09$ and $13.34\pm 0.81$ mol %, respectively. Separation of carbamylated phosphatidylethanolamine from native phosphatidylethanolamine by thin-layer chromatography was achieved in a solvent system of chloroform/methanol/ammonia (65/35/5, v/v/v). Treatment of erythrocyte lipid/Triton X-100 micelles, constructed from solvent extracts of ghost membranes from normal individuals, with phospholipase D released a chromogenic species which reacted with diacetyl. The results provide preliminary evidence for the presence of carbamylated phospholipid in the native erythrocyte membrane. The level of carbamylated phosphatidylethanolamine in the native erythrocyte membrane was estimated to be $2.85\pm 0.65$ mol %. It is suggested that the increased level of membrane carbamylation in uremics may contribute to the decreased erythrocyte survival time observed in these individuals.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1995 .T73. Source: Dissertation Abstracts International, Volume: 59-08, Section: B, page: 4096. Adviser: R. J. Thibert. Thesis (Ph.D.)--University of Windsor (Canada), 1996