Effect of L-methionine and S-adenosylmethionine on growth of an adenine mutant of Saccharomyces cerevisiae

A pink, adenine-requiring yeast utilized adenine, hypoxanthine, or S-adenosylmethionine (SAM), in quantities up to 3 mumoles per 100 ml of medium, as equivalent sources of purine for cell growth, but not methylthioadenosine or S-adenosylhomocysteine. Utilization of SAM for growth was inhibited by the presence of l-methionine in quantities greater than 0.6 mumole per 100 ml of medium. However, 6 mumoles of l-methionine had no effect on growth when adenine or hypoxanthine was the source of purine. These sources also reversed the inhibitory effects of 6 mumoles of the amino acid on the utilization of SAM. The presence of 400 mumoles of the amino acid resulted in some inhibition of growth when the organisms were grown with adenine, hypoxanthine, or adenine plus SAM but had no effect on the total uptake of adenine-8-(14)C. Studies on the uptake of radioactivity from a mixture of SAM-adenine-8-(14)C and (3)H-labeled SAM-methyl indicated that these components were taken into the cells at different rates which were altered by the presence of l-methionine. The fixation of (35)S from (35)S-labeled adenosylmethionine into the cells was inhibited by the presence of the amino acid. The cells synthesized and accumulated SAM in the presence of 400 mumoles of l-methionine plus adenine even when exogenous SAM was supplied. Approximately 47% of radioactivity fixed from exogenous SAM-adenine-8-(14)C and 12% from (3)H-labeled SAM-methyl were found in reisolated SAM.     

Modulation of adenine nucleoside excretion and incorporation in adenosine deaminase deficient human lymphoma cells

The availability of a human lymphoma cell line deficient in adenosine deaminase, adenosine kinase and methylthioadenosine phosphorylase enabled us to compare the effects of nucleoside transport inhibitors on the excretion of endogenously generated adenosine, deoxyadenosine and 5'-methylthioadenosine. The nucleoside transport inhibitors nitrobenzylthioinosine and dipyridamole blocked the efflux of adenosine, but not deoxyadenosine or 5'-methylthioadenosine. The inhibitors also prevented the uptake of exogenous adenosine, but not deoxyadenosine or 5'-methylthioadenosine, by human lymphoblasts. The results show (i) that the transport inhibitors modify adenine nucleoside efflux and influx similarly, and (ii) that the effects of the compounds on the excretion and uptake of these three physiologically important adenine nucleosides are distinctly different.     

Effect of 5'-difluoromethylthioadenosine, an inhibitor of methylthioadenosine phosphorylase, on proliferation of cultured cells

5'-Methylthioadenosine (MTA) is formed from decarboxylated S-adenosylmethionine during biosynthesis of polyamines. This nucleoside is cleaved by methylthioadenosine phosphorylase (MTA Pase) to adenine and 5-methylthioribose-I-phosphate in mammalian cells. 5'-Difluoromethylthioadenosine (DFMTA), a synthetic analog of MTA, was not a substrate for MTA Pase, but was a strong competitive inhibitor of the enzyme (Ki = 0.48 microM). DFMTA caused marked accumulation of labeled MTA formed from [35S]methionine in Raji cells, which contain MTA Pase, but not in CCRF-CEM cells, which do not contain this enzyme, suggesting that it also inhibits the enzyme in intact cells. DFMTA inhibited the growth of a variety of cultured cells and its cytostatic effect was roughly proportional to the MTA Pase activity of the cells. MTA also depressed the growth of cultured cells but, in contrast with DFMTA, its inhibitory effect was greater in MTA Pase-deficient cells (CCRF-CEM) than MTA Pase-containing cells (Raji). Inhibition of growth of Raji cells by DFMTA was partially reversed by exogenous adenine, a reaction product of MTA Pase. These results suggest that the utilization of adenine formed from MTA was important for proliferation of cells containing MTA Pase under the culture conditions employed, and that DFMTA inhibited cell growth by inhibiting MTA Pase activity.     

Synthesis of purines in human lymphoblast cells deficient in methylthioadenosine phosphorylase activity

Two human lymphoblastic cell lines, deficient in methylthioadenosine phosphorylase (MTAP) activity, were found to have increased rates of de novo purine synthesis. These MTAP⁻ cell lines were K562, an undifferentiated leukemic line and CCRF-CEM, a leukemic line of T-cell origin. Another T-cell line, CCRF-HSB-2 was found to be deficient in activity. However, this line did not demonstrate elevated rates of purine synthesis. Purine metabolism in the above cell cultures was compared with MTAP⁺ human B-cell lines and two human T-cell lines (MOLT-3 and MOLT-4). In all the MTAP⁺ cell lines, the rate of de novo purine synthesis was inhibited by the presence of methylthioadenosine in the assay medium (10 μM concentration produced more than 90% inhibition). However, purine synthesis in the MTAP⁻ cells was resistant to inhibition by methylthioadenosine. Adenine in the assay medium inhibited de novo purine synthesis in MTAP⁺ and MTAP⁻ cells to a similar degree. This inhibition was dose dependent and was elicited by concentrations similar to those of methylthioadenosine. Growth of the cell lines in culture was not affected by either methylthioadenosine or adenine at the concentrations which produced inhibition of purine synthesis. These results suggest that purine synthesis in MTAP⁺ cells is inhibited by adenine formed from the phosphorolytic cleavage of methylthioadenosine by methylthioadenosine phosphorylase.     

Characterization of a defect in the pathway for converting 5'-deoxy-5'-methylthioadenosine to methionine in a subline of a cultured heterogeneous human colon carcinoma

5'-Deoxy-5'-methylthioadenosine (methylthioadenosine) is cleaved to adenine and 5-methylthioribose-1-phosphate (methylthioribose-1-P). Methylthioribose-1-P is converted to 2-keto-4-methylthiobutyrate ( ketomethylthiobutyrate ) which is transaminated to methionine. We report that one subline of a heterogeneous human colon carcinoma, DLD-1 Clone D, only forms methylthioribose-1-P from methylthioadenosine or 5'-deoxy-5'-methylthioinosine (methylthioinosine), a deaminated derivative of methylthioadenosine, whereas Clone A converts methylthioadenosine and methylthioinosine to methionine, as shown by growth studies in culture of Clone A and Clone D cells and radioactive studies utilizing [methyl-14C]methylthioadenosine or [methyl-14C]methylthioinosine in the presence of extracts of these cells lines. To characterize this defect, we utilized three protein fractions isolated from rat liver which together convert methylthioribose-1-P to ketomethylthiobutyrate . Addition of only Fraction A to Clone D sonicates restores its ability to convert methylthioadenosine to methionine. This fraction is responsible for converting methylthioribose-1-P to 5- methylthioribulose -1-phosphate; radioactive studies confirm this observation. Thus, Clone D is deficient in an enzyme contained in Fraction A; this represents a qualitative biochemical difference between the two clones derived from a single human tumor.     

Methylthioadenosine nucleoside phosphorylase deficiency in methylthio-dependent cancer cells.

The cleavage of the methylthio group from methylthioadenosine is shown to involve two enzymes, a nucleosidase which catalyses the phosphorolytic cleavage of methylthioadenosine to yield adenine and 5-methylthioribose-1-phosphate and an enzyme which uses the latter compound as substrate and catalyses the release of the methylthio group as an ether-extractable product. Three malignant murine hematopoietic cell lines which require methylthio group supplementation for proliferation in vitro are shown to lack methylthioadenosine nucleosidase activity while retaining activity of the second enzyme. Four cell lines which are methylthio-independent in vitro contain activity of both enzymes. The data suggest that the requirement for exogenous methylthio groups in certain cells is caused by the block in their biosynthetic pathway imposed by methylthioadenosine nucleoside phosphorylase deficiency. Secondarily, the data suggest that all cells require methylthio or related groups for division.     

Impaired formylation and uptake of tetrahydrofolate by rat small gut following cobalamin inactivation

The effect of inactivation of cobalamin by N2O on the intestinal absorption of folate was studied using rat everted gut sacs. Further, in view of uncertainties about the presence of methionine synthetase in gut [1], this enzyme was measured. Everted gut sacs were incubated with [2-14C]tetrahydrofolate, and the subsequent appearance of labelled formyl- and methyl [14C] tetrahydrofolate in everted segments of small intestine of rats was studied. Considerable methionine synthetase activity was present in washed everted gut sacs but not in gut segments in the absence of such treatment. Methionine synthetase activity declined after exposure to N2O, which oxidizes and inactivates cob(I)alamin. Folate uptake by gut sacs was not affected by 24 h exposure of the animals to N2O but fell significantly after 7 days exposure. There was a significant fall in the amount of formyltetrahydrofolate formed after cobalamin inactivation and this was reversed by supplying either methionine, methylthioadenosine or sodium formate. Serine had no effect. The data support the hypothesis that methionine and methylthioadenosine act by supplying single carbon units at the formate level of oxidation.     

Adenosine metabolism in kidney slices under normoxic conditions

The effect of adenosine in rat kidney under normoxic conditions has been studied. It is demonstrated that adenosine modulates cell nucleotide levels. HPLC analysis of the purine compounds inside the cell indicates that adenosine improves the ATP/ADP ratio, whereas it diminishes the adenine content. This behaviour is not due to mediation by specific receptors, as agonists at P1 purinoceptors did not have any effect. Further evidence using inosine as well as dipyridamole and deoxycoformycin indicates that all effects are dependent on the previous uptake of adenosine. The origin of free adenine in the kidney has been investigated, and it appears to come from the phosphorolysis of 5'-methylthio-adenosine. This report is the first to describe the activity of methylthioadenosine phosphorylase (E.C. 2.4.2.28) in the kidney. It is concluded that 1) extracellular adenosine improves guinea pig renal function by increasing the ATP level and the ATP/ADP ratio; and 2) there exists a functional pathway in the kidney that produces adenine and AMP coming from methionine and ATP. This latter pathway probably produces spermine and spermidine, which are likely to be important for renal function.     

Cleavage of adenosine 5''-monophosphate during uptake by Streptomyces griseus.

Unlabeled adenine brought about a (delayed) decrease in radioactivity that had been taken up by phosphate-limited resting cells of Streptomyces griseus from [14C]adenine-labeled adenosine 5'-monophosphate (AMP). Inorganic phosphate, on the other hand, stimulated adenine uptake from AMP, presumably by activating an energy-dependent active transport mechanism. Unlabeled phosphate rapidly diluted the uptake of radioactivity from [32P]AMP. Adenine inhibited uptake of [32P]AMP but not that of [32P]orthophosphate; adenine is thought to act by inhibiting the cleavage of AMP. The uptake of 32P and 14C from double-labeled AMP showed marked differences; 32P was taken up much faster into both cells and nucleic acids. These data indicate that uptake of AMP components takes place after extracellular dephosphorylation of the nucleotide.     

Biochemical genetic analysis of the role of methylthioadenosine phosphorylase in a murine lymphoid cell line

The enzyme methylthioadenosine phosphorylase functions in both purine and polyamine metabolism is dividing mammalian cells. To determine the effects of the loss of this enzyme on cell growth and metabolism, we selected two methylthioadenosine phosphorylase-deficient mutant clones of the transplantable murine T lymphoma cell line R1.1. The first had 3.5% of wild type methylthioadenosine phosphorylase activity. The second was completely enzyme-deficient. The loss of the enzyme did not alter the growth rate, cloning efficiency, or tumor-forming ability of the T lymphoma cells. The methylthioadenosine phosphorylase-deficient clones excreted substantial amounts of methylthioadenosine into the culture medium (0.13 and 0.32 nmol/h/mg of protein, respectively) and were unable to utilize the methylthioadenosine phosphorylase substrate 2',5'-dideoxyadenosine as a purine source when de novo purine synthesis was blocked. Spermine levels were 10-20% lower in the enzyme-deficient clones than in wild type cells. The loss of methylthioadenosine phosphorylase rendered the mutants exquisitely sensitive to the antiproliferative effects of methylthioadenosine. Methylthioadenosine at 3-6 microM inhibited their growth by 50%. The toxic effects of methylthioadenosine were not attributable to inhibition of purine, pyrimidine, or polyamine synthesis.     

5-Methylthioribose. Its effects and function in mammalian cells

The growth responses of 5-deoxy-5-methylthioribose on a 5'-deoxy-5'-methylthioadenosine phosphorylase containing cell line (BW5147) and the methylthioadenosine phosphorylase-deficient cell line (L1210D) were examined. Methylthioribose was shown to dramatically affect these cells, increasing their growth rate, saturation density, and viability. It was also found that methylthioribose could satisfy the methylthio dependence of the enzyme-deficient cell line, L1210D. A model is proposed to explain the selective growth of methylthioadenosine phosphorylase-deficient cells in medium lacking a methylthio donor but containing fetal calf serum. It is hypothesized that cellularly exported methylthioadenosine is degraded to methylthioribose in the presence of medium containing serum of high methylthioadenosine phosphorylase activity (i.e. fetal calf serum). The resultant methylthioribose can then be used to satisfy the methylthio requirement of these cells. To test this theory, various purified preparations of bovine liver methylthioadenosine phosphorylase were used to artificially increase the specific activity of methylthioadenosine phosphorylase in horse serum. In each case, it was demonstrated that only medium containing serum of enzyme activity nearly equal to that of the glutathione-stimulated fetal calf serum activity, supported the growth of methylthio-dependent cells in the absence of methylthio compounds. The data suggest that the degradation of methylthioadenosine and subsequent formation of methylthioribose represents an essential process in the growth of mammalian cells.     

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K_m′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [¹⁴C]ATP or [¹⁴C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P_i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl₂, suggesting that Mg²⁺ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.     

Comparative transport activity of intact cells, membrane vesicles, and mesosomes of Bacillus licheniformis

Sodium ion was shown to stimulate strongly the transport of l-glutamic acid into cells of Bacillus licheniformis 6346 His(-). Lithium ion had a slight capacity to replace Na(+) in this capacity, but K(+) was without effect. Three of five amino acids tested. l-glutamic acid, l-aspartic acid, and l-alanine, were concentrated against a gradient in the cells. Intracellular pools of these amino acids were extractable with 5% trichloroacetic acid. Pools of l-histidine and l-lysine could not be detected. No evidence of active transport of lysine into cells could be detected, and histidine was taken up in the absence of chloramphenicol but not in its presence. The uptake of glutamic acid by membrane vesicle preparations was strongly stimulated by reduced nicotinamide adenine dinucleotide (NADH) and to a lesser extent by succinate. The presence of phenazine methosulfate increased uptake in the presence of succinate. Either l- or d-lactate and adenosine triphosphate were without effect. None of these compounds stimulated the uptake of glutamic acid by mesosomes, although some mesosome preparations contained separable membrane which was very active. NADH strongly stimulated the uptake of aspartic acid and alanine by membrane vesicles but had only a slight effect on the uptake of histidine and lysine. No evidence of active transport of any of the amino acids into mesosomes could be detected either in the presence or absence of NADH. NADH stimulation of the uptake of glutamic acid by membrane vesicles was destroyed by exposure to light of 360 nm; this inactivation was reversible by vitamin K(2(5)) or K(2(10)). Sodium ion stimulated transport of glutamic acid by membrane vesicles.     

Uptake of AMP, ADP, and ATP in Escherichia coli W

The uptake activity ratio for AMP, ADP, and ATP in mutant (T-1) cells of Escherichia coli W, deficient in de novo purine biosynthesis at a point between IMP and 5-aminoimidazole-4-carboxiamide-1-β-D-ribofuranoside (AICAR), was 1:0.43:0.19. This ratio was approximately equal to the 5'-nucleotidase activity ratio in E. coli W cells. The order of inhibitory effect on [2-3H]ADP uptake by T-1 cells was adenine > adenosine > AMP > ATP. About 2-fold more radioactive purine bases than purine nucleosides were detected in the cytoplasm after 5 min in an experiment with [8-1?C]AMP and T-1 cells. Uptake of [2-3H]adenosine in T-1 cells was inhibited by inosine, but not in mutant (Ad-3) cells of E. coli W, which lacked adenosine deaminase and adenylosuccinate lyase. These experiments suggest that AMP, ADP, and ATP are converted mainly to adenine and hypoxanthine via adenosine and inosine before uptake into the cytoplasm by E. coli W cells.     

Uptake and metabolism of adenosine by pig aortic endothelial and smooth-muscle cells in culture.

1. Adenosine, a potent vasodilator, is transported very efficiently by pig aortic endothelium in monolayer culture (approx. 50pmol/min per 10(6) cells at 2 micrometer). Uptake proceeds by diffusion at high (millimolar) substrate concentrations, and by two discrete transport processes (Km approx. 3 micrometer and 250 micrometer) at lower concentrations. Over 90% of the adenosine taken up at 10 micrometer or 100 micrometer is rapidly converted into adenine nucleotides (mainly ATP). 2. The high-affinity process is selectively inhibited by dipyridamole and by nitrobenzylthioinosine. Adenine preferentially inhibits the lower-affinity process, papapaverine inhibits both transport processes, and inosine has no significant effect. 3. Pig aortic smooth-muscle cells in culture show no high-affinity transport system for adenosine; uptake is much slower at low concentrations than that by endothelium (approx. 5pmol/min per 10(6) cells at 2 micrometer). Over 80% of the incorporated adenosine at 10 micrometer or 100 micrometer is rapidly converted into adenine nucleotides. 4. The uptake of adenosine by smooth-muscle cells is powerfully inhibited by adenine, but dipyridamole is much less potent than in endothelium. 5. We conclude that endothelial cells are mainly responsible for the removal of circulating adenosine.     

Imparied formylation and uptake of tetrahydrofolate by rat small gut following cobalamin inactivation

The effect of inactivation of cobalamin by N₂O in the intestinal absorption of folate was studied using rat everted gut sacs. Further, in view of uncertainties about the presence of methionine synthetase in gut [1], this enzyme was measured. Everted gut sacs were incubated with [2-¹⁴C]tetrahydrofolate, and the subsequent appearance of labelled formyl- and methyl[¹⁴C]tetrahydrofolate in everted segments of small intestine of rats was studied. Considerable methionine synthetase activity was present in washed everted gut sacs but not in gut segments in the absence of such treatment. Methionine synthetase activity declined after exposure to N₂O, which oxidizes and inactivates cob(I)alamin. Folate uptake by gut sacs was not affected by 24 h exposure of the animals to N₂O but fell significantly after 7 days exposure. There was a significant fall in the amount of formlytetrahydrofolate formed after cobalamin inactivation and this was reversed by supplying either methionine, methylthioadenosine or sodium formate. Serine had no effect. The data support the hypothesis that methionine and methylthioadenosine act by supplying single carbon units at the formate level of oxidation.     

On the mechanisms of ATP-induced and succinate-induced redistribution of cations in isolated rat liver cells

1. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ecto-ATPase of the cells. 2. In addition to its direct action, ATP enhanced succinate-dependent calcium uptake in a cooperative fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinate stimulates uptake into mitochondria. 3. ATP was able to lower Na+ and K+ gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration counteracted this effect though calcium uptake was then stimulated. 4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially counteracted the ATP effects on these gradients. 5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model to study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinate-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.     

Catabolism and lability of S-adenosyl-L-methionine in rat liver extracts.

The fate of S-adenosyl-L-methionine was studied in rat liver extracts by analysing the distribution of radioactivity from labelled adenosylmethionine in decomposition products, which were separated from each other by chromatographic and electrophoretic means. Marked non-enzymic degradation to adenine, pentosylmethionine, methylthioadenosine and homoserine was evident at pH 6.9-7.8. Enzymic cleavage to methylthioadenosine was stoichiometric with the accumulation of spermidine and could be totally prevented by inhibiting S-adenosyl-L-methionine decarboxylase. The results rule out the existence of adenosylmethionine cyclotransferase in rat liver and indicate that only two quantitatively significant enzymic processes are involved in hepatic adenosylmethionine degradation. Excluding nonenzymic decomposition, more than 99% of adenosylmethionine is demethylated and exclusively catabolized further by S-adenosyl-L-homocysteine hydrolase. Less than 1% of adenosylmethionine is decarboxylated and immediately utilized totally for polyamine biosynthesis.     

Schistosoma mansoni: Uptake in vitro and incorporation of adenine by adults

The rates of adenine uptake and incorporation into nucleic acids by adult male and female Schistosoma mansoni were determined during periods of up to 10 days in vitro, and comparisons were made between paired and separated worms. Adenine uptake by separated males and females exceeded that exhibited by equivalent paired worms. The rate of incorporation of adenine into nucleic acids was higher in separated females than in paired females. In contrast, the state of pairing had little effect on adenine incorporation by male S. mansoni. There was no correlation between rates of adenine uptake and incorporation and the reproductive activity of S. mansoni adults in vitro. Uptake and incorporation rates appeared to reflect the changing somatic requirements of both male and female worms.     

The effect of heme on intracellular iron pools during differentiation of friend erythroleukemia cells

The inhibition of cellular iron uptake by hemin described previously in reticulocytes was studied in murine erythroleukemia (Friend) cells that can be induced to differentiate in culture by dimethyl sulfoxide (DMSO). Hemin had no effect on iron uptake into noninduced cells. After the induction by DMSO, hemin inhibited iron uptake into Friend cells and this effect of hemin became more pronounced with the further progress of differentiation. The reduction of cellular iron accumulation was caused mainly by inhibition of iron incorporation into heme, iron uptake into the non-heme pool was influenced by hemin treatment. Inhibition of heme synthesis by isonicotinic acid hydrazide (INH) caused an accumulation of iron in mitochondria in DMSO-induced cells but not in uninduced cells. On the basis of these results, a specific system transporting iron to mitochondria induced by DMSO treatment is suggested as a target for the inhibitory action of hemin. In Friend cells of the Fw line which are deficient in ferrochelatase, heme has no effect on iron uptake. The addition of INH to the Fw cells does not enhance the iron accumulatoni in mitochondria.