Oxidation Of Krebs Cycle Intermediates Research Articles

Isolation and characterization of tightly coupled mitochondria from wild type and nap mutant Neurospora crassa.

A fast and reproducible procedure was elaborated for isolation of tightly coupled mitochondria from wild type and nap-mutant Neurospora crassa cells harvested at different growth stages. The isolated mitochondrial preparations had controlled metabolic states and were tightly coupled, i.e., displayed good respiratory control and had close to the theoretically expected maximal ADP/O ratios upon oxidation of Krebs cycle intermediates and exogenous NADH. They contained the fully competent respiratory chain with all three points of energy conservation. Oxidation of all examined substrates by mitochondria from both wild type and mutant cells was mediated by two alternative terminal oxidative systems, albeit to varying extent, with the more pronounced engagement of the alternative oxidase in the stationary growth phase and with a minor contribution of this non-phosphorylating pathway in the substrate oxidation by mutant mitochondria. Oxidation of NAD-dependent substrates by mitochondria from the two cell types was accommodated via both rotenone-sensitive and rotenone-insensitive pathways, while the level of rotenone-insensitive pathway in mutant cells was lower than in wild type cells. It is suggested that a more limited contribution of alternative non-phosphorylating oxidative pathways to the total respiration in mutant cells, as compared with wild type cells, could, at least partially, explain an elevated ATP level in these cells. However, the absence of principal differences in the arrangement of the respiratory chain in mitochondria of wild type and mutant cells implies that the elevated ATP level in the nap mutant is largely related to reduced ATP expenses for transport processes in these cells.

Biosynthesis and metabolism of steroid hormones by human adrenal carcinomas.

Over a 15-year period, our university-based laboratory obtained 125 adrenal tumors, of which 15 (12%) were adrenal cortical carcinomas. Of these, 6 (40% of the carcinomas) occurred in patients with clear clinical manifestations of steroid hormone excess. Adrenal cortical carcinoma cells derived from the surgically resected tumors in 4 of these patients were isolated and established in primary culture. Radiotracer steroid interconversion studies were carried out with these cultures and also on mitochondria isolated from homogenized tissues. Large tumors had the lowest steroidogenic activities per weight, whereas small tumors had more moderately depressed enzyme activities relative to cells from normal glands. In incubations with pregnenolone as substrate, 1 mM metyrapone blocked the synthesis of corticosterone and cortisol and also the formation of aldosterone. Metyrapone inhibition was associated with a concomitant increase in the formation of androgens (androstenedione and testosterone) from pregnenolone. Administration of metyrapone in vivo before surgery in one patient resulted in a similar increase in plasma androstenedione, though plasma testosterone levels were not significantly affected. In cultures of two of four tumors examined, dibutyryl cAMP stimulated 11ss-hydroxylase activity modestly; ACTH also had a significant stimulatory effect in one of these tumors. Unlike results obtained with normal or adenomatous adrenal cortical tissues, mitochondria from carcinomatous cells showed a lack of support of either cholesterol side-chain cleavage enzyme complex or steroid 11ss-hydroxylase activity by Krebs cycle intermediates (10 mM isocitrate, succinate or malate). This finding is consistent with the concept that these carcinomas may tend to function predominantly in an anaerobic manner, rather than through the oxidation of Krebs cycle intermediates.

Oxidation of proline and pyruvate by flight muscle mitochondria of the Colorado beetle, Leptinotarsa decemlineata say

Mitochondria isolated from the flight muscles of the Colorado beetle Leptinotarsa decemlineata show high rates of respiration for proline and pyruvate. The rate of oxidation of Krebs cycle intermediates is comparatively low. The amount of alanine formed during in vitro oxidation of proline is equal to the amount of proline used. The results suggest that proline oxidation leads to pyruvate formation and subsequent alanine synthesis. The specific activity of NAD-dependent malic enzyme is high and is supposed to participate in pyruvate formation. In the presence of low proline concentrations, pyruvate and proline oxidations are fully additive. At proline concentrations above 1 mM both oxidations do not seem to occur simultaneously.

The transport of Krebs-cycle intermediates in Azotobacter vinelandii under various metabolic conditions

1. 1. The oxidation of Krebs-cycle intermediates in Azotobacter vinelandii is inhibited by the uncouplers carbonyl cyanide p- trifluoromethoxyphenylhydrazone and carbonyl cyanide m- chlorophenylhydrazone , and energy-transfer inhibitors such as oligomycin, N,N′- dicyclohexylcarbodiimide or Dio-9. 2. 2. Uncouplers and energy-transfer inhibitors have a synergistic inhibitory effect. 3. 3. Under aerobic conditions no rapid exchange diffusion of C 4-dicarboxylates is observed. 4. 4. Cells retain their accumulated metabolites under anaerobic conditions. Under these conditions uncouplers or energy-transfer inhibitors induce an efflux of metabolites. 5. 5. Under anaerobic conditions exchange of intracellular for extracellular dicarboxylates is observed. 6. 6. It is concluded from these results that in A. vinelandii Krebs-cycle intermediates are transported at the expense of energy. There are strong indications for a direct involvement of ATP in this energy-requiring process.

VARIANT AND INVARIANT PROPERTIES OF THE MITROCHONDRIAL FRACTION ISOLATED FROM RIPENING BANANA FRUIT

SUMMARY– The mitrochondrial characteristics of oxidation of Krebs cycle intermediates, respiratory control by ADP, succinate‐linked oxidative phosphorylation, and overall morphology, and the amount of protein in the fraction did not vary as the ripening of banana fruit progressed. However, stimulation of succinoxidase activity by calcium and succinate‐driven calcium accumulation underwent striking changes as terminal senescence ensued. The results are consistent with observations of the controlled nature of terminal senescence and to the possible involvement of ancillary work performances, such as active transport, with climacteric respiration.

Respiration and protein synthesis in Escherichia coli membrane-envelope fragments. I. Oxidative activities with soluble substrates.

This paper describes experiments conducted with membranous and soluble fractions obtained from Escherichia coli that had been grown on succinate, malate, or enriched glucose media. Oxidase and dehydrogenase activities were studied with the following substrates: nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), succinate, malate, isocitrate, glutamate, pyruvate, and alpha-ketoglutarate. Respiration was virtually insensitive to poisons that are commonly used to inhibit mitochondrial systems, namely, rotenone, antimycin, and azide. Succinate dehydrogenase and NADH, NADPH, and succinate oxidases were primarily membrane-bound whereas malate, isocitrate, and NADH dehydrogenases were predominantly soluble. It was observed that E. coli malate dehydrogenase could be assayed with the dye 2,6-dichlorophenol indophenol, but that porcine malate dehydrogenase activity could not be assayed, even in the presence of E. coli extracts. The characteristics of E. coli NADH dehydrogenase were shown to be markedly different from those of a mammalian enzyme. The enzyme activities for oxidation of Krebs cycle intermediates (malate, succinate, isocitrate) did not appear to be under coordinate genetic control.

Ricerche sul meccanismo di azione della cocliobolina B: effetti sul metabolismo ossidativo diMicrococcus pyogenesvar.aureus

Abstract STUDIES ON MECHANISM OF COCHLIOBOLIN ACTIVITY. B: EFFECTS ON OXIDATIVE METABOLISM OF MICROCOCCUS PYOGENES VAR. AUREUS. — Endogenous respiration of washed 24-hours old cells of Micrococcus pyogenes var. aureus was markedly decreased by cochliobolin, which also completely prevented pyruvate, succinate, fumarate and malate oxidation. The toxin inhibited also lactate and α-ketoglutarate utilization by Micrococcus cells, while aerobic respiration of glucose was only slightly reduced, and anaerobic breakdown increased. These phenomena were irreversible, and depended upon the pH of media and the lenght of incubation periods. In contrast, respiratory activities of cell-free homogenates and acetone powders of Micrococcus were refractory to the toxin, and the oxidation of Krebs cycle intermediates was not affected. These phenomena suggest that, although cochliobolin prevens the respiratory metabolism of Micrococcus cells, the initial locus of interference probably is not one of the common respiratory enzim...

Studies on the Oxidative Metabolism of the Chick Embryo , : 2. Oxidation of Krebs Cycle Intermediates and the Effect of Dinitrophenol on it

IN PREVIOUS reports of studies on embryonic metabolism carried out in this laboratory (Feldman et al., 1958; Feldman and Couch, 1960) a system was defined for the stimulation of oxygen uptake of chick embryo preparations. It was pointed out that the maximal oxidative rate required a balance between the breakdown of adenosine triphosphate (ATP) and its synthesis. Furthermore, it was found that oxygen uptake by chick embryo preparations proceeded slowly unless magnesium and ATP were added in vitro. The addition of low concentrations of dinitrophenol in vitro stimulated the oxygen consumption of cell-free extracts of 10-day chick embryos (Feldman et al., 1958). The stimulatory effect of dinitrophenol was also observed with cell-free extracts of embryos which had been removed from eggs injected with the drug.Very little is known of the enzymatic activity of older chick embryos, particularly the enzyme systems involved in energy metabolism. It was felt that a …

Some aspects of plant mitochondria.

The presence of a large central vacuole in plant cells has greatly hindered histochemical and biochemical investigations, because, when a homogenate is prepared, the vacuolar sap, which frequently includes large quantities of the substrate and product of an enzyme reaction and in certain cases enzyme inhibitors, becomes intimately mixed with the somewhat limited amount of protoplasm present. Bentley (1952), for instance, has shown that malonic acid is widely distributed in plants; fluoroacetic acid occurs in Dichapetalum cymosum (Marais 1944), and transaconitic acid may be present in certain members of the Ranunculaceae. Though these facts pose interesting problems in the permeability relations of cell components, they greatly complicate the analysis of homogenates. Dialysis provides one method of isolating active enzyme material from the homogenate, but the concomitant loss of cofactors has limited its application. A partial solution to the problem has been obtained by isolating particulate material by differential centrifugation. This method has made possible investigations of metabolism not amenable to study by the classical methods of enzymology and simultaneously has provided an approach to the study of the spatial relations of enzymes. The pioneer work was performed by Hill & Bhagvat (1939), who demonstrated the association of cytochrome oxidase with particulate material. Several workers (Meeuse 1950; Bhagvat & Hill 1951; Stafford 1951) have demonstrated the association of succinoxidase with particulate material isolated from homogenates prepared in dilute phosphate buffer, but cytochrome c was necessary for activity. Meeuse (1950) also found that malic oxidase was associated with particulate material, but DPN and cytochrome c were required for activity. Millerd, Bonner, Axelrod & Bandurski (1951), Stafford (1953), Price & Thimann (1953) and Davies (1953) have isolated particulate material from seedlings which was active in the oxidation of Krebs-cycle intermediates. Millerd et al . (1951) were also able to demonstrate that phosphorylation accompanied the oxidation of Krebs-cycle intermediates.

The effect of zinc on the oxidation of Krebs cycle intermediates by rat liver and kidney homogenates

Zinc ions in small concentrations (2 × 10 −5 M) inhibit rat liver and kidney succinoxidase systems by forming an inactive complex with the enzyme. Under conditions which favor oxidative phosphorylation (isotonic KCl homogenates, Mg ion, and muscle adenylate or adenosine triphosphate), inhibition by zinc occurs, but this inhibition is transitory. The rate of oxidation subsequently rises to a level even higher than shown by the control data. A later fall to a lower level of activity can be attributed to the zinc inhibition of other Krebs cycle intermediates, particularly the oxidation of α-ketoglutarate and citrate. The addition of a protein such as insulin has no protective action against zinc inhibition, and crystalline zinc insulin causes the same inhibition as does zinc sulfate. With the exception of succinoxidase, the oxidizing enzymes of rat kidney are more readily inhibited by zinc ions than are those of rat liver. Amorphous insulin, in itself, inhibits citrate oxidation by rat kidney homogenates.