Intracellular Distribution of Ribonuclease Activity during Erythroid-Cell Maturation

The erythroid-cell maturation is characterized by a series of complex morphological and biochemical events which in a relatively short period of time (70h) convert the committed haematopoietic stem cell into the mature anucleate erythrocyte. Despite our progress in the understanding of some aspects of erythropoiesis, the molecular mechanisms which control the progressive disappearance of subcellular structures such as nuclei (Tavassoli & Crosby, 1973), mitochondria (Gasko & Danon, 1972), lysosomes (Ashwood-Smith & Young, 1962), polyribosomes (Burka & DeBellis, 1967) as well as RNA (Burka, 1969) and proteins (Koch et al., 1973) during cell development remain largely unknown. The velocity sedimentation through a Ficoll gradient has been successfully used in this laboratory for the separation of the erythroblasts at different stages of cell maturation and has shown that cessation of DNA and RNA synthesis in the polychromatic erythroblast (Denton & Arnstein, 1973) seems to trigger changes in activities of several enzymes, i.e. carbonicanhydrase (EC4.2.1 .I), catalase (EC 1.11.1.6), glucose 6-phosphate dehydrogenase (EC 1.1.1.49), etc. (Denton et al., 1975) and ribonuclease (EC 3.1.4.22) (Hulea et al., 1975). The fact that most of the ribonuclease activity is found as a latent complex bound to the reticulocyte cell membrane (Burka, 1971) may be the result of a redistribution of the enzyme activity among different subcellular fractions during cell development. The present report presents evidence that the quantitative and qualitative changes in ribonuclease activity from different subcellular fractions separated from early and late stages of cell development may be related to the complex biochemical and morphological events associated with cell maturation. All chemicals were reagent grade. The bone-marrow immature erythroid cells from anaemic New Zealand White rabbits were obtained as previously described (Denton & Arnstein, 1973). The cells werehomogenized in0.25 ~-sucrose-0.05~-Tris-HC1 (pH 7.4)0.025~-KC14.005 M-MgCI, in a Teflon-glass Potter-Elvehjem homogenizer. The blood reticulocytes were lysed by suspending 1 vol. of packed cells in 3 vol. of 0.01 M-Tris-HCI (pH7.4)-0.005~-MgcI, for 30s, and then restoring the iso-osmoticity with 1 . 5 ~ sucrose-0.05~-Tris-HC1 (pH7.4)4.15~-KCI in a volume equal to that of the packed cells. The separation of subcellular fractions from bone-marrow erythroid cells and blood reticulocytes will be described in detail elsewhere. The assay of ribonuclease activity and the detection of enzyme activity after polyacrylamide-gel electrophoresis were carried out as previously described (Hulea et al., 1975). As seen from Table I the different subcellular fractions separated from early and late stages of cell maturation show a marked decrease in ribonuclease activity although not to the same extent. The highest loss of enzyme activity was recorded in the lysosonial* and ribosomal fractions. The ribonuclease activity decreases much faster in the lysosomal fraction than in the soluble one, from 10-fold at pH5.4 to 30-fold at pH7.5. The ribosomal ribonuclease activity decreases approximately at the same rate over the soluble one at all pH values investigated. It has been found also that ribonuclease activity measured at pH 5.4 decreases 2 times faster than that measured at pH7.5 in both ribosomal and soluble fractions. The changes in total ribonuclease activity (Hulea et al., 1975) in addition to those observed in the subcellular fractions may be caused by several factors such as formation of ribonuclease-ribonuclease inhibitor complexes (Goto & Mizuno, 1971), inactivation of the enzyme by membrane-binding (Burka, 1971) or physical loss of cellular organelles (Tavassoli & Crosby, 1973; Gasko & Danon, 1972). Indeed, as far