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Biomedical subjects

O T Jones

Publications and source records attributed to O T Jones.

At least 145 records · Page 8Linked to original sources
PubMed1973

Studies on the biosynthesis of cytochrome c.

A soluble cytochrome was isolated and purified from the slime mould Physarum polycephalum and identified as cytochrome c by room-temperature and low-temperature (77 degrees K) difference spectroscopy. A close similarity between P. polycephalum and mammalian cytochromes c was suggested by a comparison of the initial rates of oxidation of both proteins by mammalian mitochondria. This similarity was further emphasized by redox titrations and gel-electrophoretic studies which indicated that P. polycephalum cytochrome c has an oxidation-reduction midpoint potential of +257mV at pH7.0 and a molecular weight of 12500+/-1500 (mean+/-maximum deviation for a set of six measurements). P. polycephalum exhibits an absolute requirement for protohaemin for growth. The (59)Fe-labelled haemin was prepared by chemical synthesis from protoporphyrin. The purified product had a specific radioactivity of 0.8+/-0.02muCi/mol. Growth of P. polycephalum in the presence of [(59)Fe]haemin resulted in the incorporation of (59)Fe into the plasmodial cytochrome c. The specific radioactivity of the cytochrome c haem was 0.36+/-0.02muCi/mol. The high specific radioactivity of the cytochrome haem indicates that synthesis of the holoenzyme must proceed by direct attachment of haem to the apoprotein rather than by the intermediate formation of a protoporphyrinogen-apoprotein complex. The observed decrease in the specific radioactivity of the haem group is attributed to exchange of the (59)Fe with unlabelled iron in the plasmodia either before or during attachment of the haem group to the apoprotein.

Animals↗
PubMed1973

The electron-transport system of mitochondria from the slime mould Physarum polycephalum.

A method is described for the preparation of mitochondria from the slime mould Physarum polycephalum; the mitochondria were not coupled. P. polycephalum mitochondria oxidized added NADH via a rotenone-insensitive pathway, but the oxidation of malate plus glutamate was rotenone sensitive; both of these substrates reduced much less cytochrome b than did succinate, in both aerobic and anaerobic steady states. Spectroscopy at 77 degrees K separated three absorption maxima in the alpha-band region, at 560nm, 553nm and one at 547nm due to cytochrome c. The absorption at 553nm was increased in the aerobic steady state by the addition of 2-heptyl-4-hydroxyquinoline N-oxide, suggesting that it was due to a b-type cytochrome. All three absorption maxima appeared in the aerobic steady state after the addition of a range of substrates. The respiratory activity with different substrates and the response to inhibitors of respiration were similar to those previously described for fungus mitochondria (Weiss et al., 1970; Erickson & Ashworth, 1969). When grown under conditions of haem limitation the mitochondria contained a lower concentration of cytochromes than normal.

Adenosine Triphosphate↗
PubMed1972

The utilization of iron and its complexes by mammalian mitochondria.

Sonicated mitochondria catalyse the reduction of ferric salts, and the subsequent incorporation of Fe(2+) into haem, when provided with a reducing substrate such as succinate or NADH. The rate of haem synthesis was low under aerobic conditions and, after a short lag period, accelerated once anaerobic conditions were achieved; it was insensitive to antimycin A. The lag period was decreased by preincubating the mitochondria with NADH and Fe(3+). Newly formed Fe(2+) was autoxidized rapidly and the consequent O(2) uptake was measured with an oxygen electrode to determine the rate of enzymic formation of Fe(2+) from FeCl(3); this reaction was rapid in sonicated mitochondria provided with NADH or succinate and was insensitive to antimycin A. The reaction was very slow in intact mitochondria, suggesting a permeability barrier to Fe(3+) ions. This system was used to test the permeability of the mitochondrial membrane to various iron complexes of biological importance. Of the compounds tested only ferrioxamine G appeared to penetrate readily and the iron of this complex was reduced when intact mitochondria were supplied with succinate or NADH-linked substrates. The reduction was insensitive to rotenone or antimycin A. Both ferrioxamine G and ferrioxamine B were, however, reduced by particles. The membrane fraction of sonicated mitochondria was necessary for the reduction. The rate of ferrioxamine B reduction by sonicated mitochondria was measured by a dual-wavelength spectrophotometric assay and was found to be stimulated in conditions where the Fe(2+) produced was utilized for haem synthesis. The addition of FeCl(3) to anaerobic particles caused an oxidation of cytochrome b when this region of the respiratory chain was isolated by treatment with rotenone and antimycin A. These results suggest that the reduction of ferric iron and its complexes occurs inside the inner mitochondrial membrane in proximity to ferrochelatase. Possible sites for this reduction are the flavoproteins, succinate and NADH dehydrogenase.

Animals↗
PubMed1971

Ferrochelatase and -aminolaevulate synthetase in brain, heart, kidney and liver of normal and porphyric rats. The induction of -aminolaevulate synthetase in kidney cytosol and mitochondria by allylisopropylacetamide.

1. delta-Aminolaevulate synthetase was detected in liver and kidney mitochondria prepared from normal rats. 2. The administration of allylisopropylacetamide induced an increase in delta-aminolaevulate synthetase in both liver and kidney mitochondria and the enzyme also appeared in the cytosol fraction of both tissues. Comparison with the distribution of glutamate dehydrogenase indicated that this soluble kidney delta-aminolaevulate synthetase was truly of cytosol origin and did not arise from disrupted mitochondria. The kidney cytosol enzyme was inhibited by 50% by 50mum-protohaem. 3. delta-Aminolaevulate synthetase could not be detected in mitochondria or cytosol from heart or brain from normal or porphyric rats. 4. The administration of allylisopropylacetamide caused little or no increase in ferrochelatase or cytochrome content of liver, kidney, heart or brain mitochondria.

Acetamides↗
PubMed1970

Ferrochelatase of Rhodopseudomonas spheroides.

Extracts of Rhodopseudomonas spheroides contain two ferrochelatases: one is soluble and forms metalloporphyrins from deuteroporphyrin and haematoporphyrin; the other is particulate and forms metalloporphyrins from protoporphyrin, mesoporphyrin, deuteroporphyrin and haematoporphyrin. Neither enzyme incorporates Mg(2+) into porphyrins or Fe(2+) into porphyrin cytochrome c. By using the particulate enzyme, plots of 1/v versus 1/s when one substrate was varied and the other kept constant showed that neither substrate affected the K(m) of the other. The suggested sequential mechanism for the reaction is supported by derivative plots of slopes and intercepts. The K(m) for deuteroporphyrin was 21.3mum and that for Co(2+) was 6.13mum. The enzyme incorporated Co(2+), Fe(2+), Zn(2+), Ni(2+) and Mn(2+); Cd(2+) was not incorporated and was an inhibitor, competitive with respect to Co(2+), non-competitive with respect to deuteroporphyrin. The K(i) for Cd(2+) was 0.73mum. Ferrochelatase was inhibited by protohaem, non-competitively with respect to Co(2+) or with respect to deuteroporphyrin. Inhibition by magnesium protoporphyrin was non-competitive with respect to deuteroporphyrin, uncompetitive with respect to Co(2+). The inhibitory concentrations of the metalloporphyrins are lower than those required for the inhibition of delta-aminolaevulate synthetase by protohaem. Fe(2+) is not incorporated aerobically into porphyrins unless an electron donor, succinate or NADH, is supplied; the low aerobic rate of metalloporphyrin synthesis obtained is insensitive to rotenone and antimycin. The rate of Fe(3+) incorporation increases as anaerobic conditions are achieved.

Air↗
PubMed1969

The structural organization of haem synthesis in rat liver mitochondria.

1. Anaerobic conditions are normally necessary for incorporation of iron into haems and only ferrous iron is used. After addition of succinate to an incubation mixture containing intact or ultrasonically treated mitochondria, Fe(3+) is used, but only if no inhibitors prevent the transfer of electrons from the mitochondrial respiratory chain to oxygen. 2. A dual-wavelength spectrophotometric assay for ferrochelatase is described that has been used for the continuous assay of incorporation of metal ions into porphyrins. Constants are given for the determination of rates of formation of protohaem and cobalt protoporphyrin, mesohaem, cobalt mesoporphyrin and zinc mesoporphyrin. For cobalt mesoporphyrin formation the K(m) for Co(2+) is 11x10(-6)m and that for mesoporphyrin is 5x10(-6)m. 3. An improved method for the separation of inner and outer membranes of mitochondria is described. Mitochondria swollen in hypo-osmotic media were contracted in hyperosmotic potassium chloride solution containing ATP and the outer membranes detached by mild ultrasonic treatment. Sucrose inhibited the ATP-induced contraction and decreased the yield of outer membranes. 4. Ferrochelatase is associated with cytochrome oxidase, which is used as a marker for inner mitochondrial membranes. 5. By using as substrate porphyrin dissolved in phospholipid micelles, ferrochelatase activity of intact mitochondria was shown to be latent, and to be liberated by ultrasonic treatment. 6. No ferrochelatase was detectable in microsomes or soluble cell components.

Adenosine Triphosphate↗
PubMed1969

Multiple light-induced reactions of cytochromes b and c in Rhodopseudomonas spheroides.

Illumination of chromatophore preparations from Rhodopseudomonas spheroides causes the oxidation of a cytochrome c and a slight oxidation of a cytochrome b with a maximum at 560nm. When illuminated in the presence of antimycin A the oxidation of cytochrome c was more pronounced and cytochrome b(560) was reduced; the dark oxidation of cytochrome b(560) was biphasic in the presence of succinate, but not in the presence of NADH, a less effective reductant. Split-beam spectroscopy showed that, in addition to the reduction of cytochrome b(560), another pigment with maxima at 565 and 537nm. was reduced and was more rapidly oxidized in the dark than cytochrome b(560). This pigment, tentatively identified as cytochrome b(565), was also detected in spectra at 77 degrees k, after brief illumination at room temperature; the maxima at 77 degrees k were at 562 and 536nm. In the absence of antimycin A, light caused a transient reduction of cytochrome b(565) and an oxidation of cytochrome b(560). Dark oxidation of b(565) was rapid, even in the presence of antimycin A and succinate. Difference spectra, at 77 degrees k, of ascorbate-reduced minus succinate-reduced chromatophores or of anaerobic succinate-reduced minus aerobic succinate-reduced chromatophores suggested that two cytochromes c were present, with maxima at 547 and 549nm. When chromatophores frozen at 77 degrees k were illuminated both these cytochromes c were oxidized, indicating a close association with the photochemical reaction centre. A scheme involving two reaction centres is proposed to explain these results.

Antimycin A↗
PubMed1968

Ferrochelatase of spinach chloroplasts.

Spinach chloroplasts catalyse the incorporation of Fe(2+) into protoporphyrin, mesoporphyrin and deuteroporphyrin to form the corresponding haems. This ferrochelatase activity was detected by pyridine haemochrome formation with acetone-dried powders of chloroplasts, or from the formation of [(59)Fe]haems by intact chloroplasts. Decreasing the mitochondrial contamination of the chloroplasts by density-gradient centrifugation did not cause any loss of activity: spinach ferrochelatase appears to be principally a chloroplast enzyme. The characteristics of the enzyme were examined by using [(59)Fe]haem assay. The activity was pH-dependent: for both mesohaem and protohaem formation there were two pH maxima, a major peak at about pH7.8 and a smaller peak at about pH9.2. Lineweaver-Burk plots showed that the K(m) for Fe(2+) incorporation into protoporphyrin was 8mum and that for Fe(2+) incorporation into mesoporphyrin was 36mum. At non-saturating Fe(2+) concentrations the K(m) for protoporphyrin was 0.2mum and that for mesoporphyrin was 0.4mum. Ferrochelatase was not solubilized by treatment of chloroplasts with ultrasound but was solubilized by stirring in 1% (w/v) Tween 20 at pH10.4. Unlike the rat liver mitochondrial enzyme, chloroplast ferrochelatase was not stimulated by treatment with selected organic solvents. The spinach enzyme was inactive in aerobic conditions and it was shown by using an oxygen electrode that under such conditions the addition of Fe(2+) to buffer solutions caused a rapid uptake of dissolved oxygen, believed to be due to the oxidation of Fe(2+) to Fe(3+); Fe(3+) is not a substrate for ferrochelatase.

Centrifugation, Density Gradient↗