Abstract
This work was undertaken to improve a separation method for preparation of large amounts of erythroid cells of different age with homogeneous and minimal contamination of myeloid cells. Our method was suitably employed in the study of the decay mechanism of glucose-6-phosphate dehydrogenase (G6PDH) during the erythroid cell maturation.
Twenty fractions of erythroid cells at different advancing stages of maturation were prepared by fractionating, at unit gravity, bone marrow cells from anaemic rabbit. The specific activity of the G6PDH was assayed and plotted vs the fraction number and the typical sigmoid curve of the activity decay was drawn. The separated cells were then grouped in three sets of fractions following the three phases of the sigmoid curve and the fractions of each set were combined. From the cytochemical analysis of the three main fractions so obtained, we found a 25–30% myeloid cell contamination in the first fraction, while in the other two fractions the myeloid contamination was 10% or less. For this reason we performed a rapid separation of the first fraction on a discontinuous percoll gradient. By this method, the myeloid cell contamination of the first fraction was levelled down to the other two. The fractions, so obtained, (I, II and III in order of increasing cell maturation) showed a four fold decrease of glucose-6-phosphate dehydrogenase activity expressed both per cell number and on protein base. On the contrary the concentration of the total soluble proteins did not change significantly in the three fractions.
The three purified cellular populations were used to provide information on the protein turnover of the erythroid cells during their development. We measured, in intact cells, the rate of synthesis and degradation of total proteins and then, in cell lysates, we determined the rate of degradation of G6PDH, purified from rabbit RBC and radiolabeled by reductive methylation with C14-formaldehyde. The rates of proteolysis obtained with total proteins and methyl-G6PDH clearly indicate that the proteolytic machinery of the erythroblasts reduces its activity during the cell maturation.
Similar content being viewed by others
References
Borsook H, Ratner K, Tattrie B: Studies of erythropoiesis II. A method of segregating immature from mature adult rabbit erythroblasts. Blood 34: 32–41, 1989
Shortman K, Seligman K: The separation of different cell classes from lymphoid organs. III. The purification of erythroid cells by pH-induced density changes. J Cell Biol 42:783–788,1969
Williams AF: DNA synthesis in purified populations of avian erythroid cells. J Cell Sci 10: 27–33, 1972
Pertoft H, Laurent TC: Sedimentation of cells in colloidal silica (Percoll). In: TG Pretlow, TP Pretlow (eds) Cell Separation: Methods and Selected Application. Academic Press, New York, 1982, pp 115–143
Peterson EA, Evans WH: Separation of bone marrow cells by sedimentation at unit gravity. Nature 214: 824–825, 1967
Denton MJ, Arnstein HRV: Characterization of developing adult mammalian erythroid cells separated by velocity sedimentation. Br J Haematol 24: 7–17, 1973
Light ND, Tanner MJ: Changes in surface-membrane components during the differentiation of rabbit erythroid cells. Biochem J 164: 565–578, 1977
Miller MG, Philips RA: Separation of cells by velocity sedimentation. J Cell Physiol 73: 191–202, 1969
Harrison FL, Beswick TM, Chesterton C: Separation of haemopoietic cells for biochemical investigation. Biochem J 194: 789–796, 1981
Narita H, Ikura K, Yanagawa S, Sasaki R, Chiba H: 2,3-Bisphosphoglycerate in developing rabbit erythroid cells. J Biol Chem 255: 5230–5235, 1980
Ninfali P, Palma F, Fornaini G: Rabbit bone marrow glucose-6-phosphate dehydrogenase during erythroid cell development. Mol and Cell Biochem 75, 85–92, 1987
Sanderson RF, Bird K: Cell separation by counter flow centrifugation. In: DM Prescott (ed) Methods in Cell Biology, Academic Press, New York, 1977, Vol 15, pp 1–14
De Vitte T, Plas A, Koekman E, Blankenborg G, Salden M, Wessels J, Haanen C: Separation of human bone marrow by counterflow centrifugation monitored by DNA-flowcytometry. Br J Haematol 58: 249–258, 1984
Reisner Y, Itsicovitch L, Meshorer A, Sharon N. Hemopoietic stem cell transplantation using mouse bone marrow and spleen cells fractionated by lectins. Proc Natl Acad Sci USA 75: 2933–2936, 1978
Reisner Y, Kapoor N, O'Reilly R, Good RA: Allogencic bone marrow transplantation using stem cells fractionated by lectins: VI, in vitro analysis of human and monkey bone marrow cells fractionated by sheep red blood cells and soybean agglutinin. Lancet 20/27: 1320–1324, 1980
Rennie CM, Thompson S, Parker AC, Maddy A: Human erythrocyte fractionation in ‘percoll’ density gradients. Clin Chim Acta 98: 119–125, 1979
De Flora A, Morelli A, Benatti U, Giuliano F: An improved procedure for rapid isolation of glucose-6-phosphate dehydrogenase from human erythrocytes. Arch Biochem Biophys: 169, 364–369, 1975
Dottavio-Martin D, Ravel JM: Radiolabeling of proteins by reductive alkylation with 14C-formaldehyde and sodium cyanoborohydride. Anal Biochem 87: 562–565, 1978
Prouty WF, Karnowsky MJ, Goldberg AL: Degradation of abnormal proteins in escherichia coli. J Biol Chem 250: 1112–1122,1975
Beutler E: Red cell metabolism. A manual of Biochemical Methods, Grune & Stratton, New York, 1975, 2nd Edn, pp 66–69
Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal Biochem 72: 248–254, 1976
Luzzatto L, Battistuzzi G: Glucose-6-phosphate dehydrogenase. Adv Human Genet 14: 217–329, 1984
Luzzatto K, Testa U: In: Human erythrocyte glucose-6-phosphate dehydrogenase: structure and function in normal and mutant subjects. Piomelli S, Yachin S, eds. Current Topics in Hematology Vol. 1978, pp 1–70, Alan R Liss, New York
Rapoport SM (1986): In: The Reticulocyte, pp23–24 C.R.C. press, Inc Boca Raton, Florida
Magnani M, Stocchi V, Dacha M, Fornaini G: Hexokinase in developing rabbit erythroid cells. Biochim Biophys Acta 802:346–351,1984
Denton MJ, Spencer N, Arnstein HRV: Biochemical and enzymic changes during erythrocyte differentiation. Biochem J 146: 205–211, 1975
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ninfali, P., Palma, F., Baronciani, L. et al. Glucose-6-phosphate dehydrogenase activity and protein turnover in erythroblasts separated by velocity sedimentation at unit gravity and percoll gradient centrifugation. Mol Cell Biochem 106, 151–160 (1991). https://doi.org/10.1007/BF00230181
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00230181