Presence Of Arsenate Research Articles

Mutational and Physiological Enhancement of Photosynthetic Energy Conversion in Rhodopseudomonas capsulata

Photosynthetic growth of Rhodopseudomonas capsulata, a nonsulfur purple bacterium, is severely inhibited by inorganic arsenate when this anion is present equimolar to orthophosphate in the culture medium. Studies on an arsenate-resistant mutant indicate that its resistance can be explained by a significantly increased photophosphorylation capacity of the energy-converting machinery, which can compensate for the decrease in adenosine triphosphate regeneration rate caused by esterification of arsenate in place of phosphate. Repeated subculture of the mutant in the presence of arsenate, under certain conditions, yields cell populations that have strikingly elevated contents of membrane-bound reaction-center bacteriochlorophyll, cytochromes, and photo-phosphorylation coupling factor. Membranes from such cells show unusually high photophosphorylation activity, and can be expected to provide a useful experimental material for more refined analysis of the mechanism of the energy-conversion process.

Kinetics of glucosephosphate isomerase activity on fructose in the presence of arsenate. Reactivity of alcoholic groups with arsenate

1. 1. The kinetics of glucosephosphate isomerase activity ( d-glucose -6-P ketol-isomerase, EC 5.3.1.9) on fructose in the presence of arsenate, and the effect of phosphate, fructose-6- P, and erythrose-4- P on this activity, have been studied. 2. 2. The results suggest that arsenate interacts with the hydroxyl group of C-6 of the sugar resulting in, perhaps, an ester bond, and that the resulting compound, fructose-6…arsenate, may be isomerized by the enzyme with about the same V as that of the physiological substrate fructose-6- P. 3. 3. Studies on the mechanism by which the interaction proceeds seem to indicate that, subsequent to the arsenate binding, the enzyme binds fructose, and then the interaction occurs between arsenate and the hydroxyl group of fructose.

The effect of NAD + on the catalytic efficiency of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle

The catalytic center activity for the oxidation of glyceraldehyde, acetaldehyde and propionaldehyde by glyceraldehyde-3-phosphate dehydrogenase was not observed to be appreciably affected by the degree of saturation of the enzyme with NAD +. A small increased in intrinsic rate on going from E( NAD +) I to E( NAD +) 4 was noted for glyceraldehyde (1.1 fold) and a small decrease in intrinsic rate for acetaldehyde (1.4 fold) and propionaldehyde (1.2 fold). These small changes, contrasted with the approx. 1·10 7-fold decrease in ligand affinity induced by the binding of NAD +, are consistent with the induced fit model of allosterism. The initial rate of oxidation of glyceraldehyde-3-phosphate by glyceraldehyde-3-phosphate dehydrogenase, in the presence of arsenate, decreased rapidly within the first few percent of the reaction. Investigation of the end product inhibition of this reaction suggests that the accumulation of 1-arseno-3-phosphoglycerate is partly responsible for the observed reduction in rate, and thus that the spontaneous hydrolysis of this mixed anhydride may not be rapid relative to the rate of catalysis.

Alkaline phosphodiesterase activity of goat brain cortex ribosomes.

Abstract—The ribosome preparation of goat cerebral cortex was found to possess acid and alkaline phosphodiesterase (PDase) activities, the alkaline PDase activity was greater. The alkaline PDase activity increased sharply between pH 8 and 9 and showed maximum activity between 35° and 40°. The alkaline PDase lost about half of its activity on heating the ribosome preparation at 83° for 5 min. Storage at low temperature or freezing and thawing did not cause significant loss of enzymic activity, but prolonged dialysis beyond 24 hr in the cold against magnesium‐cacodylate buffer caused some loss of activity. Copper, cobalt, inorganic phosphate ions, EDTA, urea and detergents were found to be inhibitors of the PDase activity. Differential inactivation in the presence of arsenate and zinc ions suggests the distinctness of the PDase activity from the phosphomonoesterase (PMase) activity of the ribosome preparation. The enzyme appeared firmly bound to ribosomal particles and attempts to solubilize it were unsuccessful.

The reaction between NAD + and rabbit-muscle glyceraldehydephosphate dehydrogenase

1. 1. The A 280 mμ : A 260 mμ ratio of NAD +-free rabbit-muscle glyceraldehydephosphate dehydrogenase was found to be 2.00–2.03. 2. 2. Ultracentrifugation showed that 4 moles of NAD + may be bound to the enzyme per mole (mol. wt. 145 000). The first two molecules are bound stoicheiometrically within the experimental error ( K D < 0.05 μM), while the third and fourth molecules are bound with dissociaton constants of 4 and 35 μM, respectively. 3. 3. The first three molecules of NAD + bound to the enzyme cause the formation of a band at 360 mμ of about equal intensity for each molecule. The fourth molecule causes little further increase of absorption at 360 mμ. 4. 4. A plot of the rate of reduction of NAD + by glyceraldehyde in the presence of arsenate against the NAD + concentration shows a sharp break in the curve at 4 moles NAd + per mole enzyme. 5. 5. Stopped-flow experiments showed that when up to 1 mole of NAD + is added to the enzyme, maximum absorbance increase at 360 mμ is reached within 3 msec. This corresponds to a second-order reaction constant of more than 10 8 M −1·sec −1. With 2 moles of NAD + per mole of enzyme, 81% of the reaction is over in 3 msec, and with 3 or more moles NAD + about 75%. The reaction requires about 1 sec for completion. 6. 6. Prior treatment with NAD + speeds the change of absorbance obtained with subsequent additions of NAD +. The final value obtained, however, is unaltered. 7. 7. For the muscle enzyme under our experimental conditions, a model in which the binding of 1 NAD + molecule to one protomer affects the conformation of a second protomer, either before or after binding with NAD +, appear more appropriate than the allosteric model, which requires that the binding of any one ligand molecule is intrinsically independent of the binding of any other.

Studies on Glucose Isomerizing Enzyme of Bacteria

d-Glucose-isomerizing enzyme from Escherichia intermedia HN-500, which converts d-glucose to d-fructose in the presence of arsenate, was purified by treating with manganous sulfate, rivanol, and DEAE-Sephadex column chromatography. About 180-fold purified enzyme preparation was obtained by the above procedures. The purified preparation was free from the activities of d-glucose-, d-galactose-, glucose-6-phosphate-, mannitol-, and sorbitol-dehydrogenases and was homogeneous on polyacrylamide gel in zone electrophoresis. Optima of pH and temperature for the enzyme were found to be pH 7.0 and 50°C, respectively. The enzyme was completely inactivated by heating at 60°C for ten minutes and stable in the pH range of 7.0~9.0 at 30°C. Activation energy for the isomerizing enzyme was calculated to be 15,300 calories per mole degree from Arrhenius' equation. Either in the absence or presecne of arsenate, d-mannose, d-xylose, d-mannitol and d-sorbitol could not be isomerized by the purified enzyme at all, but the pre...

Crystallization and properties of pea glucosephosphate isomerase

Glucosephosphate isomerase ( d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) of pea was isolated in the crystalline state in 14.5% yield from pea extract after 1500-fold purification. The molecular weight of the enzyme was estimated at 110 000 by ultracentrifugal analysis. The K m and molecular activity of the enzyme were determined as 2.7·10 −4 M ( Glc-6-P ) and 165 000 in Tris-HCl buffer (pH 8.5) at 37°. The isomerization reaction was equilibrated in 65% Glc-6-P and 35% Fru-6-P at pH 7.5 at 37°. 6-Phosphogluconate competitively inhibited the reaction and its K i was 1.3·10 −5 M. The pH optimum of the reaction was found to be 8.5 in Tris-HCl buffer and 9.5 in glycine-NaCl-NaOH buffer. The maximal reaction rate was observed at 50° and the activation energy was calculated as 8100 cal. Sulfhydryl groups are not thought to be involved in its active center because the reaction was not inhibited by p- chloromercuribenzoate and monoiodoacetate. The enzyme was able to isomerize glucose into fructose in the presence of arsenate.

Studies on carbohydrate metabolizing enzymes: XVI. Specificity of laminaribiose phosphorylase from Astasia ocellata

Cell-free extracts of the protozoon Astasia ocellata show laminaribiose phosphorylase activity. The enzyme catalyzes the reversible phosphorolysis of laminaribiose (and higher laminarisaccharides) to glucose and α- d-glucose 1-phosphate. At equilibrium, synthesis of laminaribiose is favored. For synthesis, α- d-glucose 1-phosphate is the only known glucosyl donor. Possible acceptor substrates are β- d-glucopyranose and di- or trisaccharides (or derivatives) which contain a nonreducing β- d-glucopyranosyl residue, the transferred glucose residue being attached by a (1 → 3)-linkage. In the presence of arsenate, laminaribiose is completely converted into glucose.

Uncoupling of respiratory-chain phosphorylation by arsenate

1. The specific uncoupling by arsenate of oxidative phosphorylation in rat-liver mitochondria has been studied in EDTA-containing medium. The K m for arsenate for stimulation of the oxidation of succinate in phosphate-free medium is about 1 mM. 2. Arsenate-stimulated respiration is inhibited by low concentrations of phosphate, in the absence of ADP. The effects of arsenate and phosphate are competitive. A phosphate:arsenate ratio of 0.1 inhibits completely. In the presence of ADP, an arsenate:phosphate ratio of 5–10 is necessary for a decrease of the P:O ratio by 50 %. 3. In the absence of phosphate, ADP stimulates the arsenate-induced respiration by 60 %. Atractyloside has no effect on the ADP-independent respiration, but stops the stimulation by ADP, whether added before or after the ADP. 4. Low concentrations of aurovertin inhibit the arsenate-induced respiration to the same extent as oligomycin. However, with 9 μg aurovertin per mg protein, in the presence of arsenate, the respiration is greater than with low concentrations. This extra respiration is inhibited by oligomycin or by phosphate. 5. The arsenate-induced ATPase amounts to only about 15 nmoles phosphate per mg protein per min ( cf. 20 for endogenous ATPase, and 300 for 2,4-dinitrophenol-induced ATPase, in the same units). The arsenate-induced ATPase is inhibited by oligomycin, but not by azide. 6. It is concluded that stimulation by ADP of arsenate-induced respiration is not due to removal by ADP of endogenous phosphate. Nor is inhibition by phosphate due to removal of ADP. 7. It is concluded that the effects of arsenate provide strong evidence in favour of the existence of a ∼P intermediate of oxidative phosphorylation, either containing phosphate alone (X∼P) or containing ADP as well as phosphate (ADP-X∼P). 8. The different effects of oligomycin on dinitrophenol- and arsenate-induced respiration support the proposition that there are at least two non-phosphorylated high-energy intermediates (or one intermediate with two sites) in the sequence of energy-conserving reactions leading from the oxidoreduction reaction of the respiratory chain to ATP.

The side activities of d-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating) from rabbit muscle: NADH-X formation

1.1. The formation of NADH-X from NADH in the presence of glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12) at low pH is observed not only in the presence of pyrophosphate, citrate or phosphate, but also in the presence of arsenate.2.2. Stimulation of NADH-X formation by acetyl phosphate is dependent on the presence of enzyme-bound NAD+; however, added NAD+ is inhibitory.3.3. Inhibition of NADH-X formation by sulphydryl reagents is prevented by acetyl phosphate in the presence of pyrophosphate or citrate, but not in the presence of arsenate or phosphate.4.4. In the absence of other polyvalent anions, acetyl phosphate alone can stimulate the conversion of NADH into NADH-X at low pH.5.5. In the presence of acetyl phosphate, the relative rate of formation of NAD+ and NADH-X from NADH is dependent on the pH and the anions present. 1. The formation of NADH-X from NADH in the presence of glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12) at low pH is observed not only in the presence of pyrophosphate, citrate or phosphate, but also in the presence of arsenate. 2. Stimulation of NADH-X formation by acetyl phosphate is dependent on the presence of enzyme-bound NAD+; however, added NAD+ is inhibitory. 3. Inhibition of NADH-X formation by sulphydryl reagents is prevented by acetyl phosphate in the presence of pyrophosphate or citrate, but not in the presence of arsenate or phosphate. 4. In the absence of other polyvalent anions, acetyl phosphate alone can stimulate the conversion of NADH into NADH-X at low pH. 5. In the presence of acetyl phosphate, the relative rate of formation of NAD+ and NADH-X from NADH is dependent on the pH and the anions present.

In vitro hybridization of lactate dehydrogenase in the presence of arsenate and nitrate ions

It is well established that in vitro hybridization of lactate dehydrogenase (LDH) can be accomplished most readily by freezing and thawing a mixture of isozymes in neutral phosphate buffer containing salts of the various halides (except fluoride). Al-though recombination of LDH subunits has been observed to occur in saturated NaCl or during reversal of urea denaturation in phosphate buffer, neither of these methods approaches the reliability and efficiency of the “salt-freeze” technique. The mechanism of in vitro hybridization remains unclear, but the ionic environment is of critical importance. We now report that hybridization of LDH isozymes can be accomplished both in neutral NaHAsO 4-HCl and in NaNO 3-NaOH systems with the same degree of efficiency and reliability as in the classical phosphate system. Furthermore, the ability of various ions to promote in vitro hybridization parallels to some degree their ability to alter the structure (e.g., denature) of various types of macromolecules. However, a detailed examination of the properties of LDH, such as thermal stability, in environments conducive to hybridization reveals no obvious general relationship between the ability of a given ion to promote hybridization and its ability to alter protein structure.

Enhancement of ribonucleoside synthesis in Ehrlich ascites tumor cells by arsenate and iodoacetate.

Ehrlich ascites tumor ceils in vitro synthesize ribonucleosides, which appear mainly in the incubation medium, by the transfer of ribose from a donor ribonucleoside to an acceptor base. In the present study, it was found that the rates of synthesis of inosine and uridine in this system were markedly enhanced in the presence of arsenate or iodoacetate. The exchange of isotope between extracellular inosine and hypoxanthine-8-C14was similarly enhanced by arsenate, but the more rapid exchange between uridine and uracil-2-C14was unaffected. Arsenate did not cause changes in the rates of uridine breakdown that would account for the enhanced rate of nucleoside synthesis and did not promote the release of nucleoside-synthesizing enzymes from the tumor cells into the incubation medium. Because lactate formation during the uridine-supported synthesis of inosine was markedly inhibited by arsenate and iodoacetate, the increase in ribonucleoside synthesis appears to be indirect and to be related to inhibition of ribose phosphate catabolism.

Possible partial reactions of the photophosphorylation process in chromatophores from Rhodospirillum rubrum

Abstract 1. 1. Chromatophores from Rhodospirillum rubrum were used to study the photosynthetic formation of ATP in relation to ATP-P 1 exchange, ADP-ATP exchange and ATPase. 2. 2. ATP-P 1 exchange was greatly stimulated by light (approx. 1000 ft-candles). Ascorbate affected the ATP-P 1 exchange activity in light in a similar manner to the photophosphorylating activity, both activities being greateest in the presence of 67 mM ascorbate. 3. 3. ADP-ATP exchange in light was stimulated more by PMS than by ascorbate. For both light ADP-ATP exchange and photophosphorylation, the optimal concentration of PMS was approx. 0.4 mM. 4. 4. In the presence of 3.3 mM ATP, ATPase activities in light and dark were depressed by the presence of more than 5 mM ascorbate. The inhibition by ascorbate was competitive with the neutralization caused by a high concentration of ATP. 5. 5. Photophosphorylation and ATP-P 1 and ADP-ATP exchange in light and dark were maximal at around pH 8, whereas ATPase activities in light and dark were maximal at pH 8.5 or more. 6. 6. Photophosphorylation activity and ATP-P i exchange and ATPase activity in light and dark were inactivated by prolonged sonication of the chromatophores. However, with prolonged sonication ADP-ATP exchanges with ascorbate in light and dark and with PMS in dark increased in activity, whereas ADP-ATP exchange with PMS in light was scarcely affected. 7. 7. ATPase activity was significantly stimulated by arsenate, 2,4-dinitrophenol and pyrophosphate, and depressed by quinacrine hydrochloride and quinine hydrochloride, even in the presence of arsenate and 2,4-dinitrophenol. 8. 8. ATP-P i and ADP-ATP exchange, which are induced by light in the presence of PMS (“in light” minus “in dark”: light-induced), were inhibited by quinacrine hydrochloride and 2,4-dinitrophenol to almost the same extent as photophosphorylation. 9. 9. ATP-P i exchange in dark was significantly inhibited by arsenate, quinacrine hydrochloride, quinine hydrochloride and 2,4-dinitrophenol, but ADP-ATP exchange in dark was not. The latter was markedly inhibited by pyrophosphate. 10. 10. Light-induced ATP-P i exchange with PMS was less inhibited by o -phenanthroline than photophosphorylation with PMS; in the presence of 0.67 mM o -phenanthroline, these two reactions were inhibited 10% and 48%, respectively. 11. 11. Photophosphorylation with ascorbate and light-induced ATP-P i exchange with ascorbate were inhibited by more than 1·10 −3 μg/ml of antimycin A. The minimal concentration required for complete inhibition was approx. 3.3·10 −2 μg/ml. In the presence of lower concentrations of antimycin A, light-induced ATP-P i exchange was inhibited less than photophosphorylation. 12. 12. From these findings, the roles of ATP-P i exchange, ADP-ATP exchange and ATPase activity are discussed in relation to the photosynthetic formation of ATP.

ADENOSINETRIPHOSPHATASE OF LIVER MITOCHONDRIA IN VIRUS HEPATITIS OF MICE.

A study of mitochondrial ATP-ase has demonstrated that in MHV-3 Craig virus hepatitis of mice there is an early lesion of the mitochondrial structure. The beginning of this lesion precedes generalized damage of the hepatic cells. After 24 hours of infection an appearance of Mg^++-stimulated ATP-ase and reduction of DNP-induced ATP-ase was observed. Experiments with mitochondria treated with deoxycholate confirm the existence of this lesion. The reutilization of inorganic P linked to the oxidation of respiratory substrates shows that up to 24 hours of infection this capacity is reduced using ß-hydroxybutyrate in the presence of arsenate. Phosphorylation in the presence of succinate is altered only in the more advanced phases of infection. It seems probable that the earliest lesion of the respiratory chain phosphorylation induced by the viral infection must be at the level of the NAD- flavin region.

ALKALINE PHOSPHOMONOESTERASE ACTIVITY OF GOAT BRAIN CORTEX RIBOSOMES.

— The ribosome preparation of goat cerebral cortex was found to possess acid and alkaline phosphodiesterase (PDase) activities, the alkaline PDase activity was greater. The alkaline PDase activity increased sharply between pH 8 and 9 and showed maximum activity between 35° and 40°. The alkaline PDase lost about half of its activity on heating the ribosome preparation at 83° for 5 min. Storage at low temperature or freezing and thawing did not cause significant loss of enzymic activity, but prolonged dialysis beyond 24 hr in the cold against magnesium-cacodylate buffer caused some loss of activity. Copper, cobalt, inorganic phosphate ions, EDTA, urea and detergents were found to be inhibitors of the PDase activity. Differential inactivation in the presence of arsenate and zinc ions suggests the distinctness of the PDase activity from the phosphomonoesterase (PMase) activity of the ribosome preparation. The enzyme appeared firmly bound to ribosomal particles and attempts to solubilize it were unsuccessful.

THE ACTION OF DIO-9: AN INHIBITOR AND AN UNCOUPLER OF OXIDATIVE PHOSPHORYLATION.

Abstract 1. 1. The antibiotic Dio-9 inhibits the acceptor-controlled respiration of intact rat-liver mitochondria in a phosphate-containing medium. Essentially the same effects have been found with glutamate, β-hydroxybutyrate, α-ketoglutarate, succinate and the ascorbate-tetramethyl-p-phenylenediamine system as substrate. 2. 2. Inhibition of respiration by Dio-9 in a phosphate-containing system cannot be reversed by 2,4-dinitrophenol. However, if 2,4-dinitrophenol is added in uncoupling concentrations (0.1 mM) prior to the antibiotic there is no inhibition of respiration by Dio-9. 3. 3. The inhibition of respiration by Dio-9 is preceded by a short burst of increased oxygen uptake. 4. 4. The inhibition of respiration by Dio-9 is dependent upon the presence of inorganic phosphate or arsenate in the incubation medium. In the absence of phosphate, Dio-9 only stimulates the respiration which is further accelerated by dinitrophenol. 5. 5. Dio-9 stimulates respiration in the presence of oligomycin or 2-n-heptyl-4-hydroxyquinolineN-oxide in the absence of phosphate but not in its presence. 6. 6. Arsenate-stimulated respiration is only slightly inhibited by Dio-9. However, the increased respiration produced by 2,4-dinitrophenol in the presence of arsenate is not obtained in the presence of Dio-9. 7. 7. Dio-9 has a much smaller effect on sonicated mitochondria or on the Keilin and Hartree heart-muscle preparation. 8. 8. Dio-9 causes a minor swelling of rat-liver mitochondria which is potentiated by inorganic phosphate. 9. 9. The antibiotic has little effect on the 2,4-dinitrophenol-induced ATPase of freshly prepared mitochondria although it inhibits the ATPase of aged mitochondria. 10. 10. The possible mode of action of Dio-9 is discussed.

Relationship between adenosine triphosphate and cation transport in the human red cell.

Fresh human blood collected in heparin and glucose was treated with glycolytic poisons such as iodoacetate and 2-deoxyglucose to retard glycolysis and reduce the high energy phosphate stores of the red cell. Under these circumstances K+ began to leak out of the cells and Na+ began to leak in. In the presence of arsenate, glycolysis proceeded but high energy phosphate stores were not maintained and cation leakage also occurred. In a final experiment, both glycolysis and high energy phosphate stores were continued normally but ouabain was added to inhibit the transport ATPase. This prevented the coupling of ATP breakdown to active cation transport and allowed K+ to leak out of the red cell and Na+ to leak in.

Detection of phosphate in the presence of arsenate

Detection of phosphate in the presence of arsenate

A titrimetric method for determining arsenite in presence of arsenate

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Modification of the release of cellular constituents by irradiated Escherichia coli

A release of cellular constituents occurred when x-irradiated (60,000 r) suspensions of Escherichia coli B/r were incubated in a phosphate buffer-glucose solution at 37 °C. ATP accounted for less than 10% of the total 260-mμ absorbing material released by such cells. The other constituents (showing maximum absorption at 260 mμ) lost from the exposed cells were nucleic acid fragments varying in size from free bases to more complex nucleic acid fragments. No release of peptides above that found with unirradiated cells under similar conditions was observed. The release of nucleic acid fragments was inhibited under each of the following conditions: ( a) the absence of phosphate in the suspending fluid; ( b) the absence of an exogenous metabolite; ( c) the presence of arsenate; ( d) a low temperature of incubation, i.e., in an ice bath. Ultraviolet-light-exposed cells, suspended in a glucose-phosphate solution at 37 °C., released 260-mμ absorbing material, and this release was dependent on the same conditions as those found for the x-ray exposed cells.