84
Biochimica et Biophysica Acta, 5 8 4 ( 1 9 7 9 ) 8 4 - - 9 3 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28859
THE RATE LIMITING STEP IN THE RETICULOCYTE UPTAKE OF T R A N S F E R R I N AND T R A N S F E R R I N IRON EFFECTS OF SOME INCUBATION VARIABLES
J A I M E M A R T f N E Z - M E D E L L f N * a n d L I L I A B E N A V I D E S **
Ddpartment de Pddiatrie, Centre de Recherche Pddiatrique, H6pital Sainte-Justine, Montrdal, Qudbec (Canada) and Departamento de Biologia, Facultad de Ciencias, Universidad Nacional, Aut6noma de Mdxico, Mdxico 20, D.F. (Mexico) (Received August 16th, 1978)
Key words: Transferrin; Iron uptake; Iron metabolism; (Reticulocyte)
Summary Reticulocytes incubated in an isotonic NaC1 saline medium containing glucose, glutamine and amino acids, were able to detach both iron atoms from all the transferrin incorporated by them. In the absence of these metabolites, although transferrin uptake was the same, the reticulocytes failed to remove completely the iron from the transferrin which they incorporated. It has been shown before that there is unspecific as well as specific binding of transferrin to the reticulocyte. By incubating the cells in the presence of a high concentration of bovine serum albumin, we have been able to prevent the unspecific attachment of transferrin. At least 94% of the iodinated transferrin was capable of donating its iron to the reticulocytes.
Introduction The uptake of iron from transferrin by reticulocytes can be conveniently represented as occurring in two steps: (1) Transferrin uptake by the cell. Transferrin present in the medium binds to transferrin-specific reticulocyte membrane receptors [1--7], and is inter-
* To w h o m c o r r e s p o n d e n c e should be addressed at: D e p a r t a m e n t o de Biologia, Facultad de Ciencias, U.N.A.M., M~xico 20, D.F., Mexico, ** Present address: D e p a r t a m e n t o de Sistemas Biol6gicos, Divisi6n de C.B.S., U.A.M.-X, Mexico, D.F.
85 nalized [8--12], in a temperature and energy-dependent process [ 12,13], probably by pinocytosis [14,15]. The internalization of transferrin has been disputed [3,4,16--19]. (2) Cell-mediated iron release from transferrin. This energy-dependent process has been correlated with reticulocyte intracellular ATP concentration [20]. Although the mechanism of iron release is still unknown, the possibility of a cell-mediated destabilization of the bicarbonate-iron-transferrin ternary complex at the level of the bicarbonate molecule has been proposed [21--25]. Although these two steps have been studied, mainly by in vitro cell incubations, many of their mechanisms still remain to be settled. In the present paper, the overall rate-limiting step of the process was studied under a variety of cell-incubation conditions. The parameters considered were: presence or absence of energy-generating sources and amino acids; and addition of a protein, other than transferrin, (bovine serum albumin) to the medium. Materials and Methods Rabbit transferrin was purified as previously described [9]. Na12SI, carrier free, and SgFeC13, 2 - 4 0 Ci/g Fe, were purchased from New England Nuclear. Red blood cells (20--30% reticulocytes) were obtained from chronically bled anemic rabbits supplemented with iron (Imferon, kindly supplied by Fissons, Ltd., Canada) [26]. In some experiments, after centrifugation of the blood, only the reticulocyte-enriched upper fourth volume of the packed red cell b u t t o n was collected, resulting in a reticulocyte concentration of 40--55%. Transferrin was saturated with SgFe [9] as demonstrated by titration [27], and iodinated with 12sI using lactoperoxidase (Calbiochem) coupled to CNBractivated Sepharose 4B (Pharmacia) [28]. Approximately 0.8 iodine atoms were incorporated for each transferrin molecule. Red blood cells were washed in isotonic NaC1 saline, 0.160 M, before using them. They were incubated as a 25% cell suspension in the described media in the presence of doubly labelled transferrin. Aliquots from the cell incubation mixture were withdrawn at indicated times, and the reaction was stopped by a 20-fold dilution with cold isotonic saline. The cells were washed three times in saline at 4 ° C, and assayed for SgFe and 12sI in a two-channel automatic gamma counting system. Incubation media. The media assayed are described below. Salt concentration was varied as stated in order to maintain osmolarity constant, as measured in a Model 3 W osmometer (Advanced Instruments Inc.). Isotonicity was taken to be 0.3 osmolar, which corresponds to that of the rabbit sera measured. The pH of Tris (Sigma)-HC1 buffers was adjusted at the temperature to be used. Transferrin concentration was always 2 0 . 1 0 -6 M, except where otherwise stated. Isotonic NaC1 media, final concentration; medium A, 0.152 M NaCI, 0.01 M Tris-HC1 (pH 7.5); medium B, 0.144 M NaC1, 0.01 M Tris-HC1 (pH 7.5), 6.11 • 10 -a M glucose; and an amino acid mixture (fully described below); medium C, 0.136 M NaC1, 0.01 M Tris-HCl (pH 7.5), 6.11 • 10 -a M glucose; an amino acid mixture; and 25.5 mg/ml of albumin. A m i n o acid mixture. An amino acid mixture was prepared, containing all the
86 amino acids necessary for hemoglobin synthesis in their proper ratios, with the exception of glutamine, which is in excess. Final concentrations (mM) are as follows, L-alanine, 0.846; L-arginine, 0.188; L-aspartic acid, 0.721; L-cysteine, 0.063; L-glutamine, 2.69; glycine, 0.627; L-histidine, 0.596; L-isoleucine, 0.125; L-leucine, 1.098; L-lysine, 0.753; L-methionine, 0.063; L-phenylalanine, 0.502; L-proline, 0.345; L-serine, 0.690; L-threonine, 0.502; L-tryptophan, 0.094; L-tyrosine, 0.188; L-valine, 0.909. A 10-fold concentrated mixture is prepared by dissolving the amino acids in warm distilled water. After adjusting the pH 7.5 with 1 M NaOH, the mixture kept frozen at --20°C in 1 ml aliquots. Before using, the aliquots may be warmed in a bath to dissolve any precipitate formed. The 1-fold amino acid concentration is calculated to be approximately 12 • 10 -3 osmolar. C o n c e n t r a t e d b o v i n e s e r u m a l b u m i n solution. 8 g of albumin (Cohn fraction V, Sigma Chemical Co.) were dissolved in 100 ml of 0.02 M citrate buffer (pH 5.1), and passed through an SP Sephadex C-50 (Pharmacia) column (0.9 X 30 cm), previously equilibrated in the same buffer. Any bovine serum transferrin present is retained by the column under these conditions [9,29], while albumin passes through. The column effluent was concentrated (Aquacide III, Calbiochem), adjusted to pH 7.5 with 1 M NaOH, and dialyzed against 0.01 M Tris-HC1 (pH 7.5). After dialysis, protein concentration was adjusted to 85 mg/ ml with the above Tris-HC1 buffer, using an wl~ ~2s0,m of 6.6 [30]. Results
Reticulocytes incubated in an isotonic simple medium have an initial iron and transferrin uptake similar to those incubated in an energy-generating medium (Fig. 1), b u t very rapidly their capacity to remove iron from transferrin decreases, such that by 90 min of incubation they have incorporated only a b o u t 60% of the total iron in the control (Fig. 1, Table I). The capacity for iron removal from transferrin of reticulocytes incubated in the energy-generating medium (Fig. 1) is such, that they incorporate iron at an initial linear rate equivalent to 1.56 • l 0 s atoms/min per reticulocyte. Assuming its full utilization for hemoglobin synthesis, 4 . 5 . 1 0 -8 mol of hemoglobin chains are formed/ml of packed red cells (23% reticulocytes) per h. This justifies the presence in our enriched medium, of an amino acid concentration sufficient to sustain that level of protein synthesis for at least 4 h. In Fig. 1 and Table I we can also see that as soon as iron removal from transferrin becomes the rate-limiting step, the amount of transferrin b o u n d to the cell starts to increase until it reaches a new, slightly higher plateau. This indicates that either its uptake or its release, or both, have been modified, until the rates approach a new equilibrium. The presence of albumin in the incubation medium lowers the total uptake of transferrin by the reticulocytes without modifying the rate of iron uptake (Figs. 2 and 3). Transferrin attached to the cells in excess of that incorporated in the presence of albumin, must be considered nonspecifically bound, as albumin is able to compete for its cell-binding sites, and it does n o t deliver its iron to the reticulocyte. The initial cell uptake of transferrin and transferrin iron was further analysed
87
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Biochimica et Biophysica Acta, 5 8 4 ( 1 9 7 9 ) 8 4 - - 9 3 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 28859
THE RATE LIMITING STEP IN THE RETICULOCYTE UPTAKE OF T R A N S F E R R I N AND T R A N S F E R R I N IRON EFFECTS OF SOME INCUBATION VARIABLES
J A I M E M A R T f N E Z - M E D E L L f N * a n d L I L I A B E N A V I D E S **
Ddpartment de Pddiatrie, Centre de Recherche Pddiatrique, H6pital Sainte-Justine, Montrdal, Qudbec (Canada) and Departamento de Biologia, Facultad de Ciencias, Universidad Nacional, Aut6noma de Mdxico, Mdxico 20, D.F. (Mexico) (Received August 16th, 1978)
Key words: Transferrin; Iron uptake; Iron metabolism; (Reticulocyte)
Summary Reticulocytes incubated in an isotonic NaC1 saline medium containing glucose, glutamine and amino acids, were able to detach both iron atoms from all the transferrin incorporated by them. In the absence of these metabolites, although transferrin uptake was the same, the reticulocytes failed to remove completely the iron from the transferrin which they incorporated. It has been shown before that there is unspecific as well as specific binding of transferrin to the reticulocyte. By incubating the cells in the presence of a high concentration of bovine serum albumin, we have been able to prevent the unspecific attachment of transferrin. At least 94% of the iodinated transferrin was capable of donating its iron to the reticulocytes.
Introduction The uptake of iron from transferrin by reticulocytes can be conveniently represented as occurring in two steps: (1) Transferrin uptake by the cell. Transferrin present in the medium binds to transferrin-specific reticulocyte membrane receptors [1--7], and is inter-
* To w h o m c o r r e s p o n d e n c e should be addressed at: D e p a r t a m e n t o de Biologia, Facultad de Ciencias, U.N.A.M., M~xico 20, D.F., Mexico, ** Present address: D e p a r t a m e n t o de Sistemas Biol6gicos, Divisi6n de C.B.S., U.A.M.-X, Mexico, D.F.
85 nalized [8--12], in a temperature and energy-dependent process [ 12,13], probably by pinocytosis [14,15]. The internalization of transferrin has been disputed [3,4,16--19]. (2) Cell-mediated iron release from transferrin. This energy-dependent process has been correlated with reticulocyte intracellular ATP concentration [20]. Although the mechanism of iron release is still unknown, the possibility of a cell-mediated destabilization of the bicarbonate-iron-transferrin ternary complex at the level of the bicarbonate molecule has been proposed [21--25]. Although these two steps have been studied, mainly by in vitro cell incubations, many of their mechanisms still remain to be settled. In the present paper, the overall rate-limiting step of the process was studied under a variety of cell-incubation conditions. The parameters considered were: presence or absence of energy-generating sources and amino acids; and addition of a protein, other than transferrin, (bovine serum albumin) to the medium. Materials and Methods Rabbit transferrin was purified as previously described [9]. Na12SI, carrier free, and SgFeC13, 2 - 4 0 Ci/g Fe, were purchased from New England Nuclear. Red blood cells (20--30% reticulocytes) were obtained from chronically bled anemic rabbits supplemented with iron (Imferon, kindly supplied by Fissons, Ltd., Canada) [26]. In some experiments, after centrifugation of the blood, only the reticulocyte-enriched upper fourth volume of the packed red cell b u t t o n was collected, resulting in a reticulocyte concentration of 40--55%. Transferrin was saturated with SgFe [9] as demonstrated by titration [27], and iodinated with 12sI using lactoperoxidase (Calbiochem) coupled to CNBractivated Sepharose 4B (Pharmacia) [28]. Approximately 0.8 iodine atoms were incorporated for each transferrin molecule. Red blood cells were washed in isotonic NaC1 saline, 0.160 M, before using them. They were incubated as a 25% cell suspension in the described media in the presence of doubly labelled transferrin. Aliquots from the cell incubation mixture were withdrawn at indicated times, and the reaction was stopped by a 20-fold dilution with cold isotonic saline. The cells were washed three times in saline at 4 ° C, and assayed for SgFe and 12sI in a two-channel automatic gamma counting system. Incubation media. The media assayed are described below. Salt concentration was varied as stated in order to maintain osmolarity constant, as measured in a Model 3 W osmometer (Advanced Instruments Inc.). Isotonicity was taken to be 0.3 osmolar, which corresponds to that of the rabbit sera measured. The pH of Tris (Sigma)-HC1 buffers was adjusted at the temperature to be used. Transferrin concentration was always 2 0 . 1 0 -6 M, except where otherwise stated. Isotonic NaC1 media, final concentration; medium A, 0.152 M NaCI, 0.01 M Tris-HC1 (pH 7.5); medium B, 0.144 M NaC1, 0.01 M Tris-HC1 (pH 7.5), 6.11 • 10 -a M glucose; and an amino acid mixture (fully described below); medium C, 0.136 M NaC1, 0.01 M Tris-HCl (pH 7.5), 6.11 • 10 -a M glucose; an amino acid mixture; and 25.5 mg/ml of albumin. A m i n o acid mixture. An amino acid mixture was prepared, containing all the
86 amino acids necessary for hemoglobin synthesis in their proper ratios, with the exception of glutamine, which is in excess. Final concentrations (mM) are as follows, L-alanine, 0.846; L-arginine, 0.188; L-aspartic acid, 0.721; L-cysteine, 0.063; L-glutamine, 2.69; glycine, 0.627; L-histidine, 0.596; L-isoleucine, 0.125; L-leucine, 1.098; L-lysine, 0.753; L-methionine, 0.063; L-phenylalanine, 0.502; L-proline, 0.345; L-serine, 0.690; L-threonine, 0.502; L-tryptophan, 0.094; L-tyrosine, 0.188; L-valine, 0.909. A 10-fold concentrated mixture is prepared by dissolving the amino acids in warm distilled water. After adjusting the pH 7.5 with 1 M NaOH, the mixture kept frozen at --20°C in 1 ml aliquots. Before using, the aliquots may be warmed in a bath to dissolve any precipitate formed. The 1-fold amino acid concentration is calculated to be approximately 12 • 10 -3 osmolar. C o n c e n t r a t e d b o v i n e s e r u m a l b u m i n solution. 8 g of albumin (Cohn fraction V, Sigma Chemical Co.) were dissolved in 100 ml of 0.02 M citrate buffer (pH 5.1), and passed through an SP Sephadex C-50 (Pharmacia) column (0.9 X 30 cm), previously equilibrated in the same buffer. Any bovine serum transferrin present is retained by the column under these conditions [9,29], while albumin passes through. The column effluent was concentrated (Aquacide III, Calbiochem), adjusted to pH 7.5 with 1 M NaOH, and dialyzed against 0.01 M Tris-HC1 (pH 7.5). After dialysis, protein concentration was adjusted to 85 mg/ ml with the above Tris-HC1 buffer, using an wl~ ~2s0,m of 6.6 [30]. Results
Reticulocytes incubated in an isotonic simple medium have an initial iron and transferrin uptake similar to those incubated in an energy-generating medium (Fig. 1), b u t very rapidly their capacity to remove iron from transferrin decreases, such that by 90 min of incubation they have incorporated only a b o u t 60% of the total iron in the control (Fig. 1, Table I). The capacity for iron removal from transferrin of reticulocytes incubated in the energy-generating medium (Fig. 1) is such, that they incorporate iron at an initial linear rate equivalent to 1.56 • l 0 s atoms/min per reticulocyte. Assuming its full utilization for hemoglobin synthesis, 4 . 5 . 1 0 -8 mol of hemoglobin chains are formed/ml of packed red cells (23% reticulocytes) per h. This justifies the presence in our enriched medium, of an amino acid concentration sufficient to sustain that level of protein synthesis for at least 4 h. In Fig. 1 and Table I we can also see that as soon as iron removal from transferrin becomes the rate-limiting step, the amount of transferrin b o u n d to the cell starts to increase until it reaches a new, slightly higher plateau. This indicates that either its uptake or its release, or both, have been modified, until the rates approach a new equilibrium. The presence of albumin in the incubation medium lowers the total uptake of transferrin by the reticulocytes without modifying the rate of iron uptake (Figs. 2 and 3). Transferrin attached to the cells in excess of that incorporated in the presence of albumin, must be considered nonspecifically bound, as albumin is able to compete for its cell-binding sites, and it does n o t deliver its iron to the reticulocyte. The initial cell uptake of transferrin and transferrin iron was further analysed
87
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