Effects of adenosine phosphates and nicotinamide nucleotides on pyruvate carboxylase from baker''s yeast

1. Pyruvate carboxylase from baker's yeast is inhibited by ADP, AMP and adenosine at pH8.0 in the presence of magnesium chloride concentrations equal to or higher than the ATP concentration. The adenine moiety is essential for the inhibitory effect. 2. In the absence of acetyl-CoA (an allosteric activator) ADP, AMP and adenosine are competitive inhibitors with respect to ATP. In the presence of acetyl-CoA, besides the effect with respect to ATP, AMP competes with acetyl-CoA, whereas ADP and adenosine are non-competitive inhibitors with respect to the activator. 3. Pyruvate carboxylase is inhibited by NADH. The inhibition is competitive with respect to acetyl-CoA and specific with respect to NADH, since NAD(+), NADP(+) and NADPH do not affect the enzyme activity. In the absence of acetyl-CoA, NAD(+), NADH, NADP(+) and NADPH do not inhibit pyruvate carboxylase. 4. Pyruvate carboxylase is inhibited by ADP, AMP and NADH at pH6.5, in the presence of 12mm-Mg(2+), 0.75mm-Mn(2+) and 0.5mm-ATP, medium conditions similar to those existing inside the yeast cell. The ADP and NADH effects are consistent with a regulation of enzyme activity by the intracellular [ATP]/[ADP] ratio and secondarily by NADH concentration. These mechanisms would supplement the already known control of yeast pyruvate carboxylase by acetyl-CoA and l-aspartate. Inhibition by AMP is less marked and its physiological role is perhaps limited.     

Automated microanalysis of adenosine phosphates, phosphocreatine, creatine, and lactate in muscle

An automated enzymatic procedure suitable for determination of ATP, ADP, AMP, phosphocreatine, creatine, and lactate in needle biopsies of human skeletal muscle (ca. 30 mg dry wt) using a fast centrifugal analyzer (Multistat III, Instrumentation Laboratory Inc.) is presented. Coefficients of variation ranged from 0.7 to 4.2% for multiple determinations of ATP, ADP, phosphocreatine, and creatine; from 6 to 24% for lactate; and from 9 to 20% for AMP. The procedure should be usable, with appropriate modification, with other tissues and with other fast centrifugal analyzers. Muscle samples are collected into liquid freon, lyophilized, and extracted with 600 microliter of 0.65 M perchloric acid. Neutralized supernatants can be stored for up to 3 years at -80 degrees C with no significant deterioration. The procedure takes much less time than similar manual procedures and gives better reproducibility, particularly for ADP and AMP.     

Effects of calcium on transendothelial albumin transfer and electrical resistance

Nicolaysen, and more recently Kern and Malik, reported that chelation of calcium increased microvascular hydraulic conductivity and albumin permeability in isolated perfused lungs. To begin to understand how calcium affects endothelial function we examined the effect of calcium chelation on an in vitro endothelium. Chelation of calcium with ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid increased the rate of transendothelial albumin transfer by 125%. Reincubation of the endothelium in calcium-repleted medium restored the rate of transfer to its original value. Chelation of extracellular calcium abolished transendothelial electrical resistance. The transendothelial electrical resistance was also restored to normal by reincubation of the endothelium in calcium-repleted medium. Chelation of extracellular calcium caused adjacent endothelial cells to retract from one another, and normal apposition of adjacent cells was restored after reincubation in calcium-repleted medium. Chelation of extracellular calcium produced a centripetal retraction of the peripheral band of actin in individual endothelial cells, and the actin band resumed its normal location after reincubation in calcium-repleted medium. Calcium is an important determinant of endothelial integrity and alterations in calcium produce dynamic changes in endothelial barrier properties and in endothelial-cell shape.     

Effects of vesicular-arbuscular mycorrhiza on the availability of iron phosphates to plants

Summary The effect of inoculation with a mycorrhizal fungus on the growth of subterranean clover and of ryegrass was measured using three sources of phosphorus with different solubilities. These were (in order of decreasing solubility): potassium dihydrogen phosphate, colloidal iron phosphate and crystalline iron phosphate. Mycorrhizal infection increased growth more for subterranean clover than for ryegrass for all sources of phosphorus. For both species the greatest benefit from mycorrhizal inoculation was obtained with the least soluble source of iron phosphate. It is suggested that the mycorrhizas were able to explore the soil more thoroughly and hence were able to locate and use the point sources of phosphorus in the insoluble iron phosphates.     

Inhibition effect of ferric iron on the kinetics of ferrous iron

Ferric iron acted as a non-competitive inhibitor for the biological oxidation of ferrous iron and decreased the inhibitory effects of high concentrations of ferrous iron as well as the auto-inhibitive effect the bacterial cells. A previously developed kinetic model for this reaction was modified to incorporate the inhibition effects of ferric iron. © Rapid Science Ltd. 1998     

Uptake of iron by apoferritin from a ferric dihydrolipoate complex

A study was made on the uptake of iron by horse spleen apoferritin, by using as an iron source the same ferric dihydrolipoate complex which represents the major product in the anaerobic removal of ferritin-bound iron by dihydrolipoate at neutral pH. The ferric dihydrolipoate complex was chemically synthesized and used as an iron donor to apoferritin. Iron uptake was studied, at slightly alkaline pH and in anaerobic conditions, as a function of the concentration of both the iron donor and apoferritin. Isolation of ferritin from mixtures of ferric dihydrolipoate and apoferritin, and subsequent identification of the oxidation state of ferritin-bound iron, showed that the first metal atoms were taken up in the ferrous form and that this early step was accompanied by accumulation of ferric iron. Total iron uptake increased with the molar ratio of complex to apoprotein and ranged over 25-40% of the iron being supplied. The amount of ferrous iron found inside the protein did not exceed 50-60 mol iron/mol ferritin after a 48-h incubation. At this time, ferric iron represented a significant fraction of the iron found in the isolated ferritin. Analytical and spectroscopic data indicated that fractional rates and equilibria for disassembly of the ferric complex in the presence of apoferritin were independent of the concentration of the protein and of the complex itself.     

State of adenosine phosphates during dehydration of yeast

Summary The effect of cultivation and dehydration conditions on the adenosine phosphate content of yeast cells has been studied. Irrespective of the cultivation conditions the total pool of adenosine phosphates was found to increase, mainly due to accumulation of ATP, during the exponential phase of cell growth and to decrease during transition of the culture into the stationary phase. Changes in the intracellular content of adenosine phosphates were parallel with changes in the respiratory activity of yeast cells cultivated under batch conditions. Yeast cells harvested at the exponential growth phase were sensitive to dehydration, losing a notable amount of adenosine phosphates as well as respiratory capacity during drying, leading to a massive dying-off of the cells. Yeast at the stationary phase was resistant to drying, and, during this process, accumulated ATP by mitochondrial oxidation of endogenous carbohydrates. The accumulated ATP was used by the dried yeast cells as an energy source in the first minutes of reactivation. On the basis of our results we recommend that the ATP content of dried yeast cells should be used as an indicator of their capacity to recover their viability by reactivation.     

Influence of adenosine phosphates and magnesium on photosynthesis in chloroplasts from peas, sedum, and spinach

¹⁴CO₂ photoassimilation in the presence of MgATP, MgADP, and MgAMP was investigated using intact chloroplasts from Sedum praealtum, a Crassulacean acid metabolism plant, and two C₃ plants: spinach and peas. Inasmuch as free ATP, ADP, AMP, and uncomplexed Mg²⁺ were present in the assays, their influence upon CO₂ assimilation was also examined. Free Mg²⁺ was inhibitory with all chloroplasts, as were ADP and AMP in chloroplasts from Sedum and peas. With Sedum chloroplasts in the presence of ADP, the time course of assimilation was linear. However, with pea chloroplasts, ADP inhibition became progressively more severe, resulting in a curved time course. ATP stimulated assimilation only in pea chloroplasts. MgATP and MgADP stimulated assimilation in all chloroplasts. ADP inhibition of CO₂ assimilation was maximal at optimum orthophosphate concentrations in Sedum chloroplasts, while MgATP stimulation was maximal at optimum or below optimum concentrations of orthophosphate. MgATP stimulation in peas and Sedum and ADP inhibition in Sedum were not sensitive to the addition of glycerate 3-phosphate (PGA).

PGA-supported O₂ evolution by pea chloroplasts was not inhibited immediately by ADP; the rate of O₂ evolution slowed as time passed, corresponding to the effect of ADP on CO₂ assimilation, and indicating that glycerate 3-phosphate kinase was a site of inhibition. Likewise, upon the addition of AMP, inhibition of PGA-dependent O₂ evolution became more severe with time. This did not mirror CO₂ assimilation, which was inhibited immediately by AMP. In Sedum chloroplasts, PGA-dependent O₂ evolution was not inhibited by ADP and AMP. In chloroplasts from peas and Sedum, the magnitude of MgADP and MgATP stimulation of PGA-dependent O₂ evolution was not much larger than that given by ATP, and it was much smaller than MgATP stimulation of CO₂ assimilation. Analysis of stromal metabolite levels by anion exchange chromatography indicated that ribulose 1,5-bisphosphate carboxylase was inhibited by ADP and stimulated by MgADP in Sedum chloroplasts.

The appearance of label in the medium was measured when [U-¹⁴C] ADP-loaded Sedum chloroplasts were challenged with ATP, ADP, or AMP and their Mg²⁺ complexes. The rate of back exchange was stimulated by the presence of Mg²⁺. This suggests that ATP, ADP, and AMP penetrate the chloroplast slower than their Mg²⁺ complexes. A portion of the CO₂ assimilation and O₂ evolution data could be explained by differential penetration rates, and other proposals were made to explain the remainder of the observations.

     

Study of iron removal from ferric citrate medium by pure and mixed cultures of iron resistant microbes

Summary A comparative study of iron removal at 30–60 C and pH 4–9 by pure (Aeromonas sp.) and mixed culture of iron resistant microbes (FMC) showed maximum efficiency of 45% (pH-8, 40 C) and 90% (pH-9, 40C) respectively in 60–72 h using a synthetic ferric citrate medium containing 650 mg/l Fe(III) with ammonium chloride as nitrogen source.     

Kinetics of iron acquisition from ferric siderophores by Paracoccus denitrificans.

The kinetics of iron accumulation by iron-starved Paracoccus denitrificans during the first 2 min of exposure to 55Fe-labeled ferric siderophore chelates is described. Iron is acquired from the ferric chelate of the natural siderophore L-parabactin in a process exhibiting biphastic kinetics by Lineweaver-Burk analysis. The kinetic data for 1 microM less than [Fe L-parabactin] less than 10 microM fit a regression line which suggests a low-affinity system (Km = 3.9 +/- 1.2 microM, Vmax = 494 pg-atoms of 55Fe min-1 mg of protein-1), whereas the data for 0.1 microM less than or equal to [Fe L-parabactin] less than or equal to 1 microM fit another line consistent with a high-affinity system (Km = 0.24 +/- 0.06 microM, Vmax = 108 pg-atoms of 55Fe min-1 mg of protein-1). The Km of the high-affinity uptake is comparable to the binding affinity we had previously reported for the purified ferric L-parabactin receptor protein in the outer membrane. In marked contrast, ferric D-parabactin data fit a single regression line corresponding to a simple Michaelis-Menten process with comparatively low affinity (Km = 3.1 +/- 0.9 microM, Vmax = 125 pg-atoms of 55Fe min-1 mg of protein-1). Other catecholamide siderophores with an intact oxazoline ring derived from L-threonine (L-homoparabactin, L-agrobactin, and L-vibriobactin) also exhibit biphasic kinetics with a high-affinity component similar to ferric L-parabactin. Circular dichroism confirmed that these ferric chelates, like ferric L-parabactin, exist as the lambda enantiomers. The A forms ferric parabactin (ferrin D- and L-parabactin A), in which the oxazoline ring is hydrolyzed to the open-chain threonyl structure, exhibit linear kinetics with a comparatively high Km (1.4 +/- 0.3 microM) and high Vmax (324 pg-atoms of 55Fe min-1 of protein-1). Furthermore, the marked stereospecificity seen between ferric D- and L-parabactins is absent; i.e., iron acquisition from ferric parabactin A is non stereospecific. The mechanistic implications of these findings in relation to a stereospecific high-affinity binding followed by a nonstereospecific postreceptor processing is discussed.     

Effects of electron transport inhibitors and uncouplers on the oxidation of ferrous iron and compounds interacting with ferric iron in Acidithiobacillus ferrooxidans

Oxidation of Fe2+, ascorbic acid, propyl gallate, tiron, L-cysteine, and glutathione by Acidithiobacillus ferrooxidans was studied with respect to the effect of electron transport inhibitors and uncouplers on the rate of oxidation. All the oxidations were sensitive to inhibitors of cytochrome c oxidase, KCN, and NaN3. They were also partially inhibited by inhibitors of complex I and complex III of the electron transport system. Uncouplers at low concentrations stimulated the oxidation and inhibited it at higher concentrations. The oxidation rates of Fe2+ and L-cysteine inhibited by complex I and complex III inhibitors (amytal, rotenone, antimycin A, myxothiazol, and HQNO) were stimulated more extensively by uncouplers than the control rates. Atabrine, a flavin antagonist, was an exception, and atabrine-inhibited oxidation activities of all these compounds were further inhibited by uncouplers. A model for the electron transport pathways of A. ferrooxidans is proposed to account for these results. In the model these organic substrates reduce ferric iron on the surface of cells to ferrous iron, which is oxidized back to ferric iron through the Fe2+ oxidation pathway, leading to cytochrome oxidase to O2. Some of electrons enter the uphill (energy-requiring) electron transport pathway to reduce NAD+. Uncouplers at low concentrations stimulate Fe2+ oxidation by stimulating cytochrome oxidase by uncoupling. Higher concentrations lower deltap to the level insufficient to overcome the potentially uphill reaction at rusticyanin-cytochrome c4. Inhibition of uphill reactions at complex I and complex III leads to deltap accumulation and inhibition of cytochrome oxidase. Uncouplers remove the inhibition of deltap and stimulate the oxidation. Atabrine inhibition is not released by uncouplers, which implies a possibility of atabrine inhibition at a site other than complex I, but a site somehow involved in the Fe2+ oxidation pathway.     

"Hormone-like" effect of adenosine and inosine on gluconeogenesis from lactate in isolated hepatocytes

In isolated rat hepatocytes adenosine and inosine showed a dose-dependent increase in the rate of glucose synthesis from lactate with a Ka of 7.5 x 10(-8) and 9 x 10(-8) M, respectively. Absence of this action was recorded with: IMP, xanthosine, adenine, hypoxanthine, and uric acid. A reciprocal inhibition of individual gluconeogenic stimulation was found in cells incubated with glucagon or epinephrine and adenosine, but not with inosine. 5'-(N-ethyl) carboxamido adenosine was more potent than adenosine, whereas N6-(L-2-phenylisopropyl)-adenosine antagonized the stimulation of gluconeogenesis by adenosine. Neither of the analogs used modified the stimulatory role of inosine on the studied pathway. Adenosine and inosine may be involved in the short term regulation of gluconeogenesis.     

Recovery of scrap iron metal value using biogenerated ferric iron

The utility of employing biogenerated ferric iron as an oxidant for the recycling of scrap metal has been demonstrated using continuously growing cells of the extremophilic organism Acidithiobacillus ferrooxidans. A ferric iron rich (70 mol%) lixiviant resulting from bioreactor based growth of A. ferrooxidans readily solubilized target scrap metal with the resultant generation of a leachate containing elevated ferrous iron levels and solubilized copper previously resident in the scrap metal. Recovery of the copper value was easily accomplished via a cementation reaction and the clarified leachate containing a replenished level of ferrous iron as growth substrate was shown to support the growth of A. ferrooxidans and be fully recyclable. The described process for scrap metal recycling and copper recovery was shown to be efficient and economically attractive. Additionally, the utility of employing the E(h) of the growth medium as a means for monitoring fluctuations in cell density in cultures of A. ferrooxidans is demonstrated.