Rabbit red blood cell hexokinase:intracellular distribution during reticulocytes maturation

Summary The intracellular localization and isozyme distribution of hexokinase were studied during rabbit reticulocyte maturation and aging. In reticulocytes 50% of the enzyme was particulate while in the mature erythrocytes all the hexokinase activity was soluble. The bound enzyme co-sediments with mitochondria and by column chromatography it was found to be hexokinase Ia. The cytosol of reticulocytes contains hexokinase Ia (38%) and hexokinase Ib (62%) while the mature erythrocytes contain only hexokinase Ia. The amount of bound hexokinase decreases very quickly during cell maturation and aging as was shown by following in vivo reticulocyte maturation or by analysis of hexokinase compartmentation in cells of different ages, obtained by density gradient ultracentrifugations. A role for this intracellular distribution of hexokinase is suggested.     

(Na,K)-ATPase and Ouabain binding in reticulocytes from dogs with high K and low K erythrocytes and their changes during maturation

The present study demonstrated that dog reticulocytes had considerable amounts of (Na,K)-ATPase, but lost it rapidly during maturation into erythrocytes. Furthermore, reticulocytes from dogs possessing erythrocytes characterized with high (Na,K)-ATPase activity and high K, low Na concentrations (HK dogs; Maede, Y., Inaba, M., and Taniguchi, N. (1983) Blood 61,493-499) had more ouabain binding sites than cells from normal dogs (LK dogs). Our results were as follows: i) The maximal binding capacities (Bmax) for ouabain binding at equilibrium were approximately 0 and 1,500 binding sites/cell in LK and HK dog erythrocytes, respectively. ii) Reticulocytes from LK dogs possess approximately 5,700 ouabain binding sites/cell. iii) The Bmax value for ouabain in HK reticulocytes was about 10,000 sites/cell, being 2-fold that in LK reticulocytes. iv) Ouabain-sensitive fluxes of 24Na and 42K in each type of reticulocyte were compatible with the number of ouabain binding sites on the cells. v) Ouabain binding capacity, as well as (Na,K)-ATPase activity, in the reticulocytes from LK dogs fell rapidly to nearly zero during the maturation into erythrocytes. vi) Although reticulocytes from HK dogs also showed a similar regression of (Na,K)-ATPase during maturation, they retained a certain number of ouabain binding sites even after maturation, resulting in the high activity of (Na,K)-ATPase in HK erythrocyte membrane.     

Adenyl cyclase activities in rat erythrocytes during stress erythropoiesis: localization of the enzyme in the reticulocytes

NaF- and isoproterenol-stimulated adenyl cyclase activity found in preparations from rat erythrocytes is obviously localized mainly in the reticulocytes. After treatment with acetylphenyl hydrazine, both reticulocyte counts and adenyl cyclase activity increase dose-dependently. Evidence is given that adenyl cyclase activity in the red blood cell is lost during the maturation process.     

Analysis of the tetrahydrobiopterin synthesizing system during maturation of murine reticulocytes

The enzymes of tetrahydrobiopterin synthesis have been studied in murine bone marrow, in spleen, in erythrocytes, and in reticulocytes. Mice with chemically induced and with genetically conditioned reticulocytosis as found in the lactate dehydrogenase deficient strain (Ldh-1c/Ldh-1c) were used for analysis of reticulocytic enzyme activities. The activity of the biopterin synthesizing system is highest in bone marrow even though it amounts to only about 10% as compared with liver. The first enzyme of the biosynthetic pathway, GTP-cyclohydrolase, virtually disappears during the final maturation step of reticulocytes. In contrast, the activities of 6-pyruvoyltetrahydropterin synthase and of sepiapterin reductase of erythrocytes are only reduced to about one half of the reticulocyte level. The absence of biopterin in erythrocytes is therefore caused by the loss of the enzyme that initiates the pterin biosynthetic pathway.     

Maturation of rabbit reticulocytes: degradation of specific reticulocyte proteins.

As analyzed by polyacrylamide-gel electrophoresis, rabbit reticulocyte cytoplasm contains, in addition to globin, seven predominant polypeptides. The amounts of these range from 0.1 to 1.2% of globin. Rabbit erythrocytes contain only three of these nonglobin polypeptides. The loss of the four other polypeptides is correlated with maturation of the reticulocytes to erythrocytes. We also fractionated reticulocytes according to age by equilibrium centrifugation through a gradient of metrizamide and showed that younger reticulocytes contain much more of these four polypeptides than do more mature reticulocytes.The four polypeptides that are lost during differentiation are also very sensitive to in vitro destruction by chymotrypsin or trypsin under conditions where globin and the three reticulocyte nonglobin peptides that remain during reticulocyte maturation are completely resistant. Our results are consistent with the notion that the degradative rate of a reticulocyte cytoplasmic protein is determined by its susceptibility to general intracellular proteases.     

A cellular activator of catecholamine-sensitive adenylate cyclase in rat reticulocytes and erythrocytes: changes during reticulocyte development and effects on the beta receptor

Rat reticulocytes contain an isoproterenol-sensitive adenylate cyclase activity which is lost with maturation to erythrocytes despite no change in the density of β-adrenergic receptors. To explore this observation, a cytosol factor, previously shown to be important in the expression of catecholamine-sensitive adenylate cyclase in the reticulocyte, was compared to a cytosol factor obtained in a similar manner from mature erythrocytes. The cytosol factor from reticulocytes augmented isoproterenol-responsive adenylate cyclase activity in reticulocyte and erythrocyte membranes half-maximally at 0.7 ± 0.1 (SEM) and 1.1 ± 0.3 μg/ml, respectively. These concentrations of reticulocyte-derived cytosol factor were significantly lower (P < 0.01) than those concentrations of the factor from erythrocytes necessary to augment isoproterenol-responsive adenylate cyclase activity in reticulocyte (9.7 ± 2.3) and erythrocyte (7.5 ± 1.0) membranes. Cytosol factor from reticulocytes also caused greater total isoproterenol responsiveness than that from erythrocytes both in reticulocyte (784 ± 107 vs 525 ± 65 pmol/mg protein) and in erythrocyte membranes (54 ± 6 vs 36 ± 3); P < 0.05. Neither reticulocyte nor erythrocyte cytosol factor affected the concentration at which isoproterenol half-maximally stimulated adenylate cyclase in either set of membranes. However, the cytosol factor from reticulocytes markedly decreased the binding affinity of isoproterenol for β receptors in reticulocytes from 0.8 ± 0.2 to 6.9 ± 1.4 μm; P < 0.001. This reticulocyte factor had no significant effect on the binding affinity of isoproterenol for erythrocyte membranes. Erythrocyte factor did not change the binding affinity for isoproterenol in either reticulocyte or erythrocyte membranes.     

A particle-associated ATP-dependent proteolytic activity in erythroleukemia cells

An ATP-dependent proteolytic activity has been detected in both mouse erythroleukemia (Friend) cells and human (K562) erythroleukemia cells. Exposure of the Friend cells to dimethyl sulfoxide, which stimulates differentiation, increased ATP-dependent proteolysis approximately 2-fold although inducing differentiation in the K562 line had no significant effect on proteolysis. In contrast to the previously described soluble ATP-dependent proteolytic system of reticulocytes, the activity in the more primative erythroid cells is associated with a particulate fraction and is readily sedimentable by centrifugation at 100,000 X g for 1 h. Like the soluble reticulocyte system, the particulate activity requires divalent cation and is inhibited by hemin as well as vanadate. The activity was isolated on a sucrose cushion (30%) and did not appear to be associated with membranes, cytoskeleton, or polysomes. This enzymatic activity which degrades abnormal globin chains may initially reside in a particulate fraction and then become solubilized during erythroid maturation to the reticulocyte stage. Alternatively, the particulate activity may disappear with cell maturation being replaced by a distinct soluble activity. ATP-dependent proteolytic activity is eventually lost with reticulocyte maturation and further aging of erythrocytes.     

Biogenesis of erythrocyte membrane proteins. In vivo studies in anemic rabbits.

To study the process of red cell membrane protein synthesis we have followed the time course of [3-H]leucine appearance in total protein and individual peptides of the erythrocyte membrane following injection of the amino acid into phenylhydrazine-anemic rabbits. Multiple peripheral blood samples were taken from single animals over a 5-week period. Erythrocyte membrane proteins were separated by polyacrylamide gel electrophoresis in sodium dodecylsulfate and dithiothreitol; incorporation of radioactivity was determined by gel slicing and liquid scintillation spectrometry. Appearance of [3-H]leucine in circulating erythrocytes reached a peak at 1-3 days, with a steady decline thereafter. The radioactive amino acid appeared first in the lowest molecular weight peptides and last in the largest peptides; at the earliest time point (8 h), little radioactivity was observed in any of the four largest peptides present in the membranes (bands A, 1, 2 and 3). Certain smaller peptides (bands 4, 5 and 9) were the predominant species labeled at this time. By 24 h all peptides showed significant incorporation. With maturation of the red cells, label largely disappeared from bands A, 9 and several smaller peptides; this was confirmed by finding that the peptides are virtually absent from mature circulating erythrocytes. These data are interpreted as showing that red cell membrane proteins are synthesized asynchronously during the life cycle of the erythrocyte; the largest peptides are made predominantly in the earlier marrow stages of development, while certain of the smaller peptides are still being synthesized in the reticulocyte stage. Several membrane proteins appear to be specific to the reticulocyte and are lost during the process of cell maturation in the circulation.     

Increase of 2,3-bisphosphoglycerate synthase/phosphatase during maturation of reticulocytes with high 2,3-bisphosphoglycerate content

In the rabbit and in the rat, which possess erythrocytes with high concentration of 2,3-bisphosphoglycerate, the 2,3-bisphosphoglycerate synthase activity increases more than two fold during reticulocyte maturation. Isolation of the enzymes with 2,3-bisphosphoglycerate synthase activity present in extracts of reticulocytes and mature erytrocytes by ion exchange fast liquid chromatography shows that the increase in the synthase activity is due to the accumulation of the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase (EC 2.7.5.4/EC 3.1.3.13) which represents more than 80% of the synthase activity of the cell extracts. During reticulocyte maturation phosphoglycerate mutase (EC 5.4.2.1), which makes a small contribution to the 2,3-bisphosphoglycerate synthase activity in the erythroid cells, decreases in the rabbit and remains constant in the rat.     

Alteration in the protein components of catecholamine-sensitive adenylate cyclase during maturation of rat reticulocytes

The maturing rat reticulocyte was used as a model system in which to study developmental changes in the protein components of hormone-sensitive adenylate cyclase. Plasma membranes from rat erythrocytes display 10 to 20% of the adenylate cyclase activity and 30 to 50% of the beta-adrenergic receptors which are measured in membranes from rat reticulocytes, as noted by others. Reticulocyte membranes also display equal activities in response to (-)-isoproterenol in the presence of either GTP or GTP gamma S, whereas erythrocyte membrane adenylate cyclase is twice as active in the presence of isoproterenol plus GTP gamma S as in the presence of isoproterenol plus GTP. We have studied this system in greater detail by developing or applying independent assays for the catalytic protein (C) and the guanine nucleotide-binding regulatory protein (G/F) of adenylate cyclase. C was assayed in membranes by its intrinsic Mn2+-stimulated activity. It was also measured by reconstituting membranes with saturating amounts of GTP gamma S-activated G/F, yielding an operationally defined Vmax for the catalyst. By either assay, reticulocytes display about 3-fold greater C activity than do erythrocytes. G/F was assayed by its ability to confer GTP gamma S-stimulated activity upon C (which was supplied by membranes of cyc- S49 lymphoma cells). This assay indicates that reticulocyte membranes contain about 3 times as much G/F as do erythrocyte membranes. Cholera toxin and [32P]NAD were used to [32P]ADP-ribosylate the 45,000- and 52,000-dalton subunits of G/F. Total incorporation of 32P into these subunits decreased 3- to 4-fold with reticulocyte maturation. The ratio of label in the 52,000-dalton peptide to that in the 45,000-dalton peptide decreased from 0.29 in reticulocyte membranes to 0.14 in erythrocyte membranes. The apparently coordinate decrease in the amounts of C, G/F, and beta-adrenergic receptors suggest that the stoichiometry between these components is maintained during maturation, and may account for the decrease in adenylate cyclase in the membranes. However, the qualitative changes in responsiveness to hormones in the presence of GTP or GTP gamma S may be related to loss or proteolysis of the 52,000-dalton subunit of G/F.     

Maturational loss of the vitamin C transporter in erythrocytes

Erythrocytes have the same intracellular concentration of ascorbate as plasma, which is much lower than that of nucleated cells. To determine why erythrocytes are unable to concentrate ascorbate, we tested for the presence of ascorbate transporters in these cells. Human erythrocytes had very low rates of uptake of radiolabeled ascorbate, which was accounted for by the lack of ascorbate transporter SVCT2 in immunoblots. Using a cell culture model of Friend virus-infected mouse erythroblasts, immunoblots showed that the SVCT2 was present in the erythroblast stages, but was lost following extrusion of the nucleus in the formation of the reticulocyte stage. Rates of specific ascorbate transport correlated with the presence of the SVCT2. These results show that mature erythrocytes fail to concentrate ascorbate due to the loss of SVCT2 during maturation in the bone marrow.     

Ultrastructural localization of glycogen in erythrocytes and developing erythrocytic cells in normal human bone marrow

Summary The periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) reaction was employed for the ultrastructural cytochemical localization of saliva-labile glycogen in the erythrocytic cells in normal human blood and bone marrow. Particulate glycogen was demonstrated in the cytoplasm of all developmental forms of erythrocytic cells from the proerythroblast through the reticulocyte; a few particles of glycogen also were present in mature erythrocytes even in the peripheral blood. Statistical evaluation of the number of glycogen particles in mid-plane cell sections at each morphological stage of development indicated a significant and stepwise decrease during cellular maturation. This change in glycogen content may reflect both cellular utilization and mitosis during the maturational sequence.Supported by Grant No. SR01AM 12084-15 from the National Institutes of Health, Bethesda, Maryland.Appreciation is expressed to Anita Topson, Barbara Speakmon and Marjorie Griffith for their technical assistance and to Dr. Gerald King for performing the bone marrow aspirations.     

2,3-Bisphosphoglycerate,fructose, 2,6-bisphosphate and glucose 1,6-bisphosphate during maturation of reticulocytes with low 2,3-bisphosphoglycerate content

In contrast to the species with erythrocytes of high 2,3-bisphosphoglycerate content, in the sheep the concentration of 2,3-bisphosphoglycerate decreases during maturation of reticulocytes. The decrease can be explained by the drop of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios that result from the decline of phosphofructokinase, pyruvate kinase, phosphoglycerate mutase and the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase. The concentrations of fructose 2,6-bisphosphate and aldohexose 1,6-bisphosphates also decrease during sheep reticulocyte maturation in parallel to the 6-phosphofructo 2-kinase and the glucose 1,6-bisphosphate synthase activities.     

Transfer RNA in reticulocyte maturation

The disappearance of tRNA during the maturation of rabbit reticulocytes under the stress of phenylhydrazine-induced hemolysis was studied. The tRNA content of reticulocytes and of erythrocytes derived from them was compared. The results show that tRNA persists longer after reticulocyte maturation than ribosomes and than the ability to incorporate amino acids into protein. Considerable uniformity of tRNA degradation was noted with about 15% of the tRNA for most amino acids remaining after reticulocyte maturation. The half-life of tRNA in the maturing cells is estimated to be 50--60 h. There is little tRNA lacking the 3'-terminal pCpCpA moiety in cells derived from reticulocytes.     

Induction of the ATP-dependent proteolytic system in guinea pig reticulocyte lysates by triiodothyronine

The mechanism involved in the decreased numbers of several trans-membrane proteins such as sodium pump sites, sodium-lithium countertransport, sodium potassium cotransport proteins, proteins mediating the passive efflux of sodium and insulin receptors in erythrocytes from patients with hyperthyroidism is not known. The ATP-dependent proteolytic system which is involved in the loss of trans-membrane proteins during the maturation of the reticulocyte may be involved in the accelerated loss of these membrane proteins. Therefore, the effect of thyroid hormones on the ATP-dependent proteolytic activity of reticulocyte lysates was examined in this study. Reticulocytosis was induced in 14 guinea pigs by phenylhydrazine hydrochloride injections for 5 consecutive days followed by 2 days of rest. T3 (10 micrograms/100 g body weight) was injected into 7 animals on day 4 and day 6. Reticulocyte-rich blood was withdrawn on day 8. Oxygen consumption determined 24 hours after injection of T3 was 25% higher (p less than 0.01) and T3 treated animals had a 2.5 fold higher (p less than 0.01) weight loss than control animals. The ATP-dependent proteolytic activity measured in reticulocyte lysates using 125I labelled lysozyme was 3.6 fold higher in the T3 than in the control group of guinea pigs (p less than 0.01). We conclude that thyroid hormones induce the ATP-dependent proteolytic activity of reticulocyte lysates which may be responsible for the reduced number of several trans-membrane proteins found in erythrocytes from patients with hyperthyroidism.     

Transferrin receptors during rabbit reticulocyte maturation.

Experiments were performed to examine the fate of transferrin receptors in reticulocytes as these cells mature in vivo to erythrocytes. Reticulocytosis, synchronized by administration of actinomycin D, was induced in adult rabbits. Simultaneous measurements were made of haematological parameters and the interaction between transferrin and reticulocytes while the cells matured in vivo to erythrocytes. As the reticulocytes matured there was a parallel decline in their ability to take up transferrin and transferrin iron. At the same time, there was a proportionate decrease in the density of receptors for transferrin on the reticulocyte surface. The affinity of the receptors for transferrin remained unaltered during the maturation process. It was concluded that the inability of erythrocytes to take up transferrin or its iron is due primarily to the loss of transferrin receptors from the maturing reticulocyte surface.     

Transferrin receptors during rabbit reticulocyte maturation

Experiments were performed to examine the fate of transferrin receptors in reticulocytes as these cells mature in vivo to erythrocytes. Reticulocytosis, synchronized by administration of actinomycin D, was induced in adult rabbits. Simultaneous measurements were made of haematological parameters and the interaction between transferrin and reticulocytes while the cells matured in vivo to erythrocytes. As the reticulocytes matured there was a parallel decline in their ability to take up transferrin and transferrin iron. At the same time, there was a proportionate decrease in the density of receptors for transferrin on the reticulocyte surface. The affinity of the receptors for transferrin remained unaltered during the maturation process. It was concluded that the inability of erythrocytes to take up transferrin or its iron is due primarily to the loss of transferrin receptors from the maturing reticulocyte surface.     

Biogenesis of erythrocyte membrane proteins in vivo studies in anemic rabbits

To study the process of red cell membrane protein synthesis we have followed the time course of [³H]leucine appearance in total protein and individual peptides of the erythrocyte membrane following injection of the amino acid into phenylhydrazine-anemic rabbits. Multiple peripheral blood samples were taken from single animals over a 5-week period. Erythrocyte membrane proteins were separated by polycrylamide gel electrophoresis in sodium dodecylsulfate and dithiothreitol; incorporation of radioactivity was determined by gel slicing and liquid scintillation spectrometry. Appearance of [³H]leucine in circulating erythrocytes reached a peak at 1–3 days, with a steady decline thereafter. The radioactive amino acid appeared first in the lowest molecular weight peptides and last in the largest peptides; at the earliest time point (8 h), little radioactivity was observed in any of the four largest peptides present in the membranes (bands A, 1, 2 and 3). Certain smaller peptides (bands 4, 5 and 9) were the predominant species labeled at this time. By 24 h all peptides showed significant incorporation. With maturation of the red cells, label largely disappeared from bands A, 9 and several smaller peptides; this was confirmed by finding that the peptides are virtually absent from mature circulating erythrocytes. These data are interpreted as showing that red cell membrane proteins are synthesized asynchronously during the life cycle of the erythrocyte; the largest peptides are made predominantly in the earlier marrow stages of development, while certain of the smaller peptides are still being synthesized in the reticulocyte stage. Several membrane proteins appear to be specific to the reticulocyte and are lost during the process of cell maturation in the circulation.     

Analysis by partitioning in aqueous two-phase systems of the loss of transferrin-binding capacity during maturation of rat reticulocytes

A decrease in the number of binding sites for¹²⁵I-transferrin, without an apparent modification of the association constant, has been observed during the maturation of reticulocytes into erythrocytes. As an experimental model, different red cell populations from phenylhydrazinic anaemic rates (95% to 12% reticulocyte-rich) have been used. The fractionation by multiple partition in two-phase systems of these red cell populations has been applied here to show the relationship between number of transferrin receptors and rate of reticulocyte maturation.