Formation of a cytochrome c-like species from horse apoprotein and hemin catalyzed by yeast mitochondrial cytochrome c synthetase

Cytochrome c synthetase in yeast mitochondria catalyzes the formation of a yeast cytochrome c-like species from the apoprotein and hemin (Basile, G., DiBello, C., and Taniuchi, H. (1980) J. Biol. Chem. 255, 7181-7191). To test the specificity of this enzyme, 125I-labeled horse apocytochrome c was incubated with the yeast mitochondrial fraction in the presence of hemin, NADPH, and an ethanol extract of the postmitochondrial fraction. A radioactive 125I-labeled cytochrome c-like species was formed in yields of up to 26%. This 125I-labeled species is indistinguishable from horse cytochrome c by ion exchange chromatography (under the conditions which allow separation of horse and yeast cytochrome c), resistance in its reduced form to digestion by trypsin, resistance against autoxidation, reduction by cytochrome b2, and generation of the apoprotein after treatment with silver sulfate and dithiothreitol. With unlabeled horse apoprotein and [59Fe]hemin, the yield of a [59Fe-labeled horse cytochrome c-like species was up to 7% with respect to the apoprotein incubated. The yield of the 59Fe-labeled species was not altered by the addition of unlabeled FeCl3. Conversely, synthesis of the 59Fe-labeled species was not detectable after incubation of yeast mitochondria with unlabeled horse apoprotein, unlabeled hemin, and 59FeCl3. The formation of both 125I- and 59Fe-labeled cytochrome c-like species was sensitive to heat. Thus, we conclude that cytochrome c synthetase catalyzes direct bonding of heme (or hemin) to the apoprotein. Since the amino acid sequences of horse and yeast cytochromes c differ considerably, cytochrome c synthetase may recognize only a limited region(s) of the apoprotein.     

Iron utilization in rabbit reticulocytes: A study using succinylacetone as an inhibitor of heme synthesis

We have investigated the effect of succinylacetone (4,6-dioxoheptanoic acid) on hemoglobin synthesis and iron metabolism in reticulocytes. Succinylacetone, 0.1 and 1 mM, inhibited [2-¹⁴C]glycine incorporation into heme by 91.2 and 96.4%, respectively, and into globin by 85 and 90.2%, respectively. 60 μM hemin completely prevented the inhibition of globin synthesis by succinylacetone, indicating that succinylacetone inhibits specifically the synthesis of heme. Added porphobilinogen, but not δ-aminolevulinic acid, partly overcame the inhibition of ⁵⁹Fe incorporation into heme caused by succinylacetone suggesting that the drug inhibits δ-aminolevulinic acid dehydratase in reticulocytes. Succinylacetone, 10 μM, 0.1 and 1 mM, inhibited ⁵⁹Fe incorporation into heme by 50, 90 and 93%, respectively, but stimulated reticulocyte ⁵⁹Fe uptake by about 25–30%. In succinylacetone-treated cells ⁵⁹Fe accumulates in a fraction containing plasma membranes and mitochondria as well as cytosol ferritin and an unidentified low molecular weight fraction obtained by Sephacryl S-200 chromatography. Reincubation of washed succinylacetone- and ⁵⁹Fe-transferrin-pretreated reticulocytes results in the transfer of ⁵⁹Fe from the particulate fraction (plasma membrane plus mitochondria) into hemoglobin and this process is considerably stimulated by added protoporphyrin. Although the nature of the iron accumulated in the membrane-mitochondria fraction in succinylacetone-treated cells is unknown some of it is utilizable for hemoglobin synthesis, while cytosolic ferritin iron would appear to be mostly unavailable for incorporation into heme.     

Heme transport from rat liver mitochondria to the microsomes invitro

When rat liver mitochondria labeled with [⁵⁹Fe]heme were incubated with microsomes in the presence of cytosol, about 16 % of the heme in mitochondria was transported to microsomes during a 1 hr-incubation period. In the absence of cytosol, little heme was transported. DEAE-Sephadex column chromatography of the cytosol partially purified by pH 5.1 treatment and ammonium sulfate precipitation (45–65%) revealed that there were at least two proteins with a releasing activity from mitochondria via heme transport.     

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.     

Transport of heme by hemopexin to the liver: Evidence for receptor-mediated uptake

We used carefully defined heme-hemopexin complexes to investigate the role of hemopexin in the catabolism of heme in vivo. Uptake of rabbit [⁵⁹Fe]heme-[¹²⁵I]hemopexin by rat liver was rapid. The liver-associated ¹²⁵I reached a maximum 5 minutes after injection, nearly 7-fold higher than apo-hemopexin, whereas liver-associated ⁵⁹Fe increased with time. This together with an inverse relationship of [¹²⁵I]hemopexin in the liver and serum during the course of heme transport suggests that hemopexin was released from the liver back to the circulation. Saturation of uptake with heme-hemopexin, reaching about 170 pmol [¹²⁵I]hemopexin (gm liver)^?1 5 minutes after injection of 11 nmol, indicates a receptor-mediated process.We conclude that hemopexin delivers heme to the liver via interaction with a finite number of receptors and returns to the circulation.     

Interaction of Ca2+ and H+ with heme A in cytochrome oxidase

Ca²⁺ ions shift the absorption spectrum of reduced cytochromea in mitochondria by acting from the outside of the membrane. In isolated cytochrome oxidase the shift may be induced by either Ca²⁺ or H⁺, the apparent pK varying between 6.20 and 5.75 depending on the state of cytochromea ₃. Studies of the Soret band show that Ca²⁺ also shifts the spectrum of ferrocytochromea ₃ in isolated oxidase in contrast to the situation in mitochondria or isolated oxidase reconstituted into liposomes. Model studies with reduced bis-imidazole heme A reveals an analogous spectral shift induced by Ca²⁺. Esterification of the propionate carboxyls of heme A abolishes the spectral shift, suggesting that it is due to interaction of Ca²⁺ with these groups. When taken together with the data with intact mitochondria, this suggests that the propionate side chains of cytochromea are accessible to Ca²⁺ and H⁺ from the outside of the mitochondrial membrane. In the soluble enzyme both hemesa anda ₃ are accessible. Thus hemea may be located near the outside of the inner membrane whereas hemea ₃ experiences a different environment in which no Ca²⁺ shift occurs.     

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.     

Early labeled heme synthesis in normal rats and rats with iron deficiency anemia

Heme synthesis from [2-¹⁴C]glycine was studied in liver and red blood cells. In normal rats liver contained two early [¹⁴C] heme peaks maximal at 1 and 4.5 h, followed by a long plateau of heme labeling. These phases were present in both microsomes and mitochondria. Cycloheximide suppressed formation of the first but not the second heme component. All phases of hepatic heme labelling were reduced in iron-deficient rats, with better preservation of the microsomal fraction. In iron-deficient rats responding to iron therapy, the first peak merged with an enlarged and premature second component; the increase was most marked in mitochondria. Thus, labeled heme metabolism was less perturbed in microsomes than mitochondria in both of these conditions. Peripheral blood also contained a [¹⁴C]heme peak at 1 h in all experimental groups. This was highest with the increased eythroid response observed in irontreated rats. The first heme peak, present in both hepatic and erythroid cells, may represent a pool of free or unassigned heme. The later heme component may reflect formation of hemoproteins, which could be related directly or indirectly to the initial, rapid turnover heme component.     

The composition of the incubation medium influences the sensitivity of mitochondrial permeability transition to cyclosporin A

The aim of this work was to study permeability transition, and the influence of the composition of the incubation medium, on the inhibitory action of cyclosporin A. It was found that cyclosporin inhibited the opening of a nonspecific pore, as induced by the uncoupler carbonyl cyanide m-chlorophenylhydrazone, provided K⁺ was present in the incubation medium, but failed to do so if mitochondria are incubated in sucrose or Na⁺-based medium. It was also found that the sensitivity of mitochondria to the uncoupler depended on the incubation mixture, being more sensitive when sucrose was the osmotic support. Matrix Ca²⁺ release, large amplitude swelling, and drop in transmembrane electric gradient revealed permeability transition. The titration of membrane thiol groups shows them to be increased in mitochondria incubated in sucrose medium, in comparison with the values found in mitochondria incubated in KCl or NaCl medium. Our proposal is that the incubation in sucrose medium propitiated a conformational change of membrane proteins in such a way that cyclosporin was unable to bind to its target site.     

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.     

Effect of phospholipase-D on rat kidney mitochondria

Incubation of purified rat kidney mitochondrial fraction with phospholipase-D resulted in the accumulation of phosphatidic acid in the membrane due to the degradation of membrane-bound phosphatidylcholine, -serine and-ethanolamine Simultaneously with the hydrolysis of the phospholipids, cholesterol and protein were released from the mitochondrial membrane into the medium, and binding of Ca²⁺ by mitochondrial membranes increased. Phospholipase Dtreated mitochondrial fraction exhibited increased swellingin vitro in the early stages of incubation (15 min) after which the mitochondria were ruptured. Membrane-bound adenosine triphosphatase was partially inactivated and the enzyme activity was not significantly restored by incubation with sonicated dispersions of phosphatidylcholine,-serine and cholesterol. These results indicate that removal of choline, serine and ethanolamine from membrane-bound phospholipids disrupt phospholipid-cholesterol and phospholipid-protein association and affect functions of the membrane. Communication no, 2468.     

Dimethyl sulfoxide (DMSO) stimulates heme synthesis in quail embryonic yolk sac cells

Dissociated yolk sac cells from quail embryos at the definitive primitive streak stage were reaggregated, using a gyratory shaker with or without dimethyl sulfoxide (DMSO). After 24 h of incubation in the shaker, the aggregates were transferred onto a whole egg agar medium containing ⁵⁹Fe, and incubation was continued for an additional 48 h. It was clearly shown that DMSO-treated yolk sac aggregates showed a higher incorporation of radioactive iron into heme than the control culture without DMSO. The maximal stimulatory effect was observed at around 0.75% DMSO.     

Pharmacokinetics of the iron chelator desperrioxamine as affected by liposome encapsulation: potential in treatment of chronic hemosiderosis

Desferrioxamine (DF), the chelator of choice for removal of excess stored iron, is limited by its rapid excretion, metabolic breakdown, and low cell uptake. We have encapsulated DF in unilamellar and multilamellar liposomes, and have compared the short-term pharmacokinetics of nonencapsulated and encapsulated ⁵⁹Fe-labeled DF after intravenous administration. Disappearance of ⁵⁹Fe-DF from the plasma was very rapid in mice receiving multilamellar liposome-encapsulated and nonencapsulated drug, but much slower in mice receiving unilamellar liposomes. Between 1 and 24 hours after injection, nonencapsulated ⁵⁹Fe-DF never exceeded 1–5% of the injected dose (ID) in liver or < 0.7% in spleen; whereas after either multilamellar or unilamellar liposomes, the uptake in liver was 30–35% ID, and in spleen was 1–5% ID. Excretion of ⁵⁹Fe-DF was much slower with liposome encapsulation. These results indicate that liposomes can effectively deliver DF to critical organs of iron storage. Thus this drug delivery system is potentially useful for treatment of iron overload.     

Effect of Sclerin on Incorporation of Oleic Acid into Phospholipids and Activity of Phospholipase in Mitochondria

Sclerin (SCL) stimulated the oxidation and the incorporation into the phospholipids of Na-[1-¹⁴C]-oleate in mitochondria isolated from rat liver, preventing the depression of the phosphorylating functions and protecting 2,4-dinitrophenol (DNP)-activated ATPase in mitochondria during incubation with oleate. Also, SCL markedly enhanced the activity of phospholipase to hydrolyze endogenous substrates in mitochondria. The increase in the activity was due to reconstruction of phospholipids through esterification of oleate in mitochondrial membrane, but not to the de novo enzyme synthesis. It was concluded that the level of endogenous phospholipase in mitochondria during incubation reflects the energy- dependent reacylation of the lysophospholipids produced by the action of phospholipase in mitochondrial membrane.     

Selective Cytochrome c Displacement by Phosphate and Ca2+ in Brain Mitochondria

In brain mitochondria, phosphate- and Ca²⁺-dependent cytocrome c (cyt c) release reveals pools that interact differently with the inner membrane. Detachment of the phosphate-dependent pool did not influence the pool released by Ca²⁺. Cyt c pools were also detected in a system of cyt c reconstituted in cardiolipin (CL) liposomes. Gradual binding of cyt c (1 nmol) to CL/2–[12-(7-nitrobenz- 2-oxa-1,3-diazol-4-yl)amino]dodecanoyl-1-hexadecan oyl-sn-glycero-3-phosphocholine (NBDC₁₂-HPC) liposomes (10 nmol) produced NBD fluorescence quenching up to 0.4 nmol of added protein. Additional bound cyt c did not produce quenching, suggesting that cyt c-CL interactions originate distinct cyt c pools. Cyt c was removed from CL/NBDC₁₂-HPC liposomes by either phosphate or Ca²⁺, but only Ca²⁺ produced fluorescence dequenching and leakage of encapsulated 8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis-pyridinium bromide. In mitochondria, complex IV activity and mitochondrial membrane potential (Δψ_m) were not affected by the release of the phosphate-dependent cyt c pool. Conversely, removal of cyt c by Ca²⁺ caused inhibition of complex IV activity and impairment of Δψ_m. In a reconstituted system of mitochondria, nuclei and supernatant, cyt c detached from the inner membrane was released outside mitochondria and triggered events leading to DNA fragmentation. These events were prevented by enriching mitochondria with exogenous CL or by sequestering released cyt c with anti-cyt c antibody.     

Intermediate steps in cellular iron uptake from transferrin. II. A cytoplasmic pool of iron is released from cultured cells via temperature-dependent mechanical wounding

Summary A previous study described a cytoplasmic, transferrin (Tf)-free, iron (Fe) pool that was detected only when cells were mechanically detached from the culture substratum at 4°C, after initial incubation with⁵⁹Fe-¹²⁵I-Tf at 37°C (Richardson and Baker, 1992a). The release of this internalized⁵⁹Fe could be markedly reduced if the cells were treated with proteases or incubated at 37°C prior to detachment. The present study was designed to characterize this Fe pool and understand the mechanism of its release. The results show that cellular⁵⁹Fe release increased linearly as a function of preincubation time with⁵⁹Fe-Tf subsequent to mechanical detachment at 4°C using a spatula. These data suggest that the⁵⁹Fe released was largely composed of end product(s) and was not an “intermediate Fe pool.” When the Fe(II) chelator, dipyridyl (DP), was incubated with⁵⁹Fe-Tf and the cells, it prevented the accumulation of⁵⁹Fe that was released following mechanical detachment at 4°C. Other chelators had much less effect on the proportion of⁵⁹Fe released. Examination of the⁵⁹Fe released showed that after a 4-h preincubation with⁵⁹Fe-Tf, approximately 50% of the⁵⁹Fe was present in ferritin. These data indicate that mechanical detachment of cells at 4°C resulted in membrane disruptions that allow the release of high M, molecules. Moreover, electron microscopy studies showed that detachment of cells from the substratum at 4°C resulted in pronounced membrane damage. In contrast, when cells were detached at 37°C, or at 4°C after treatment with pronase, membrane damage was minimal or not apparent. These results may imply that temperature-dependent processes prevent the release of intracellular contents on membrane wounding, or alternatively, prevent wounding at 37°C. The evidence also indicates that caution is required when interpreting data from expriments where cells have been mechanically detached at 4°C.     

Factors affecting iron and transferrin release from rabbit reticulocyte ghosts to cytosol

⁵⁹Fe- and ¹²⁵I-labelled transferrin-labelled rabbit reticulocyte ghosts were incubated at 37°C for 60 min with unlabelled reticulocyte and erythrocyte stroma-free haemolysates, and the ability of these haemolysates to release ⁵⁹Fe- and ¹²⁵I-labelled transferrin was investigated. Reticulocyte and erythrocyte haemolysates were equally effective in releasing ⁵⁹Fe from the ghosts, but only the reticulocyte haemolysate was able to release ¹²⁵I-labelled transferrin. The elution profiles of the post-incubation haemolysates upon AcA 44 gel filtration were similar. The ⁵⁹Fe appeared as five separate peaks and the ¹²⁵I-labelled transferrin appeared as a single, unbound peak. In the post-incubation reticulocyte haemolysate, 25% of the ⁵⁹Fe was bound to ferritin and transferrin, and 69% was associated with the haemoglobin fraction; 52.8% of the ⁵⁹Fe was present as haem-⁵⁹Fe intimately associated with haemoglobin. Another 12.5% of the ⁵⁹Fe was loosely bound to proteins in the haemoglobin fraction. The haem-⁵⁹Fe released to the haemoglobin fraction was derived from preformed haem in the reticulocyte ghost. ⁵⁹Fe release was not impaired in experiments in which haem and protein synthesis were inhibited with isonicotinic acid hydrazide and cycloheximide. When tested alone, the haemoglobin fraction was able to release ⁵⁹Fe from the ghosts to an even greater degree than reticulocyte haemolysate. It is concluded that protein in the haemoglobin fraction function as heme carriers.Less than 6% of the ⁵⁹Fe released by reticulocyte haemolysate was associated with a low molecular size protein fraction. Removal of this fraction from the unlabelled haemolysate by ultrafiltration did not impair the ⁵⁹Fe-releasing capacity of the haemolysate. However, both this fraction and the ferritin fraction were able to bind some ⁵⁹Fe from the ghosts. Ferrous and ferric chelators, as well as defatted bovine serum albumin, were also able to bind ⁵⁹Fe from the ghosts, but not to the same degree as the haemolysates.The release of ¹²⁵I-labelled transferrin from the ghosts by the reticulocyte haemolysate was affected by stimulatory and inhibitory factors. The stimulatory factor(s) was present in the non-haemoglobin components of the haemoglobin fraction. The inhibitory effect was dependent on the low molecular weight fraction.