The effect of acid pH and citrate on the release and exchange of iron on rat transferrin

The effect of acid pH and citrate on the exchange of iron between binding sites of rat transferrin has been studied. In the absence of citrate, diferric transferrin shows stepwise loss of iron atoms with the first atom of iron released at approximately pH 5.2. Citrate at physiologic concentrations (1 · 10^?3 M) or greater allows random iron removal at pH 6.5 or less. Iron dissociation from monoferric transferrin at acid pH, with or without citrate, is a random process. At pH 7.4, randomization of iron on transferrin takes from 3 to 6 h in the presence of millimolar concentrations of citrate. We conclude that at acid pH and in the presence of citrate concentrations likely to occur in vivo in the rat there is little scrambling of iron bound to transferrin.     

Humic acid and iron uptake by plants

Summary The behaviour of ferric EDTA and ferric citrate in nutrient solution and their interaction with humic acid was investigated at various hydrogen ion concentrations using the technique of membrane ultrafiltration to separate small iron species from high molecular weight products of hydrolysis and to estimate the binding of iron by humic acid. Ferric EDTA was found to be of small molecular size at all pH values between 5.0 and 7.0 whilst ferric citrate solutions contained an increasing proportion of high molecular weight material as pH was increased from 5.0 to 7.0. Some iron present in solutions of both ferric EDTA and ferric citrate was bound by humic acid at all pH values from 5.0 to 7.0. Studies were also made of the uptake of iron by wheat roots from nutrient solutions containing either ferric EDTA or ferric citrate and of the effect of humic acid on uptake. More iron was absorbed from ferric EDTA than from ferric citrate at all pH values. Increasing pH between 5.0 and 7.0 resulted in a progressive decrease in the uptake of iron in both cases. The presence of humic acid depressed iron absorption from both solutions at all pH values.     

Studies of the binding of different iron donors to human serum transferrin and isolation of iron-binding fragments from the N- and C-terminal regions of the protein.

1. Trypsin digestion of human serum transferrin partially saturated with iron(III)-nitrilotriacetate at pH 5.5 or pH 8.5 produces a carbohydrate-containing iron-binding fragment of mol.wt. 43000. 2. When iron(III) citrate, FeCl3, iron (III) ascorabate and (NH4)2SO4,FeSO4 are used as iron donors to saturate the protein partially, at pH8.5, proteolytic digestion yields a fragment of mol.wt. 36000 that lacks carbohydrate. 3. The two fragments differ in their antigenic structures, amino acid compositions and peptide 'maps'. 4. The fragment with mol.wt. 36000 was assigned to the N-terminal region of the protein and the other to the C-terminal region. 5. The distribution of iron in human serum transferrin partially saturated with various iron donors was examined by electrophoresis in urea/polyacrylamide gels and the two possible monoferric forms were unequivocally identified. 6. The site designated A on human serum transferrin [Harris (1977) Biochemistry 16, 560--564] was assigned to the C-terminal region of the protein and the B site to the N-terminal region. 7. The distribution of iron on transferrin in human plasma was determined.     

Uptake and subcellular processing of 59Fe-125I-labelled transferrin by rat liver.

The uptake of transferrin and iron by the rat liver was studied after intravenous injection or perfusion in vitro with diferric rat transferrin labelled with 125I and 59Fe. It was shown by subcellular fractionation on sucrose density gradients that 125I-transferrin was predominantly associated with a low-density membrane fraction, of similar density to the Golgi-membrane marker galactosyltransferase. Electron-microscope autoradiography demonstrated that most of the 125I-transferrin was located in hepatocytes. The 59Fe had a bimodal distribution, with a larger peak at a similar low density to that of labelled transferrin and a smaller peak at higher density coincident with the mitochondrial enzyme succinate dehydrogenase. Approx. 50% of the 59Fe in the low-density peak was precipitated with anti-(rat ferritin) serum. Uptake of transferrin into the low-density fraction was rapid, reaching a maximal level after 5-10 min. When livers were perfused with various concentrations of transferrin the total uptakes of both iron and transferrin and incorporation into their subcellular fractions were curvilinear, increasing with transferrin concentrations up to at least 10 microM. Analysis of the transferrin-uptake data indicated the presence of specific transferrin receptors with an association constant of approx. 5 X 10(6) M-1, with some non-specific binding. Neither rat nor bovine serum albumin was taken up into the low-density fractions of the liver. Chase experiments with the perfused liver showed that most of the 125I-transferrin was rapidly released from the liver, predominantly in an undegraded form, as indicated by precipitation with trichloroacetic acid. Approx. 40% of the 59Fe was also released. It is concluded that the uptake of transferrin-bound iron by the liver of the rat results from endocytosis by hepatocytes of the iron-transferrin complex into low-density vesicles followed by release of iron from the transferrin and recycling of the transferrin to the extracellular medium. The iron is rapidly incorporated into mitochondria and cytosolic ferritin.     

The effect of lysosomotrophic bases and inhibitors of transglutaminase on iron uptake by immature erythroid cells

The mechanism by which weak bases block iron uptake by immature erythroid cells was investigated using rabbit and rat reticulocytes and erythroblasts from the fetal rat liver. A large variety of bases was found to inhibit iron uptake but to have a much smaller or no effect on transferrin uptake by the cells. Quinacrine and chloroquine were active at the lowest concentrations. Dansylcadaverine, an inhibitor of transglutaminase, was also active at low concentration. However, the results do not indicate a role for transglutaminase in the iron uptake process. Instead they show that the major effect of the bases is to inhibit iron release from transferrin molecules on or within the cells. The possible mechanism of this effect was investigated by measurement of intracellular ATP levels, intracellular pH and by morphological studies utilizing fluorescent and electron microscopy. The bases caused little change in ATP levels, but elevated intracellular pH, probably due to accumulation within intracellular vesicles, which were shown to accumulate fluorescent weak bases, to swell under the action of the bases and to be the site of intracellular localization of transferrin. It is concluded that the bases tested in this work inhibit iron release from transferrin in intracellular vesicles by increasing their pH rather than by blocking transglutaminase and thereby restricting endocytosis. Reduction of transferrin uptake by the cells when it occurs is probably due to inhibition of recycling of transferrin receptors to the outer cell membrane.     

Factors influencing the uptake of iron and plutonium into cells

Uptake of 59Fe as well as 125I-labelled Fe-transferrin into HeLa cells points to the existence of a limited number of specific binding sites. This is in contrast to hepatocytes and hepatoma cells (Hep G2) where metal uptake from transferrin is very low, not saturable and cannot be prevented by an excess of the protein. Iron uptake into these cells is much higher from the citrate complex. The same is true for plutonium uptake into rat hepatocytes, while the uptake of this metal into Hep G2 cells is very small regardless of the ligand. In contrast to iron, plutonium presented as citrate is taken up into HeLa cells much better than plutonium presented as transferrin. The uptake of both metals from the citrate complex requires a high activation energy and can be prevented only by inhibition of oxidative phosphorylation. Other processes such as endocytosis, intactness of microtubuli, assembly of microfilaments or pH of the lysosomes do not seem to be of importance. Metal uptake from the citrate complex can be prevented only by the presence of other chelating agents and/or by transferrin. It can be assumed, therefore, that the metals react directly with constituents of the cell membrane, a process in which chelating agents can successfully compete if they form strong enough complexes with the metals.     

Release of iron from diferric transferrin in the presence of rat liver plasma membranes: no evidence of a plasma membrane diferric transferrin reductase

The transfer of iron from diferric transferrin to bathophenanthroline disulfonate was measured under varying conditions by spectrophotometry and EPR spectroscopy. Intact rat hepatocytes efficiently mediated the transfer of iron from human diferric transferrin to bathophenanthroline disulfonate. Isolated rat liver plasma membranes, in contrast, failed to facilitate the reaction at pH 7.4 in the presence of NADH, although the membranes were able to reduce ferricyanide and to oxidize NADH. Oxidation of NADH was stimulated by diferric transferrin. However, ferricyanide reductase and transferrin-stimulated NADH oxidase activities were apparently not linked to release of iron from transferrin. Our results, together with theoretical considerations, show that the ability (or inability) of intact cells or isolated plasma membranes to facilitate the transfer of iron from transferrin to strong diferric iron chelators does not allow interferences about the existence of an iron reduction step as part of the process of cellular uptake of iron from transferrin.     

Light microscopic, non-immunologic demonstration of iron-binding proteins in hematopoietic cells

Iron-binding proteins were localized by their saturation with iron using iron nitrilotriacetate (FeNTA), maintenance of protein-iron-binding at specific values of pH, and visualization of the iron with acid ferrocyanide (AF). Human neutrophilic cells showed strong blue granular and diffuse cytoplasmic staining. Human mid- and late-stage erythroblasts showed moderate diffuse cytoplasmic staining. Monocytes and macrophages showed reactions similar to those seen with AF technique alone. Other hematopoietic cells showed minimal or no stain positivity. Nuclear positivity was not observed in any cells. Concanavalin A (ConA) treatment of purified neutrophils reduced their FeNTA-AF positivity; supernatants from these cells showed precipitin lines of identity with anti-lactoferrin (Lf) stainable with FeNTA and AF. Cellulose acetate electrophoresis of crude neutrophil extracts treated with [59Fe]NTA showed multiple protein bands; one band co-migrated with purified Lf and showed autoradiographic positivity. Rabbit heterophils and rat neutrophils showed less FeNTA-AF positivity, consistent with less Lf in these cells than in human neutrophils. Washing smears with 0.1 M citrate, pH 6.0, between FeNTA and AF treatments eliminated only erythroblast positivity; 0.1 M citrate, pH 4.0, ablated neutrophil staining as well. Ferritin-hemosiderin staining was preserved at both values of pH. These results indicate that FeNTA-AF technique specifically visualizes neutrophil Lf, and suggest that the observed erythroblast positivity is due to transferrin (Tf).     

Growth of human tumor cell lines in transferrin-free,low-iron medium

Summary Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake, mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates (Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either transferrin (5 μg/ml) or a higher concentration of ferric citrate (500 μM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth in transferrin-free medium with a substantially lower concentration of ferric citrate (5 μM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron. Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in transferrin-free, low-iron medium (5 μM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine, inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the well-characterized receptor-mediated endocytosis process.     

Effect of extracellular hemin on hemoglobin and ferritin content of erythroleukemia cells

Mouse (MEL) and human (K-562) erythroleukemia cell lines can be induced to undergo erythroid differentiation, including hemoglobin (Hb) synthesis, by extra cellular hemin. In order to study the effect of extracellular hemin on intracellular ferritin and Hb content, we have used Mossabauer spectroscopy to measure the amount of 57Fe incorporated into ferritin or Hb and a fluorescent enzyme-linked immunosorbent assay (ELISA) to measure the ferritin protein content. When K-562 cells were cultured in the presence of a 57Fe source either as transferrin or citrate, in the absence of a differentiation inducer, all the intracellular 57Fe was detected in ferritin. When the cells were cultured in the presence of 57Fe-hemin, 57Fe was found in both ferritin and Hb. 57Fe in ferritin increased rapidly, and after 2 days it reached a plateau at 5 X 10(-14) g/cell. 57Fe in Hb increased linearly with time and reached the same value after 12 days. Addition of other iron sources such as iron-saturated transferrin, iron citrate, or iron ammonium citrate caused a much lower increase in ferritin protein content as compared to hemin. When K-562 cells were induced by 57Fe-hemin in the presence of 56Fe-transferrin, 57Fe was found to be incorporated in equal amounts into both ferritin and Hb. However, when the cells were induced by 56Fe-hemin in the presence of 57Fe-transferrin, 57Fe was incorporated only into ferritin, but not into Hb, which contained 56Fe iron. These results indicate that in K-562 cells, when hemin is present in the culture medium it is preferentially incorporated into Hb, regardless of the availability of other extra- or intracellular iron sources such as transferrin or ferritin. In MEL cells induced to differentiate by dimethylsulfoxide (DMSO) a different pattern of iron incorporation was observed; 57Fe from both transferrin and hemin was found to incorporate in ferritin as well as in Hb.     

The effects of an antibody to the rat transferrin receptor and of rat serum albumin on the uptake of diferric transferrin by rat hepatocytes

The role of high-affinity specific transferrin receptors and low-affinity, non-saturable processes in the uptake of transferrin and iron by hepatocytes was investigated using fetal and adult rat hepatocytes in primary monolayer culture, rat transferrin, rat serum albumin and a rabbit anti-rat transferrin receptor antibody. The intracellular uptake of transferrin and iron occurred by saturable and non-saturable mechanisms. Treatment of the cells with the antibody almost completely eliminated the saturable uptake of iron but had little effect on the non-saturable process. Addition of albumin to the incubation medium reduced the endocytosis of transferrin by the cells but had no significant effect on the intracellular accumulation of iron. The maximum effect of rat serum albumin was observed at concentrations of 3 mg/ml and above. At a low incubation concentration of transferrin (0.5 microM), the presence of both rat albumin and the antibody decreased the rate of iron uptake by the cells to about 15% of the value found in their absence, but to only 40% when the diferric transferrin concentration was 5 microM. These results confirm that the uptake of transferrin-bound iron by both fetal and adult rat hepatocytes in culture occurs by a specific, receptor-mediated process and a low-affinity, non-saturable process. The low-affinity process increases in relative importance as the iron-transferrin concentration is raised.     

The preparation, properties, and metabolism of 14C-bicarbonate-labelled transferrin

¹⁴C-bicarbonate-labelled transferrin was prepared in order to study the role of bicarbonate in the cell-mediated release of iron from transferrin. ¹⁴C-bicarbonate bound to transferrin only in the presence of iron and with a ratio of bound bicarbonate to bound iron of one. The transferrin-¹⁴C-bicarbonate complex was very stable in Tris-HCl buffered at pH 7.5–9.0 even in the presence of excess non-radioactive bicarbonate. However, oxalate, citrate, and phosphate promoted a rapid exchange of transferrin-bound ¹⁴C-bicarbonate with bicarbonate present in the medium.Rabbit reticulocytes effected a temperature-dependent release of ¹⁴C-bicarbonate from transferrin at the same rate at which they incorporated ⁵⁹Fe from transferrin — suggesting the existence of a coordinated mechanism in the cells for the release of both iron and bicarbonate from transferrin.     

Effect of iron (III) in the presence of various ligands on the phagocytic and metabolic activity of human polymorphonuclear leukocytes

FeCl3 or Fe(III) that attached to chelating ligands such as citrate or nitrilotriacetic acid (NTA) at a molar ratio of 1:1 had a toxic effect on PMN. Uptake of radiolabeled Staphylococcus aureus by PMN, preincubated for 2 hr at 37 degrees C in a medium containing Fe(III)-citrate or Fe(III)-NTA, was significantly lower than that of control PMN preincubated without excess iron (p less than 0.002). However, at a 1:2 molar ratio of Fe(III) to citrate or NTA, the iron was not toxic. In contrast, the iron-liganding molecules transferrin and deferoxamine protected the PMN against the noxious effect of iron at concentrations just high enough to sequester all the iron. Fe(III) increased the generation of luminol chemiluminescence by stimulated PMN, whereas the oxygen consumption of the cells was not altered in the presence of Fe(III); this suggests a catalytic effect of iron on the production by PMN of oxygen metabolites at some step beyond the formation of superoxide. No effect of iron was observed when the incubation was performed at 4 degrees C, nor when an oxygen-radical scavenger such as thiourea, mannitol, or catalase was present in the incubation medium. Also, Fe(III) had much less effect on the phagocytic function of PMN of a patient with chronic granulomatous disease. The results indicate that the Fe(III)-induced defect in the phagocytic capacity of PMN depends on the nature and the concentration of the ligand attached to the iron ion, and also suggest that the noxious effect of iron on the PMN function is a result of its ability to catalyze the generation of toxic oxygen species by these cells.     

The receptor for transferrin on murine myeloma cells: one-step purification based on its physiology, and partial amino acid sequence

The receptor for transferrin is one of the major surface proteins of proliferating lymphocytes and other cells. It binds ferrotransferrin from serum and endocytoses it into an acidic nonlysosomal intracellular compartment where iron is released, but in which apotransferrin remains tightly bound to its receptor. Recycling of the apotransferrin-receptor complex to the cell surface is associated with a return to neutral pH and concomitant loss of affinity of apotransferrin for its receptor. Apotransferrin is then free to leave the cell and initiate a new cycle. We have exploited this cycle in a novel method for the purification of the receptor for transferrin. Murine myeloma cells were lysed in nonionic detergent, and the lysate passed over a column of ferrotransferrin-agarose at pH 7.4. After washing with sodium acetate at pH 5.0, iron was removed with sodium citrate pH 5.0 and desferrioxamine. Upon returning the pH to neutrality, the receptor was eluted and found to be homogeneous by SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. The degree of purification was estimated to be at least 3,000-fold, and the calculated yield was 10 to 20%. The purified receptor was capable of binding to transferrin. The receptor was digested with trypsin, and the resulting peptides were separated by reversed-phase high performance liquid chromatography in NH4HCO3. Selected peptides were rechromatographed in 0.1% trifluoroacetic acid, and their amino acid sequences were determined.     

Iron exchange between transferrin molecules mediated by phosphate compounds and other cell metabolites.

The ability of a large number of cellular metabolites to release iron from transferrin was investigated by measuring the rate at which they could mediate iron exchange between two types of transferrin. Rabbit transferrin labelled with 59Fe was incubated with human apotransferrin in the presence of the metabolites. After varying periods of incubation the human transferrin was separated from the rabbit transferrin by immunoprecipitation. GTP, 2,3-diphosphoglycerate, ATP, ADP and citrate produced the most rapid exchange of iron between the two types of transferrin, but many other compounds showed some degree of activity. Iron exchange mediated by the organic phosphates had the characteristics of a single first-order reaction and was sensitive to changes of incubation temperature and pH. The activation energy for the exchange reaction was approx. 13 kcal/mol. The rate of iron exchange from the oxalate - iron - transferrin complex was much lower than from bicarbonate - iron - transferrin. It is concluded that several organic phosphates have the capacity of releasing iron from transferrin. These compounds may represent the means by which the iron is released during the process of cellular uptake.     

Involvement of transferrin in the reduction of iron by the transplasma membrane electron transport system

Nonpermeable electron acceptors can be reduced by a transplasma membrane electron transport system in suspensions of intact cells. Here we report that diferric transferrin is reduced by HeLa S3 cells. The reduction is recorded spectrophotometrically as the formation of the ferrous complex of bathophenanthroline disulfonate. Ferric ammonium citrate can also be used as an electron acceptor, and the presence of low concentrations of diferric transferrin greatly stimulates the reduction of trivalent iron under these conditions. Likewise very low concentrations of ferricyanide, which does not give rise to a ferrous bathophenanthroline disulfonate complex formation, have a strong stimulatory effect on the complex formation when ferric ammonium citrate is the source of ferric iron. Apotransferrin is a potent inhibitor of the reaction. The inhibition occurs at the concentration necessary for complete occupancy of the transferrin receptors. The inhibition can be demonstrated also when high concentrations of ferricyanide are used as electron acceptor. The possible mechanism behind the reported phenomena is discussed, and it is concluded that the transplasma membrane electron transport system can be involved in the process of cellular iron uptake.     

Studies on the mechanism of iron release from transferrin.

Iron release from human, rabbit, rat and sheep transferrin, chicken conalbumin and human lactoferrin was measured by the change in absorbance of solutions of the iron-protein complexes or by the release of 59Fe from the protein conjugated to agarose. Several phosphatic compounds and iron chelators were able to mediate the process (ATP, GTP, 2,3-diphosphoglycerate, inositol hexaphosphate, pyridoxal 5-phosphate, cytidine 5-triphosphate, pyrophosphate, inorganic phosphate, citrate, EDTA, oxalate, nitrilotriacetate). The greatest rate of iron release was found with pyrophosphate and the least with inorganic phosphate. Different rates of iron release were obtained with the different proteins, greatest with human transferrin and least with lactoferrin. With each of the proteins and the mediators there was a linera relationship between the H+ concentration and the rate of iron release. At any given pH the rate of iron release increased to a maximal rate as the mediator concentration was raised. It is concluded that iron release from transferrin under the conditions of these experiments involves an initial interaction between H+ and the iron-transferrin complex followed by release of the iron under the action of the mediator.     

Regulation of HeLa cell transferrin receptors

HeLa cells were found to have a single class of non-interacting receptors specific for transferrin. Both apotransferrin and diferric transferrin competed equally with 125I-diferric transferrin for receptor binding. Transferrin binding was temperature-dependent and reversible. Binding of transferrin to cells exhibited a KD of 27 nM with a maximum binding capacity of 1.8-3.7 x 10(6) molecules/cell. Cells grown in the presence of diferric transferrin or in the presence of ferric ammonium citrate exhibited a concentration- and time-dependent decrease in 125I-diferric transferrin binding. The decrease in binding activity reflected a reduction in receptor number rather than an alteration in ligand receptor affinity. Growth of cells in saturating concentrations of apotransferrin did not cause a decrease in receptor number. When iron-treated cells were removed to media free of ferric ammonium citrate, the receptor number returned to control values by 40 h. When receptors were removed with trypsin, cells grown and maintained in ferric ammonium citrate-supplemented media demonstrated a rate of receptor reappearance 47% that of control cells grown in ferric ammonium citrate-free media. Cells grown in media supplemented with diferric transferrin or ferric ammonium citrate exhibited an increase in cytosolic iron content. The transferrin receptor number returned to normal after cells were removed to unsupplemented media, despite persistent elevation of cytosolic iron content. Increased iron content did not appear to be the sole factor determining receptor number.     

Iron uptake and heme synthesis by isolated rat liver mitochondria. Diferric transferrin as iron donor and the effect of pyrophosphate

Isolated rat liver mitochondria accumulate iron from fully saturated transferrin at neutral pH. With 5 microM iron as diferric transferrin, accumulation at 30 degrees C amounts to approx. 40 pmol/mg protein per h. With access to a suitable porphyrin substrate, 70-80% of the amount of iron accumulated is recovered in heme. Mobilization of iron and synthesis of heme both depend on a functioning respiratory chain. Vacant iron-binding sites on mono- and apotransferrin compete with the mitochondria for iron mobilized from transferrin. Pyrophosphate at concentrations in the range 10-50 microM enhances mobilization of iron, counterbalances the inhibitory effect of mono- and apotransferrin and enhances metallochelatase activity. The results emphasize the putative suitability of pyrophosphate as an intracellular iron-transport ligand in situ.     

Intravesicular pH and iron uptake by immature erythroid cells

The intravesicular pH of intact rabbit reticulocytes was measured by two methods; one based on the intracellular:extracellular distribution of DMO (5, 5, dimethyl + oxazolidin-2,4-dione), methylamine, and chloroquine and the other by quantitative fluorescence microscopy of cell-bound transferrin. The latter method was also applied to nucleated erythroid cells from the fetal rat liver. A pH value of approximately 5.4 was obtained with both methods and in both types of cells. Treatment of the cells with lysosomotrophic agents, metabolic inhibitors, and ionophores elevated the intravesicular pH and inhibited iron uptake from transferrin. When varying concentrations of NH4Cl were used, a close correlation was observed between the inhibition of iron uptake and elevation of the intravesicular pH. At pH 5.4 iron release from rabbit iron-bicarbonate transferrin in vitro was much more rapid than from iron-oxalate transferrin. The bicarbonate complex donates its iron to rabbit reticulocytes approximately twice as quickly as the oxalate complex. It is concluded that the acidic conditions within the vesicles provide the mechanism for iron release from the transferrin molecule after its endocytosis and that the low vesicular pH is dependent on cellular metabolism.