Hemoglobin synthesis in the maturing rabbit reticulocyte.

Abstract

A method is described for fractionating rabbit reticulocytes by buoyant density centrifugation in an albumin (BSA) gradient. The progressively lighter cells contained more ribosomes and were more active in protein synthesis than the denser cells. Cytological, physical, and biochemical properties of the fractions showed that the procedure separates the cells according to their degree of physiological maturity. Fractions were examined for their protein synthesizing activity and ribosomal distribution by sucrose gradient analysis. With increasing cell maturation the percent of ribonucleoprotein present as polysomes declined slightly, the pentamer persisted as the major ribosomal aggregate, and the specific activity of the polysomes decreased. The loss of hemoglobin synthesis during reticulocyte maturation is closely correlated with the loss of total ribosomal material and with an increasing percentage of inactive polysomes. A fraction of the most immature reticulocytes was labeled with H[superscript 3]-leucine and transfused into a normal rabbit for in vivo maturation. BSA-gradient analysis of blood samples taken at various time intervals confirmed that the position of the cells in a BSA gradient is a function of their age. A reticulocyte cell-free amino acid incorporating system is described which is capable, after an initial rapid incorporation, of a linear rate of protein synthesis for at least two hours. This system was used to further investigate the differences between reticulocyte fractions from a BSA gradient. Crossed incubations of ribosomes and supernatants showed that not only the ribosomes become less active but that supernatant factors limit amino acid incorporation in the more mature cells. The nature of the supernatant effect is discussed. Preliminary cell-free studies using polysomes and 80S ribosomes, isolated from a sucrose gradient, showed that 80S ribosomes are inactive by themselves. However, in the presence of polysomes they participate in amino acid incorporation. The results indicate that the rate of protein synthesis is a function of the concentrations of both monosomes and polysomes.