Reaction mechanism of electron transfer from FeII(CN)6(4-) or W(IV)(CN)8(4-) to the cupric ions in human copper, zinc superoxide dismutase

The electron transfer reactions from FeII(CN)6(4-) and W(IV)(CN)8(4-) to the cupric ions in human copper, zinc superoxide dismutase were followed by the micro-stopped-flow method. The kinetic rate data clearly indicate that FeII(CN)6(4-) or W(IV)(CN)8(4-) first forms an adduct with the enzyme through the interaction with Arg143 of the active cavity and then an electron from FeII(CN)6(4-) or W(IV)(CN)8(4-) of the adduct transfers to the cupric ion in the enzyme. The dissociation constants of the adducts of FeII(CN)6(4-) and W(IV)(CN)8(4-) were 4.0(+/-0.3) x 10(-3) and 2.2(+/-0.3) x 10(-3) M, respectively. In spite of the difference between the standard redox potentials of FeIII(CN)6(3-)/FeII(CN)6(4-) (468 mV) and W(V)(CN)8(3-)/W(IV)(CN)8(4-) (556 mV), the electron transfer rate constant (0.148(+/-0.005) s(-1) of FeII(CN)6(4-) at 25 degrees C is very similar to that of W(IV)(CN)8(4-) (0.072(+/-0.011) s(-1)). The entropy values of the adduct formations and the activation energies of the electron transfer rates were determined by the temperature dependence of the dissociation constants of the adducts and the electron transfer rates. The enthalpy values of the formation of adducts are almost zero, so that the driving forces to form the adducts are mainly derived from the entropy. The activation energy of the electron transfer rate of FeII(CN)6(4-) is very similar to that of W(IV)(CN)8(4-). The formation of the adduct between FeII(CN)6(4-) and the enzyme was inhibited by the presence of various anions (ClO4-, SO4(2-), SCN-, and N3-). The bulky anions SO4(2-) and ClO4- behave as competitive inhibitors for FeII(CN)6(4-); these anions should interact mainly with Arg143, as it has a positive charge at the entrance of the active cavity. The competitive inhibition constants of ClO4-, SO4(2-), and SCN- were 0.010, 0.012, and 0.008 M. The azide ion, which is smaller than SO4(2-) or ClO4-, shows mixed inhibition, because N3- can interact with Arg143 (competitive inhibition) and also directly binds to the cupric ion in h-SOD (noncompetitive inhibition). The competitive and noncompetitive inhibition constants of N3- were 0.004 and 0.016 M, respectively.     

X-ray microanalysis of non-aldehyde-fixed glycogen contrast stained with OsVIIIO4, OsVIIIFeIII, or OsVIFeII complex in vitro

Summary Fibrin-enrobed, commercially produced glycogen was treated, without prior glutaraldehyde fixation, to a form of post-fixation with solutions of Os^VIIIO₄ or with a mixture of either Os^VIIIO₄ plus K₃Fe^III(CN)₆ or K₂Os^VIO₄ plus K₄Fe^II(CN)₆.Only the last mixture gave constrast staining of the glycogen in unstained ultrathin sections. The first mixture rendered the glycogen just barely visible but the glycogen contrast was increased by lead staining. The glycogen treated with the Os^VIIIO₄ solution was not contrast stained and was just observable after lead staining.Qualitative X-ray microanalysis of the glycogen in the ultrathin sections confirmed the presence of osmium and iron in the glycogen treated with both mixtures. The glycogen treated with Os^VIIIO₄ alone was difficult to analyse.Quantitative X-ray microanalysis showed that, in the glycogen treated with the Os^VIIIO₄ mixture plus K₃Fe^III(CN)₆, the mean atomic osmium to iron ratio was 15. In the glycogen treated with K₂Os^VIO₄ plus K₄Fe^II(CN)₆ this ratio was 117. However, the mean net osmium intensity in the latter case was 15 times higher than in the former case and for the iron even 40 times higher.The Unit for Analytical Electron Microscopy was established by collaboration between the Erasmus University of Rotterdam (W. C. de Bruijn), the University of Leiden and the Organization for Health Research TNO. The analytical microscope was purchased with funds from the Netherlands Organization for Pure Scientific Research (ZWO).     

Indirect determination of the rate of iron uptake into the apoprotein of the ribonucleotide reductase of E. coli

The second-order rate constant k(apo) for uptake of FeII by the apoprotein of Ribonucleotide Reductase R2 has been measured by letting that reaction compete with the uptake of FeII by ferrozine (rate constant k(Fz)). The rate of the FeII/ferrozine reaction was studied at high ferrozine concentrations, and an effective first-order rate constant k(Fz) for the disappearance of FeII determined in the presence of bovine serum albumin as a viscogen. Solutions of apoprotein and ferrozine in various ratios were mixed with FeII solutions in a stopped-flow apparatus, and the growth of the 562 nm FeII(ferrozine)3 absorbance monitored. Attempts to fit the data to a variety of kinetic schemes imply that uptake of the second FeII by apo is slower than uptake of the first, suggesting that the rate-determining step in the activation of R2 is a conformational change after the uptake of the first iron. The resulting value of k(apo) is 1.8(1) x 10(6) M(-1) x s(-1).     

Effect of endrin on the hepatic distribution of iron and calcium in female sprague-dawley rats

The distribution of iron and calcium in hepatic subcellular fractions of female rats treated with endrin (1, 2, 3, 4, 10, 10-hexachloro-6, 7-epoxy-1, 4, 4α, 5, 6, 7, 8, 8α-octahydroendo, endo-1, 4:5, 8-dimethanonaphthalene) was determined. Endrin in corn oil was administered orally to rats in single doses of 3, 4.5, or 6 mg/kg, and the animals were killed at 0, 12, 24, 48, or 72 hr posttreatment. Iron and calcium were determined by atomic absorption spectroscopy. The administration of endrin increased the iron content of mitochondria and decreased the iron content of microsomes and nuclei. Significant increases occurred in the calcium content of mitochondria, microsomes, and nuclei. Thus, the results indicate that with respect to the subcellular distribution of iron and calcium, endrin produces differential effects. Vitamin E succinate administration partially prevented the endrin-induced hepatic alterations in iron and calcium homeostasis. Endrin also produced dose- and time-dependent increases in the liver and spleen weight/body weight ratios, while decreasing the thymus weight/body weight ratios. The altered distribution of calcium and iron may contribute to the broad range of effects of endrin.     

Glycogen, its chemistry and morphological appearance in the electron microscope. III. Identification of the tissue ligands involved in the glycogen contrast staining reaction with the osmium (VI)-iron(II) complex

Synopsis By application of appropriate blocking reactions (acetylation, de-amination, methylation and NaHSO₃-treatment) it is demonstrated that the tissue ligands involved in the selective glycogen contrast staining reaction with the Os^VI. Fe^II complex (known to be present in the combination K₂OsO ₄ ^K ₄Fe(CN)₆) are the glycogen C₂–C₃ di-hydroxyl groups. Deliberate conversion of the diols into di-aldehydes and (di-)carboxyl groups by the application of specific oxidative agents followed, by application of the Os^VI.Fe^II-complex results morphologically in identical selective contrast staining of glycogen.By applying appropriate blocking reactions to such pre-oxidized aldehyde fixed glycogen, evidence is accumulated that K₂OsO₄ and K₃Fe(CN)₆ are unable to oxidize diols, whereas OsO₄ and H₂O₂ are able to convert diols into carboxyl groups.From these results it is concluded that in the combination K₂OsO ₄ ^K ₄Fe(CN)₆ the Os^VI.Fe^II complex reacts with unchanged diols in the glycogen, whereas the OsO₄ in the combination OsO ₄ ^K ₄Fe(CN)₆ can petentially create carboxyl groups in the aldehydefixed glycogen.The addition of urea to the two glycogen contrasting combinations (K₂OsO ₄ ^K ₄Fe(CN)₆ or OsO ₄ ^K ₄Fe(CN)₆), also emphasizes that, although morphologically both combinations produceidentical contrast stained glycogen, chemically the contrast staining is apparently obtained in a different way, as urea prevented the contrast for mation in the glycogen by the combination K₂OsO ₄ ^K ₄Fe(CN)₆, but not by the combination OsO ₄ ^K ₃Fe(CN)₆.     

An inverse relation between mitochondrial hexokinase content and phosphoglucomutase activity of rat tissues

Summary The hexokinase: fumarase ratios of mitochondria isolated from ten tissues of the rat were determined, and compared with the tissue content of phosphoglucomutase and phosphorylase, taken as representatives of enzymes concerned with glycogen metabolism. A generally inverse relationship was found between the mitochondrial hexokinase: fumarase ratio and phosphoglucomutase levels. The cytochrome: fumarase ratios were relatively invariant in these same mitochondria. The results are interpreted as indicating a specialization of mitochondria, with increased amounts of hexokinase being associated with the mitochondria in tissues exhibiting less dependence on glycogen metabolism, as judged from phosphoglucomutase levels.     

Ultrastructural study of liver cells from rooks living in ecologically unfavorable areas

The ultrastructure of liver cells was studied in rooks (Corvus frugilegus) living in radioactive and chemical contamination areas. The ultrastructure of liver cells from rook as well as jackdaw (Corvus monedula) and hooded crow (Corvus cornix) (Corvidae family) from a conventionally clean area was studied as control. Control hepatocytes proved to contain a great number of mitochondria, many of which were swollen and had clear matrix and disorganized cristae. The cristae nearly lacked glycogen and had abundant lipid droplets, which often tightly contacted mitochondria. The cytoplasm of hepatocytes in birds from both ecologically unfavorable areas had numerous mitochondria with the same ultrastructure. In contrast to control, the hepatocyte cytoplasm: (1) contained a lot of glycogen; (2) there were many lipid droplets, which directly contacted glycogen granules; and (3) had more abundant peroxisomes. In addition to normal erythrocytes, the sinusoids contained erythrocytes with mitochondria, vesicles, and lipid droplets in their cytoplasm. Analysis of many micrographs of lipid droplets contacting glycogen granules, mitochondria, peroxisomes, and cisterns of smooth endoplasmic reticulum allowed us to propose that glycogen is synthesized via gluconeogenesis from glycerol and products of fatty acid oxidation in the liver cell cytoplasm of rooks from ecologically unfavorable areas as distinct from control.     

Iron uncouples oxidative phosphorylation in brain mitochondria isolated from vitamin E-deficient rats

Few, if any, studies have examined the effect of vitamin E deficiency on brain mitochondrial oxidative phosphorylation. The latter was studied using brain mitochondria isolated from control and vitamin E-deficient rats (13 months of deficiency) after exposure to iron, an inducer of oxidative stress. Mitochondria were treated with iron (2 to 50 microM) added as ferrous ammonium sulfate. Rates of state 3 and state 4 respiration, respiratory control ratios, and ADP/O ratios were not affected by vitamin E deficiency alone. However, iron uncoupled oxidative phosphorylation in vitamin E-deficient mitochondria, but not in controls. In vitamin E-deficient mitochondria, iron decreased ADP/O ratios and markedly stimulated state 4 respiration; iron had only a modest effect on these parameters in control mitochondria. Thus, vitamin E may have an important role in sustaining oxidative phosphorylation. Low concentrations of iron (2 to 5 microM) oxidized mitochondrial tocopherol that exists in two pools. The release of iron in brain may impair oxidative phosphorylation, which would be exacerbated by vitamin E deficiency. The results are important for understanding the pathogenesis of human brain disorders known to be associated with abnormalities in mitochondrial function as well as iron homeostasis (e.g., Parkinson's disease).     

Selective dehydrogenation (oxidation) of 3,4-dimethoxybenzyl alcohol by a non-heme iron lignin-peroxidase reaction mimic.

In pyridine, bis(2,2'-bipyridine)iron(II) (Fe(bpy)2+(2)) activates hydrogen peroxide for the efficient and selective catalytic dehydrogenation (oxidation) of veratryl alcohol (model-substrate monomer for lignin; 3,4-(MeO)2PhCH2OH). Several other complexes (FeII(OPPh3)2+(4), FeII(O2bpy)2+(2), FeII(MeCN)2+(4), FeII(PA)2, FeIIICl3) are effective catalysts for the dehydrogenation of veratryl alcohol and benzyl alcohol, but their selectivity (relative reactivity with 3,4-(MeO)2PhCH2OH vs. PhCH2OH) is less than the 6.1 ratio that is observed for the optimized FeII(bpy)2+(2)/H2O2/pyridine (py) system. The reactivities have been determined for several other methoxybenzyl alcohols that are model substrates for lignin (e.g., 4-MeOPhCH2OH and (MeO)3PhCH2OH).     

Electron staining of the cell surface coat by osmium-low ferrocyanide

In aldehyde-fixed liver and renal cortex of rat and mouse several variations of postfixation with osmium tetroxide plus potassium ferrocyanide ( FeII ) were tried. Depending on the ferrocyanide concentration different staining patterns were observed in TEM. -Osmium-High Ferrocyanide [40 mM (approximately 1%) OsO4 + 36 mM (approximately 1.5%) FeII , pH 10.4], stains membranes and glycogen. Cytoplasmic ground substance, mitochondrial matrices and chromatin are partially extracted, cell surface coats remain unstained. Membrane contrast, but extraction too, are higher with solutions containing cacodylate- than phosphate-buffer. -Osmium-Low Ferrocyanide [40 mM (approximately 1%) OsO4 + 2 mM (approximately 0.08%) FeII , pH 7.4], stains cell surface coats and basal laminae, but not glycogen, except for special cases. The trilaminar structure of membranes is poorly delineated. Signs of cytoplasmic extraction are not visible. The surface coat staining is stronger and more widespread with solutions containing phosphate- instead of cacodylate-buffer; it is enhanced by section staining with lead citrate. The cell surface coat stain does not traverse tight junctions nor permeate membranes.     

Ovarian nests in cultured Russian sturgeon Acipenser gueldenstaedtii and North American paddlefish Polyodon spathula comprised of previtellogenic oocytes

Ovarian nests in the ovaries of sexually maturing Russian sturgeon Acipenser gueldenstaedtii and North American paddlefish Polyodon spathula were investigated. They comprised early previtellogenic, diplotene stage oocytes and somatic cells. In the nucleoplasm, these oocytes contained chromatin in the form of grains, threads and lampbrush chromosomes, primary nucleoli and multiple nucleoli. Two stages of oocytes in nests were distinguished by differences in the distribution of mitochondria with distorted cristae and lipid droplets in the ooplasm. These stages were as follows: pre‐early stage 1 (PE 1) and early stage 1 (EP 1) previtellogenic oocytes. In PE 1 oocytes few mitochondria with distorted cristae and lipid droplets were distributed randomly. The ooplasm of PE 1 oocytes was not differentiated into homogeneous and granular compartments. In EP 1 oocytes, mitochondria with distorted cristae were more numerous and grouped in the vicinity of the nucleus, lipid droplets accumulated near these mitochondria. In the nucleoplasm of EP 1 oocytes several low electron‐dense spherical bodies, possibly Cajal bodies, were present.     

Effect of endrin on the hepatic distribution of iron and calcium in female Sprague-Dawley rats.

The distribution of iron and calcium in hepatic subcellular fractions of female rats treated with endrin (1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4 alpha,5,6,7,8,8 alpha- octahydroendo,endo-1,4:5,8-dimethanonaphthalene) was determined. Endrin in corn oil was administered orally to rats in single doses of 3, 4.5, or 6 mg/kg, and the animals were killed at 0, 12, 24, 48, or 72 hr post-treatment. Iron and calcium were determined by atomic absorption spectroscopy. The administration of endrin increased the iron content of mitochondria and decreased the iron content of microsomes and nuclei. Significant increases occurred in the calcium content of mitochondria, microsomes, and nuclei. Thus, the results indicate that with respect to the subcellular distribution of iron and calcium, endrin produces differential effects. Vitamin E succinate administration partially prevented the endrin-induced hepatic alterations in iron and calcium homeostasis. Endrin also produced dose- and time-dependent increases in the liver and spleen weight/body weight ratios, while decreasing the thymus weight/body weight ratios. The altered distribution of calcium and iron may contribute to the broad range of effects of endrin.     

Studies on the utilization of ferritin iron in the ferrochelatase reaction of isolated rat liver mitochondria.

The utilization of ferritin as a source of iron for the ferrochelatase reaction has been studied in isolated rat liver mitochondria. 1. It was found that isolated rat liver mitochondria utilized ferritin as a source of iron for the ferrochelatase reaction in the presence of succinate plus FMN (or FAD). 2. Under optimal experimental conditions, i.e., approx. 50 micromol/1 FMN, 37 degrees C, pH 7.4 and 0.5 mmol/l Fe(III) (as ferritin iron), the release process, as shown by the formation of deuteroheme, amounted to approx. 0.5 nmol iron/min per mg protein. 3. The release process could not be elicited by ultrasonically treated mitochondria, lysosomes, microsomes or cytosol, i.e., the release of iron from ferritin was due to mitochondria and was a function of the in situ orientation of the mitochondrial inner membrane. 4. The release of iron from ferritin by the mitochrondria might be of relevance not only for the in situ synthesis of heme in the hepatocyte, but also with respect to the mechanism(s) by means of which iron is mobilized for transport to the erythroid tissue.     

Effects of bromadiolone on some organs and tissues (liver, kidney, spleen, blood) of coypu (Myocastor coypus)

Bromadiolone damaged the erythrocytes, resulting in a probable saturation of transferrin, a deposit of iron in the connective tissue and in a few cells of the proximal tubules of the kidneys and an increased storage of ferritin in the spleen. In the hepatocytes, mitochondria were distorted, their lipid inclusions being granular; a large depletion of glycogen may be considered a reflection of an elevated phosphorylase a ascribable to the proliferation of the smooth endoplasmic reticulum. In the kidneys, pyelonephritis may be irrelevant to the poisoning of the animals. Bromine could not be detected using microanalytical methods.     

Subcellular localization of hexokinases I and II directs the metabolic fate of glucose

Background

The first step in glucose metabolism is conversion of glucose to glucose 6-phosphate (G-6-P) by hexokinases (HKs), a family with 4 isoforms. The two most common isoforms, HKI and HKII, have overlapping tissue expression, but different subcellular distributions, with HKI associated mainly with mitochondria and HKII associated with both mitochondrial and cytoplasmic compartments. Here we tested the hypothesis that these different subcellular distributions are associated with different metabolic roles, with mitochondrially-bound HK''s channeling G-6-P towards glycolysis (catabolic use), and cytoplasmic HKII regulating glycogen formation (anabolic use).

Methodology/Principal Findings

To study subcellular translocation of HKs in living cells, we expressed HKI and HKII linked to YFP in CHO cells. We concomitantly recorded the effects on glucose handling using the FRET based intracellular glucose biosensor, FLIPglu-600 mM, and glycogen formation using a glycogen-associated protein, PTG, tagged with GFP. Our results demonstrate that HKI remains strongly bound to mitochondria, whereas HKII translocates between mitochondria and the cytosol in response to glucose, G-6-P and Akt, but not ATP. Metabolic measurements suggest that HKI exclusively promotes glycolysis, whereas HKII has a more complex role, promoting glycolysis when bound to mitochondria and glycogen synthesis when located in the cytosol. Glycogen breakdown upon glucose removal leads to HKII inhibition and dissociation from mitochondria, probably mediated by increases in glycogen-derived G-6-P.

Conclusions/Significance

These findings show that the catabolic versus anabolic fate of glucose is dynamically regulated by extracellular glucose via signaling molecules such as intracellular glucose, G-6-P and Akt through regulation and subcellular translocation of HKII. In contrast, HKI, which activity and regulation is much less sensitive to these factors, is mainly committed to glycolysis. This may be an important mechanism by which HK''s allow cells to adapt to changing metabolic conditions to maintain energy balance and avoid injury.     

Intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.     

The proton stoichiometry of electron transport in Ehrlich ascites tumor mitochondria.

Initial rate measurements of the stoichiometric relationships between H+ ejection, K+ and Ca2+ uptake, and electron transport were carried out on mitochondria from Ehrlich ascites tumor cells grown in mice. With succinate as substrate and N-ethylmaleimide to prevent interfering H+ reuptake via the phosphate carrier, close to 8 H+ were ejected per oxygen atom reduced (H+/O ejection ratio = 8.0); with the NAD-linked substrates pyruvate or pyruvate + malate, the H+/O ejection ratio was close to 12. The average H+/site ratio (H+ ejected/2e-/energy-conserving site) was thus close to 4. The simultaneous uptake of charge-compensating cations, either K+ (in the presence of valinomycin) or Ca2+, was also measured, yielding average K+/site uptake ratios of very close to 4 and Ca2+/site ratios close to 2. It was also demonstrated that each calcium ion enters the respiring tumor mitochondria carrying two positive electric charges. These stoichiometric data observed in mitochondria from Ehrlich ascites tumor cells thus are in complete agreement with similar data on normal rat liver and rat heart mitochondria and suggest that the H+/site ratio of mitochondrial electron transport may be 4 generally. It was also observed that the rate of deltaH+ back-decay in anaerobic tumor mitochondria following oxygen pulses is some 6- to 8-fold greater than in rat liver mitochondria tested at equal amounts of mitochondrial protein.     

Properties of mitochondria isolated from cyanide-sensitive and cyanide-stimulated cultures of Acanthamoeba castellanii

1. Mitochondria isolated from cultures of Acanthamoeba castellanii exhibit respiratory control and oxidize alpha-oxoglutarate, succinate and NADH with ADP:O ratios of about 2.4, 1.4 and 1.25 respectively. 2. Mitochondria from cultures of which the respiration was stimulated up to 50% by 1mm-cyanide (type-A mitochondria) and from cyanide-sensitive cultures (type-B mitochondria) had similar respiratory-control ratios and ADP:O ratios. 3. State-3 rates of respiration were generally more cyanide-sensitive than State-4 rates, and the respiration of type-A mitochondria was more cyanide-resistant than that of type-B mitochondria. 4. Salicylhydroxamic acid alone had little effect on respiratory activities of either type of mitochondria, but when added together with cyanide, irrespective of the order of addition, inhibition was almost complete. 5. Oxidation of externally added NADH by type-A mitochondria was mainly via an oxidase with a low affinity for oxygen (K(m)[unk]15mum), which was largely cyanide-sensitive and partially antimycin A-sensitive; this electron-transport pathway was inhibited by ADP. 6. Cyanide-insensitive but salicylhydroxamic acid-sensitive respiration was stimulated by AMP and ADP, and by ATP after incubation in the presence of MgCl(2). 7. Addition of rotenone to mitochondria oxidizing alpha-oxoglutarate lowered the ADP:O ratios by about one-third and rendered inhibition by cyanide more complete. 8. The results suggest that mitochondria of A. castellanii possess branched pathways of electron transport which terminate in three separate oxidases; the proportions of electron fluxes via these pathways vary at different stages of growth.     

Intracellular iron trafficking: role of cytosolic ligands

Iron acquired by cells is delivered to mitochondria for metabolic processing via pathways comprising undefined chemical forms. In order to assess cytosolic factors that affect those iron delivery pathways, we relied on microscopy and flow-cytometry for monitoring iron traffic in: (a) K562 erythroleukemia cells labeled with fluorescent metal-sensors targeted to either cytosol or mitochondria and responsive to changes in labile iron and (b) permeabilized cells that retained metabolically active mitochondria accessible to test substrates. Iron supplied to intact cells as transferrin-Fe(III) or Fe(II)-salts evoked concurrent metal ingress to cytosol and mitochondria. With either supplementation modality, iron ingress into cytosol was mostly absorbed by preloaded chelators, but ingress into mitochondria was fully inhibited only by some chelators, indicating different cytosol-to-mitochondria delivery mechanisms. Iron ingress into cytosol or mitochondria were essentially unaffected by depletion of cytosolic iron ligands like glutathione or the hypothesized 2,5 dihydroxybenzoate (2,5-DHBA) siderophore/chaperone. These ligands also failed to affect mitochondrial iron ingress in permeabilized K562 cells suspended in cytosol-simulating medium. In such medium, mitochondrial iron uptake was >6-eightfold higher for Fe(II) versus Fe(III), showed saturable properties and submicromolar K(1/2) corresponding to cytosolic labile iron levels. When measured in iron(II)-containing media, ligands like AMP, ADP or ATP, did not affect mitochondrial iron uptake whereas in iron(III)-containing media ADP and ATP reduced it and AMP stimulated it. Thus, cytosolic iron forms demonstrably contribute to mitochondrial iron delivery, are apparently not associated with DHBA analogs or glutathione but rather with resident components of the cytosolic labile iron pool.     

The effect of copper excess on iron metabolism in sheep.

Sheep were treated with large amounts of copper (20 mg of CuSO4,5H2O/kg body wt. per day) for 9 weeks to examine the effect of copper excess on iron metabolism. In addition to confirming that massive haemolysis and accumulation of copper occurs in the liver, kidney and plasma after 7 weeks of exposure to excess copper, it was observed that excess copper produced an increased plasma iron concentration and transferrin saturation within 1 week. Further, iron preferentially accumulated in the spleen between 4 and 6 weeks of copper treatment, producing 3-fold increases in the iron content of both the ferritin and non-ferritin fractions. A 3-4 fold increase was also observed in the amount of ferritin that could be isolated from the spleen. The copper treatment had little or no effect on the concentration of iron in the liver and bone marrow. The following properties of erythrocytes were also unaffected by copper treatment: size, haemoglobin content and pyruvate kinase activity, although the erythrocyte concentration of copper increased after 6 weeks. Copper accumulated in the spleen between 6 and 9 weeks, probably owing to the phagocytosis of erythrocytes containing high concentrations of copper. The data suggest that copper excess influences iron metabolism, initially by causing a compensated haemolytic anaemia, and later by interfering with re-utilization of iron from ferritin in the reticuloendothelial cells of the spleen.