Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes

Rats were injected with⁵⁹Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2–7.4 in the former case and 6.4–6.7 in the latter. Red cells were then centrifuged (5) and approximately 7–10% of the packed cells from the top and 7–10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2–7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4–6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4–6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.     

Separation of rabbit red cells by density in a bovine serum albumin gradient and correlation of red cell density with cell age after in vivo labeling with 59-Fe

Rabbit red cells are separated by centrifuging them for one hour at 33,000 G in density gradient tubes of bovine serum albumin. The separation represented an equilibrium situation since rebanding experiments showed that the cells from a layer would again seek that density layer when recentrifuged in a new gradient tube. When rabbits were injected with ⁵⁹Fe, the radioactive red cells at one day were nearly all light, but these labeled cells moved into the more dense layers over the next few days. This not only shows that the separation by density is discriminating but that some red cells became dense very quickly. Bearing in mind the problems of interpreting radioactive iron data because of the possibility of reutilization, it is tentatively concluded that dense red cells are not necessarily senescent red cells since these dense cells appear to persist for the normal life span of the rabbit red cell.     

Modulators of macrophage transferrin or transferrin-like protein

The effects of endotoxin or testosterone on the amount of transferrin ⁵⁹Fe (or like protein) in the murine macrophages was investigated. The mouse peritoneal macrophages were laden with ⁵⁹Fe tagged red cells following the injection of mice with either agent. After harvesting the cells, they were lysed and the transferrin iron was released with 40% trichloroacetic acid. The supernatant (extract transferrin iron) and the pellet (other iron proteins iron) were separated by centrifugation and their radioactivities counted. The results were expressed in percentage. The endotoxin group had a geometric mean of transferrin ⁵⁹Fe of 0.14% compared to 0.28% for the control, p < 0.001. The geometric mean for transferrin ⁵⁹Fe of the testosterone treated group was 0.51% compared to 0.35% for the control, p < 0.05. Therefore, the endotoxin seems to contract the transferrin pool whereas testosterone seems to expand it.     

The Wavy Erythropoiesis of Developing Chick Embryos. Isolation of Each Wave by a Differential Lysis and Identification of the Constituent Erythroid Types

Erythroid carbonic anhydrase (CA) activity of chick embryos from the third day of incubation to the egg hatching has been determined. Five minor activity peaks with maxima at 3, 6, 9, 15 and 17 days of development and a major one with maximum at 19 days have been found. The correlation between the peak distribution and the timing of release into the blood stream of waves of newly produced erythroid cells has been demonstrated on the basis of the following observations: 1) a linear correlation exists between red cell maturation and increase of CA activity; 2) chick red cells undergo lysis in the "Ørskov" medium when their CA activity exceeds a threshold value (23±3 Units/10⁹ red cells); and 3) the lysis kinetics of red cells in the Ørskov medium is proportional to their CA content. We have thus been able to distinguish the immature erythroid forms from the mature ones on the basis of their behaviour in the Ørskov medium. In the blood of developing chick embryos, we have found waves of newly produced red cells at about 2, 4, 7, 10, 16 and 18 days of development.
The same experimental criteria allowed us to detect the waves of red cell production in the erythropoietic organs. One wave has been detected in the blood islands at about 2 days; four waves in the yolk sac at about 5, 6, 11 and 15 days; two waves in the spleen at about 18 and 20 days; two waves in the bone marrow at about 19 days of incubation and 1 day after hatching.
Primitive erythroid cells are produced in the first two waves: that of blood islands at 2 days and that of yolk sac at 5 days. Definitive red cells are produced in the other waves with the exception of the second wave of spleen and of the second wave of bone marrow, which are constituted by red cells of adult type.     

Melittin lysis of red cells

Summary This paper describes experiments designed to explore interactions between human red blood cell membranes and melittin, the main component of bee venom. We found that melittin binds to human red cell membranes suspended in isotonic NaCl at room temperature, with an apparent dissociation constant of 3×10^–8 m and maximum binding capacity of 1.8×10⁷ molecules/cell. When about 1% of the melittin binding sites are occupied, cell lysis can be observed, and progressive, further increases in the fraction of the total sites occupied lead to progressively greater lysis in a graded manner. 50% lysis occurs when there are about 2×10⁶ molecules bound to the cell membrane. For any particular extent of melittin binding, lysis proceeds rapidly during the first few minutes but then slows and stops so that no further lysis occurs after one hour of exposure of cells to melittin. The graded lysis of erythrocytes by melittin is due to complete lysis of some of the cells, since both the density and the hemoglobin content of surviving, intact cells in a suspension that has undergone graded melittin lysis are similar to the values observed in the same cells prior to the addition of melittin. The cells surviving graded melittin lysis have an increased Na and reduced K, proportional to the extent of occupation of the melittin binding sites. Like lysis, Na accumulation and K loss proceed rapidly during the first few minutes of exposure to melittin but then stops so that Na, K and hemoglobin content of the cells remain constant after the first hour. These kinetic characteristics of both lysis and cation movements suggest that melittin modifies the permeability of the red cell membrane only for the first few minutes after the start of the interaction. Direct observation of cells by Nomarsky optics revealed that they crenate, become swollen and lyse within 10 to 30 sec after these changes in morphology are first seen. Taken together, these results are consistent with the idea that melittin produces lysis of human red cells at room temperature by a colloid osmotic mechanism.     

Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes. Relevance to their fractionation by centrifugation.

Rats were injected with 59Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2-7.4 in the former case and 6.4-6.7 in the latter. Red cells were then centrifuged (5) and approximately 7-10% of the packed cells from the top and 7-10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2-7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4-6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4-6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.     

Studies of murine erythroid cell development. Synthesis of heme and hemoglobin

Techniques of cell separation were used to isolate murine erythroid precursors at different states of maturation. Cells were studied before and after short-term incubation in the presence or absence of erythropoietin. Complementary results were obtained by direct examination of the cell fractions and by the short-term culture experiments. Indices of heme synthesis, including incorporation of 59Fe or [2-14C]glycine into heme and activity of delta-aminolevulinic acid synthetase, were already well developed in the least mature cells, chiefly pronormoblasts. Activity then rose moderately in the cell fractions consisting primarily of basophilic and polychromatophilic normoblasts, and fell off with further increases in cell maturity. On short-term culture in the presence of erythropoietin, activity declined with increasing cell maturation except in the least mature fraction where the original level of activity was maintained. By contrast, synthesis of labeled hemoglobin ([3H]leucine) was very low in the least mature cell fractions and rose progressively with increasing cell maturity. The rate of hemoglobin synthesis increase in cells at all stages of maturation when cultured in the presence of erythropoietin. Despite the different patterns observed for heme synthesis and hemoglobin synthesis, both synthetic activities were consistently higher in cells cultured with erythropoietin as compared to controls. These findings suggest that erythropoietin stimulates biochemical differentiation of erythroid precursors at various stages of maturation. They also demonstrate an asynchronism between heme synthesis and hemoglobin syhthesis; heme synthesis is already well developed in the least mature erythroid cells and begins to diminish as the capacity for hemoglobin synthesis continues to rise.     

AN EARLY RESPONSE OF THE MORPHOLOGICALLY RECOGNIZABLE ERYTHROID PRECURSORS TO BLEEDING

⁵⁵Fe autoradiography of the peripheral red blood cells has been used to study the proliferation of the recognizable erythroid precursors in bled animals. The transit time of the recognizable erythroid precursors present in the bone marrow and labelled with ⁵⁵Fe 6 hr before bleeding, remains unchanged, but the number of red cells produced by these precursors is significantly greater than normal. It is deduced that the increased red cell production is brought about by an increase in the number of divisions that the cells undergo during maturation and that a shortening in the red cell cycle time is implied. The possibility that the transit time of the progeny of cells differentiating into pro-erythroblasts after bleeding may be shorter than the transit time of the precursors already differentiated before bleeding, is briefly discussed.     

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.     

A radioautographic study of the incorporation of iron 55 by the ameloblasts in the zone of maturation of rat incisors

This study was designed to study the time course of the incorporation of 55Fe into the ameloblasts of maturation in rat incisors. Male Sherman rats (100 +/- 5 gm) were injected intravenously with 0.9 mCi of 55Fe and sacrificed in pairs by perfusion at various time intervals from 5 min to 7 days after injection. The incisors were demineralized in 4.13% disodium EDTA, postfixed in 1% osmium tetroxide in veronal acetate buffer, and embedded in Epon. Incisors from control rats injected with only physiological saline were treated in the same way. Sections from blocks of tissue in the zone of maturation were prepared for light microscope radioautographic observations. The greatest incorporation of iron occurred at 9 mm within the zone; at this site the ameloblasts contained few pigment granules. About 5 mm deeper into the zone the activity fell off to zero, as observed at 2.5 hr after injection of 55Fe. Between 1 day and 7 days after injection the 55Fe labeling was found over the cells containing many pigment granules, while the initial labeling over the cells within 9 mm of the zone had diminished. These data have shown that at any given time, from 30 min to 4 hr, the iron enters the maturation ameloblasts over a wide extent of the zone, reaching a maximum at about 9 mm from the onset of maturation. However, at longer times (1 day to 7 days) the labeling curve shifts and shows the greatest activity beyond 9 mm within the zone.     

Transferrin receptors,iron transport and ferritin metabolism in friend erythroleukemia cells

Four aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of erythroid differentiation by dimethyl sulfoxide. (1) The binding of ¹²⁵I-labeled transferrin was determined over a range of transferrin concentrations from 0.5 to 15 μM. Scatchard analysis of the binding curves demonstrated equivalent numbers of transferrin binding sites per cell: 7.78 ± 2.41 · 10⁵ in non-induced cells and 9.28 ± 1.57 · 10⁵ after 4 days of exposure to dimethyl sulfoxide. (2) The rate of iron transport was determined by measuring iron uptake from ⁵⁹Fe-labeled transferrin. Iron uptake in non-induced cells was approx. 17 000 molecules of iron/cell per min; 24 h after addition of dimethyl sulfoxide it increased to 38 000, and it rose to maximal levels of approx. 130 000 at 72 h. (3) Heme synthesis, assayed qualitatively by benzidine staining and measured quantitatively by incorporation of ⁵⁹Fe or [2-¹⁴C]glycine into cyclohexanone-extracted or crystallized heme, was not detected until 3 days after addition of dimethyl sulfoxide, when 12% of the cells were stained by benzidine and 6 pmol ⁵⁹Fe and 32 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. After 4 days, 60% of the cells were benzidine positive and 34 pmol ⁵⁹Fe and 90 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. (4) The rate of incorporation of ⁵⁹Fe into ferritin, measured by immunoprecipitation of ferritin by specific antimouse ferritin immunoglobulin G, rose from 4.4 ± 0.6 cells to 18.4 ± 1.3 pmol ⁵⁹Fe/h per 10⁸ cells 3 days after addition of dimethyl sulfoxide, and then fell to 11.6 ± 3.1 pmol 4 days after dimethyl sulfoxide when heme synthesis was maximal. These studies indicate that one or more steps in cellular iron transport distal to transferrin binding is induced early by dimethyl sulfoxide and that ferritin may play an active role in iron delivery for heme synthesis.     

RENEWAL OF TASTE BUD CELLS IN RAT CIRCUMVALLATE PAPILLAE

The life span of taste bud cells in rat circumvallate papillae was measured by autoradiography after labeling them with a pulse of [³H]thymidine. Specimens of circumvallate papillae were taken daily 1·5-18·5 days after the isotope was administered; thereafter, specimens were taken on alternate days until 25·5 days. For each time interval, the number of labeled cell nuclei was counted in 200-450 taste buds and plotted as the ratio of labeled cells/taste bud v. time after injection of [³H]TdR. In all, 6958 taste buds were counted. The total number of labeled cells (dark plus light) per taste bud reached peaks at 6·5, 13·5 and 20·5 days. The curve for the number of labeled dark cells/bud had essentially the same shape as that for total cells. The number of labeled light cells/bud reached a modest peak at 6·5 days and slowly declined to a plateau for the remainder of the experiment. The data show that an average of 2 days elapsed after injection before labeled dark cells entered the bud and they spent an average of 7 days in the non-proliferating taste bud compartment; thus, the life span of the dark cell was 9 days. The life span of the light cell was difficult to estimate quantitatively, but this cell type was labeled at a much slower rate than dark cells and is assumed to have a significantly longer tenure in the taste bud.     

Hepatic subcellular metabolism of heme from heme-hemopexin: incorporation of iron into ferritin

The subcellular distribution and metabolic fate of [⁵⁹Fe]heme-[¹²⁵I]-labeled hemopexin after receptor-mediated interaction with the liver was examined in the rat. After intravenous injection, [⁵⁹Fe]heme from the complex and ⁵⁹Fe from hepatic catabolism of this heme accumulate in the liver and undergo changes in their subcellular distribution over 2 hours. The amounts of [⁵⁹Fe]heme and particularly of ⁵⁹Fe increase in the cytosol while remaining constant or decreasing in membranous fractions. In contrast, [¹²⁵I]-labeled hemopexin associated with the liver during heme transport is always a small fraction of the dose and is not measurably catabolized under these conditions.Gel filtration of the cytosol showed that ⁵⁹Fe increased linearly with time in a high molecular weight fraction which was identified immunologically as ferritin. We conclude that heme transported by hemopexin is metabolized by the liver and the iron conserved.     

Translocation of iron from soybean cotyledons

Soybean seeds, Glycine max L. Merrill, were produced by plants treated from anthesis to seed maturity with ⁵⁹Fe supplied as ferric ethylenediaminedi (o-hydroxyphenylacetate). Seed coats accounted for 7.4% of dry seed weight and had Fe concentrations 5 times greater than the embryos. After germinating 2 days, cotyledons contained 69.6% and radicles 5.0% of original seed Fe. Fractions of seed Fe unavailable to seedlings were: 19.8% in seed coats, 1.7% in germination paper, 0.1% in the water under germinating seeds, and 3.8% unaccounted for. Every 3 days seedlings received nutrient solution without Fe or with 10 μm ferric ethylenediaminedi (o-hydroxyphenylacetate) and developed as deficient Fe or normal Fe plants. The deficient Fe cotyledons on day 18 retained 13% of the labeled Fe originally present. Cotyledons of normal Fe plants retained 50 to 70% of their original Fe. Moreover, cotyledons of the normal Fe plants accumulated externally supplied Fe and finally contained twice the quantity of Fe originally present. Stem exudate collected above cotyledons of deficient Fe plants contained 5.3 μm⁵⁹Fe. Electrophoresis of exudate showed that most of the ⁵⁹Fe migrated anodically as a single band and was in the position of ferric citrate.     

Maturation of nascent DNA fragment is disturbed after treatment of cultured mouse FM3A cells with 8-methoxypsoralen plus near-ultraviolet radiation

By the method of sedimentation in 5–20% alkaline sucrose gradient, the process of maturation of the nascent DNA fragment was studied with cultured mouse FM3A cells treated with 8-methoxypsoralen plus near-ultraviolet radiation. This treatment is known to cause crosslinks of the chromosomal DNA strands. The profile of the newly-replicated DNA, labeled for 10 min with [³H]thymidine immediately after treatment, was the same as that of the untreated cells, where the incorporated radioactivity was present in the intermediate DNA fragment (about 50–80 S). But, when the treated cells were labeled after several hours of incubation, the labeled DNA became much shorter due to inhibition of maturation of the initial DNA fragment (the Okazaki fragment) to the intermediate DNA. With the use of aphidicolin, a specific inhibitor of eukaryotic DNA polymerase α, it became apparent that, in addition to formation of the crosslinks, further DNA replication is required to cause this inhibition of DNA maturation. Aphidicolin also suppressed the inhibition of incorporation of [³H]thymidine into cellular DNA after treatment, but inhibition of this incorporation resumed after its removal.     

Effect of primary leaves on 59Fe uptake by roots and 59Fe distribution in the shoot of iron sufficient and iron deficient bean (Phaseolus vulgaris L.) plants

A study has been made on the effect of primary leaves on iron (Fe) distribution in the shoot. Bean (Phaseolus vulgaris L.) seedlings were precultured in nutrient solution with 8×10^-5 M FeEDTA for 4 days, and then grown further with either 8×10^-5 M FeEDTA (+Fe) or without Fe supply (-Fe) for another 5 days. Thereafter, both +Fe and -Fe plants were treated in three different ways: undisturbed; one primary leaf removed; or one primary leaf shaded, starting two hours before supply ⁵⁹FeEDTA to the roots. The +Fe plants were supplied with 8×10^-5 M ⁵⁹FeEDTA, and the -Fe plants with only 1×10^-6 M ⁵⁹FeEDTA. After 1 to 8 hour uptake periods, plants were harvested and ⁵⁹Fe in different organs was determined. Removal or shading of one primary leaf did not affect ⁵⁹Fe uptake by roots and ⁵⁹Fe translocation to the shoot in +Fe plants. In the -Fe plants, however, removal of one primary leaf decreased ⁵⁹Fe uptake by roots, whereas shading of one primary leaf had no effect on ⁵⁹Fe uptake but slightly enhanced ⁵⁹Fe translocation from roots to the shoot. The quantity of ⁵⁹Fe in primary leaves was positively correlated with quantity of ⁵⁹Fe in the stem in the -Fepplants, but not in the +Fe plants. In both, the +Fe and -Fe plants, the quantity of ⁵⁹Fe in the shoot apex was positively correlated with ⁵⁹Fe in primary leaves. The results suggest that irrespective of the Fe nutritional status of plants, the source of Fe for the shoot apex is Fe retranslocated from primary leaves.     

Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles

We have developed a new procedure for introducing macromolecules into cultured mammalian cells based on osmotic lysis of pinocytic vesicles. Cells are first incubated in culture medium containing 0.5 M sucrose, 10% polyethylene glycol 1000 and the macromolecule to be transferred. Cells are then placed in medium diluted with 0.66 parts water. Most pinocytic vesicles formed in the presence of sucrose burst in hypotonic medium, thereby releasing the enclosed macromolecule. L929 cells remain fully viable after a single hypertonic sucrose treatment, and a majority survives four successive rounds of osmotic lysis. This procedure, termed osmotic lysis of pinosomes, has been used to transfer substantial amounts of horseradish peroxidase, antiricin antibodies and dextran 70,000 into the cytosol of L929 cells. Direct comparison of the degree of ricin resistance conferred by transfer of antiricin antibodies revealed pinosome lysis to be equal, if not superior, to injection mediated by red blood cells.     

Iodine metabolism of the endostyle of larval lampreys,ammocoetes of Lampetra japonica

Summary Experiments were carried out to study the iodine metabolism of the endostyle of the larval lamprey which is considered to be homologous to the thyroid gland. Larval lampreys, ammocoetes of Lampetra japonica were intraperitoneally injected with 200 c of Na ¹²⁵I; their endostyles were removed 30 minutes, 1, 2, 4, 6, 8 and 24 hours after the treatment. Type 1 and type 4 cells (Marine) were almost inactive in binding iodine. Silver grains appeared within 30 minutes after the injection over the apical cell membrane including the surfaces of microvilli and cilia of type 2 c and type 3 cells. These grains increased in number until 2 hours. A few of apical small vesicles of the same cells were labeled 1 to 2 hours after the injection. Small dense granules large dense bodies, and multivesicular bodies in the type 2 c and type 3 cells were labeled especially at 6 to 24 hours. The ratio in number of the labeled dense granules, or bodies to the unlabeled ones tended to increase markedly with time. Large or small vacuoles, dense or light in the cytoplasm of some type 5 cells which lack indications of protein-synthesis sign in the cytoplasm were labeled 6 to 24 hours after the injection of ¹²⁵I, and the number of the labeled vacuoles increased with time. From these facts, we conclude that: (1) iodination of the thyroglobulin of type 2c and type 3 cells takes place almost entirely at the apical cell membrane region, (2) the thyroglobulin-like protein contained in the apical small vesicles of type 2c and type 3 cells is slightly iodinated, (3) although it is difficult to determine whether the dense granules and bodies, which might be lysosomes, are secretory substances or reabsorbed materials, the possibility of the occurrence of reabsorption and hydrolysis of the thyroglobulin in the type 2c and type 3 cells should be considered, and (4) reabsorption of the thyroglobulin from the endostylar lumen by some type 5 cells should be also considered.     

Changes in alanine and glutamine transport during rat red blood cell maturation

Alanine and glutamine transport have been studied during red blood cell maturation in the rat. Kinetic parameters of Na⁺-dependent L-alanine transport were:K _m 0.43 and 1.88 mM andV _max 158 and 45 nmoles/ml ICW/min for reticulocytes and erythrocytes, respectively. During red cell maturation in the rat there is a loss of capacity and affinity of the system ASC for L-alanine transport. The values for Na⁺-dependent L-glutamine transport in reticulocytes wereK _m 0.51 mM andV _max 157 nmoles/ml ICW/min. On the other hand, a total loss of L-glutamine transport mediated by both N and ASC systems is demonstrated in mature red cells. This seems to indicate that during rat red cell maturation the system N disappears. Furthermore, the system ASC specificity in mature cells changes, and glutamine enters the red cell by non-mediated diffusion processes.     

Ferric pyridoxal isonicotinol hydrazone can provide iron for heme synthesis in reticulocytes

We have examined whether reticulocytes depleted of transferrin might incorporate ⁵⁹Fe from ⁵⁹Fe-labelled pyridoxan isonicotinoyl hydrazone (PIH). Transferrin-depleted reticulocytes showed a time-, temperature- and concentration-dependent incorporation of ⁵⁹Fe when incubated with 20–200 μM ⁵⁹Fe-PIH. The amount of ⁵⁹Fe incorporated with 200 μM ⁵⁹Fe-PIH is equal to or higher than that taken up from transferrin at 20 μM ⁵⁹Fe concentration. After 60 min about 60% of the ⁵⁹Fe taken up by the cells is recovered in heme while the remainder is probably still bound to PIH. 1 mM succinyl acetone (a specific inhibitor of heme synthesis) inhibits PIH-mediated incorporation of ⁵⁹Fe into heme by about 79% indicating that ⁵⁹Fe from ⁵⁹Fe-PIH is incorporated into de novo synthesized protoporphyrin. As is the case with transferrin, erythrocytes do not incorporate ⁵⁹Fe from ⁵⁹Fe-PIH. Pretreatment of reticulocytes with pronase does not inhibit their ability to incorporate ⁵⁹Fe from ⁵⁹Fe-PIH, suggesting that, unlike the uptake of Fe from transferrin, membrane receptors are not involved in the uptake of Fe-PIH by the cells.