Carbamylation of Brain Proteins with Cyanate in vitro and in vivo

Abstract

Unequivocal demonstration of the incorporation of [14C]cyanate into brain proteins as a carbamyl derivative rather than via decomposition to CO2 is presented; this is based on the isolation of radioactive hydantoins and on the loss of radioactivity on hydrolysis of the hydantoins. The influence of conditions on the extent of carbamylation of brain proteins in vitro by cyanate are presented. The optimum pH is about 7 and the carbamylation is dependent on temperature, concentration and time. There was no detectable difference in the rate and/or extent of cyanate incorporation in vitro between normal and previously carbamylated animals. Experiments in vivo show a linear increase in the carbamylation of brain proteins with intraperitoneal injection of cyanate up to the highest concentration tested (2 μmol/g). The extent of carbamylation increases progressively with repeated injections for about 3 days. After this time with further injections there is increased incorporation, but at a lower rate. Experiments in vivo show that animals who have been previously carbamylated with non‐radioactive cyanate incorporate about 25% less cyanate than control animals when given a pulse of [14C]cyanate. Fractionation of the major cell components revealed cyanate incorporation into all fractions but a higher incorporation in the supernatant fraction. When brain was fractionated by differential extraction there was incorporation in all the fractions, including the water, salt and solvent‐insoluble residue. Although some differences were detected, additional studies are needed to determine whether carbamylation affects protein turnover. The higher sensitivity of experiments in vivo for discrimination of protein carbamylation is discussed in relation to the present work and to recent work from others. Copyright © 1974, Wiley Blackwell. All rights reserved