T H E INCORPORATION OF NI~-GLYCINE BY AVIAN ERYTHROCYTES AND RETICULOCYTES IN VITRO BY V. ALLFREY Am)

The present report summarizes a group of tracer experiments designed to test (1) whether desoxyribonucleic acid (DNA) and its associated histone are in a state of continuous nitrogen turnover in the avian red cell, (2) whether the rate of glycine incorporation into hemoglobin of the nucleated reticulocyte exceeds that observed in the mature erythrocyte, and (3) whether a difference in rate of synthesis exists between the hemoglobin of the erythro° cyte nucleus and hemoglobin in the cytoplasm. The measure of DNA and histone turnover, and of hemoglobin synthesis, applied was the rate of incorporation of heavy nitrogen into these compounds, following the incubation of avian blood cells in the presence of N16-1abeled glycine. The incubation procedure used was essentially that recommended by London, Shemin, and Rittenberg (1), in which a suspension of freshly drawn blood (containing added N15-glycine) is incubated under aerobic conditions at 38°C. Following incubation, the DNA and histone were isolated, and the hemoglobin was crystallized both from whole cells, and from nuclear and cytoplasmic fractions prepared in non-aqueous media (2). The N 15 concentrations (atom per cent N 15 excess) of these substances were determined in the mass spectrometer after the usual co.nversion of organic nitrogen to N, gas. I t was found that the incorporation of N15-glycine into histone and DNA by in vitro suspensions of chicken erythrocytes and reticulocytes was negligible. The rates of glycine incorporation into hemoglobin indicate that the nucleated reticulocyte is more than twice as active in this respect as the erythrocyte. In both cases the rate of nitrogen incorporation into hemoglobin is low, and is only about a tenth of that measured in the heme prosthetic grou p. Because of the low over-all N 15 uptake into hemoglobin, differences which might have existed between nuclear and cytoplasmic hemoglobins could not be detected.