Stimulation Of O2 Uptake Research Articles

Nickel uptake in Bradyrhizobium japonicum.

Free-living Bradyrhizobium japonicum grown heterotrophically with 1 microM 63Ni2+ accumulated label. Strain SR470, a Hupc mutant, accumulated almost 10-fold more 63Ni2+ on a per-cell basis than did strain SR, the wild type. Nongrowing cells were also able to accumulate nickel over a 2-h period, with the Hupc mutant strain SR470 again accumulating significantly more 63Ni2+ than strain SR. These results suggest that this mutant is constitutive for nickel uptake as well as for hydrogenase expression. The apparent Kms for nickel uptake in strain SR and strain SR470 were found to be similar, approximately 26 and 50 microM, respectively. The Vmax values, however, were significantly different, 0.29 nmol of Ni/min per 10(8) cells for SR and 1.40 nmol of Ni/min per 10(8) cells for SR470. The uptake process was relatively specific for nickel; only Cu2+ and Zn2+ (10 microM) were found to appreciably inhibit the uptake of 1 microM Ni, while a 10-fold excess of Mg2+, Co2+, Fe3+, or Mn2+ did not affect Ni2+ uptake. The lack of inhibition by Mg2+ indicates that nickel is not transported by a magnesium uptake system. Nickel uptake was also inhibited by cold (53% inhibition at 4 degrees C) and slightly by the ionophores nigericin and carbonyl cyanide m-chlorophenylhydrazone. Other ionophores did not appreciably affect nickel uptake, even though they significantly stimulated O2 uptake. The cytochrome c oxidase inhibitors azide, cyanide, and hydroxylamine did not inhibit Ni2+ uptake, even at concentrations (of cyanide and hydroxylamine) that inhibited O2 uptake. The addition of oxidizable substrates such as succinate or gluconate did not increase nickel uptake, even though they increased respiratory activity. Nickel update showed a pH dependence with an optimum at 6.0. Most (approximately 85%) of the 63Ni2+ taken up in 1 min by strain SR470 was not exchangeable with cold nickel.

Differential effect of glucagon on gluconeogenesis in periportal and pericentral regions of the liver lobule.

The effect of glucagon on gluconeogenesis was measured in periportal and pericentral regions of the liver lobule by monitoring changes in rates of O2 uptake on the surface of the perfused liver with miniature O2 electrodes after infusion of lactate. When lactate (2 mM) was infused into livers from starved rats perfused in the anterograde direction, O2 uptake was increased 2.5-fold more in periportal than in pericentral regions, reflecting increased energy demands for glucose synthesis. Under these conditions, glucagon infusion in the presence of lactate increased O2 uptake exclusively in periportal regions of the liver lobule. Thus, when perfusion is in the physiological anterograde direction, the metabolic actions of glucagon predominate in periportal regions of the liver lobule under gluconeogenic conditions in the starved state. When livers were perfused in the retrograde direction, however, glucagon stimulated O2 uptake exclusively in pericentral regions. Thus glucagon only stimulates gluconeogenesis in 'upstream' regions of the liver lobule irrespective of the direction of flow.

Lactate oxidation byTreponema pallidum

Whole cells ofTreponema pallidum consumed O2 with lactate in a glucose-depleted medium.d(−) Lactate caused marked stimulation of O2 uptake at a rate similar to that with glucose, whereasl(+) lactate resulted in no increase over the reduced rate observed upon glucose depletion. Lactate oxidation was specific for α-hydroxy straight-chain acids of 3,4, and 5 carbons. O2 uptake during lactate oxidation proceeded independently of pyruvate oxidation and required NAD. The product of lactate oxidation was pyruvate.d(−) Lactate-stimulate O2 uptake was sensitive to chlorpromazine and resistant to amytal and cyanide. Glucose did not inhibit the oxidation of lactate as shown by the additive effect of both substrates on O2 uptake. Oxidation of glucose, but not lactate, provided energy necesary for motilibty or maintenance of virulence. A mixture of lactate isomers was formed from glucose with thel(+) isomer concentration remaining constant and thed(−) isomer concentration varying inversely with dissolved O2 concentration. The function of lactate as an oxidizable substrate is apparently quite distinct from that of glucose.

Effect of the herbicide glyphosate on respiration and hydrogen consumption in soil

The effect of glyphosate on soil respiration and Hz oxidation in an agricultural soil was investigated. The effects of the pure herbicide and commercial formulation, Roundup® (Monsanto Company), were compared in soil under both aerobic and anaerobic conditions. Both formulations stimulated O2 uptake as well as aerobic and anaerobic CO2 evolution. Roundup caused more stimulation than glyphosate under aerobic incubation conditions; the formulations had an equal effect on anaerobic CO2 evolution. Hydrogen oxidation was inhibited by both formulations in aerobic and anaerobic soil. Aerobic H2 oxidation was inhibited to the same extent by both formulations; Roundup had a stronger inhibitory effect on anaerobic H2 oxidation than did glyphosate.

Light-driven reduction of oxygen as a method for studying electron transport in the green photosynthetic bacterium Chlorobium limicola

The use of O2 uptake as a valid assay for non-cyclic photosynthetic electron flow in membranes from Chlorobium limicola is discussed. It is recommended that methyl viologen, catalase and superoxide dismutase should be added to the experimental medium. The addition of methyl viologen more than doubled the rate of O2 uptake observed on illumination with 1 mM sulphide as donor. Superoxide dismutation was shown to be efficient under the experimental conditions by means of standard additions of potassium superoxide dissolved in dimethylsulphoxide. The highest rates of light stimulated O2 uptake were obtained with sulphide as electron donor, and approached 50 μmol O2 · h-1 · mg bacteriochlorophyll c -1 with 0.2 mM sulphide. The presence of 5 mM 2-mercaptoethanol or 3 mM sulphite as electron donor led to lower light stimulated rates of O2 uptake, while 5 mM thiosulphate had little effect. The rates were insensitive to uncoupler. The light stimulated O2 uptake with 0.2 mM sulphide as donor was 20–30% inhibited by 10 μM antimycin A and 50 μM cyanide.

Isolated perfused head of rainbow trout. I. Gas transfer, acid-base balance, and hemodynamics.

Branchial gas transfer, acid-base balance, and hemodynamics were critically evaluated and compared in Ringer-perfused and blood-perfused heads of rainbow trout. Blood perfusion stimulated O2 uptake and CO2 excretion across the gills to values more representative of intact fish. The stimulatory effect of blood on gas transfer was due to increased O2 carrying capacity (O2 uptake) and the presence of erythrocytic carbonic anhydrase (CO2 excretion). Adding carbonic anhydrase to Ringer enhanced CO2 excretion in a manner similar to blood. During Ringer perfusion, arteriovenous pH gradients were abnormal (arterial pH less than venous pH). Perfusion with blood or addition of carbonic anhydrase to Ringer reversed the pH gradients to typical in vivo levels. Branchial vascular resistance to flow was abnormally high in both Ringer- and blood-perfused preparations, primarily as a result of low dorsal aortic pressure. Input pressure increased during blood perfusion and was similar to ventral aortic pressure in vivo. Perfusion with Ringer may have caused irreversible deterioration of gill function as indicated by decreased arterial Po2 and O2 extraction effectiveness after a rapid switch from Ringer to blood perfusion. The results are discussed with reference to the suitability of perfused trout head preparations for studying gill gas transfer, acid-base balance, and hemodynamics. Comparisons are made between the perfused head preparation and intact fish as well as with other types of perfused gill preparations.

The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations.

An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence.

Contribution of matrix carbonic anhydrase to citrulline synthesis in isolated guinea pig liver mitochondria.

Synthesis of citrulline from ornithine, NH4+, and HCO3- by isolated pig liver mitochondria is inhibited by acetazolamide, a specific inhibitor of carbonic anhydrase, at the same concentrations which inhibit the mitochondrial matrix carbonic anhydrase. At an acetazolamide concentration sufficient to give complete inhibition of matrix carbonic anhydrase, the rate of citrulline synthesis is reduced by 71%, but no further decrease in citrulline is observed at higher concentrations of acetazolamide. Stimulation of O2 uptake by ornithine under conditions of maximal citrulline synthesis is also inhibited by acetazolamide. At pH 6.7, the ratio of citrulline synthesis is depressed relative to the rates observed over the range 7.2-7.7, and acetazolamide inhibits this rate by only 20%. These results support the hypothesis that the physiological role of carbonic anhydrase in liver mitochondria is to supply HCO3- as the substrate for the enzyme carbamoyl phosphate synthetase I, which provides the intermediate carbamoylphosphate in the rate-limiting step of citrulline synthesis. Since the uncatalyzed rate of CO2 hydration is rapid enough that it should not be rate-limiting for the carbamoylphosphate synthetase reaction, carbonic anhydrase appears to regulate access of HCO3- in the synthetase and so should be considered as one of the enzymes participating in the biosynthetic pathway leading to urea formation in the hepatocyte.

The mitochondrial localization of hexokinase in pea leaves

Up to 80% of total cellular hexokinase (EC 2.1.7.4) activity in pea (Pisum sativum L.) leaves was found to be associated with particulate fractions. Fractionation on sucrose density gradients showed this particulate activity to be associated exclusively with mitochondria. In the presence of glucose and ATP, the bound mitochondrial hexokinase could support rates of O2 uptake of up to 30% of normal ADP-stimulated rates. This stimulation of O2 uptake by hexokinase was completely sensitive to oligomycin, indicating that it resulted from an increase in the supply of ADP for mitochondrial oxidative phosphorylation. Spectrophotometric measurements of the mitochondrial hexokinase activity showed that ADP could support rapid rates of activity provided oxidizable substrates were also present to support the conversion of ADP to ATP in oxidative phosphorylation. Carboxyatractyloside, an inhibitor of adenine-nucleotide uptake by mitochondria, inhibited this ADP-supported activity, but had no effect on hexokinase activity in the presence of added ATP, demonstrating that the hexokinase enzyme was located external to the inner mitochondrial membrane. Oligomycin also inhibited ADP-supported activity but had no effect on ATP-supported hexokinase activity. Glucose (Km 53 μM) was the preferred substrate of pea-leaf mitochondrial hexokinase compared with fructose (Km 5.1 mM). Hexokinase was not solubilised in the presence of glucose-6-phosphate.

Chloride accumulation in plant cells as a homeostatic system: Energy supply as a dependent variable

During the accumulation of KCl by excised tissue of the carrot storage root, Cl−, influx falls by 90% or more. The possibility that this reduction is caused by depletion of respiratory substrate and consequent limitation of Cl− influx by energy supply is disproved. If energy were limiting, then after accumulating KCl (i) the rate of O2 uptake would be less; (ii) the ATP content might be less; (iii) external glucose and/or sucrose would stimulate O2 uptake and Cl− influx; and (iv) respiratory uncouplers would stimulate O2 uptake less. In each case the opposite was found. The ratio of O2 uptake in KCl-loaded tissue to that in nonloaded tissue was 1.25±0.21 (7); the ATP concentration was the same after KCl loading; glucose did not stimulate O2 uptake (except indirectly in the same way as a nonmetabilized glucose analogue) nor did glucose or sucrose stimulate Cl− influx; and the maximal O2 uptake in the presence of the uncoupler ClCCP was the same after KCl loading.

Carbonyl cyanide-m-chlorophenyl hydrazone-resistant Escherichia coli mutant that exhibits a temperature-sensitive unc phenotype.

Two spontaneous Escherichia coli mutant strains which are resistant to an oxidative phosphorylation uncoupler, carbonyl cyanide-m-chlorophenyl hydrazone, were isolated. Strain CM22 (ccr-2) was resistant to another uncoupler, pentachlorophenol, and to the inhibitors of proton-translocating ATPase, namely tributyltin and sodium azide. Carbonyl cyanide-m-chlorophenyl hydrazone or pentachlorophenol administered to cell suspensions of strain CM22 did not cause a pH change induced by H+ influx, and a similar result was obtained with everted particles. The respiratory rate of strain CM22 with succinate was twice that of wild-type strain KH434. When carbonyl cyanide-m-chlorophenyl hydrazone was administered, a stimulation of O2 uptake was observed in wild-type strain KH434 but not in the mutant strain CM22. Strain CM22 did not grow on succinate at 42 degrees C. Isolation of a true revertant at a frequency of 10(-8) demonstrated that the pleiotropic phenotype was induced by a single mutation. P1 transduction indicated that the mutant allele, ccr-2, was cotransduced with the ilv genes at a frequency of about 55%.

Oxidation of acetate, acetyl CoA and acetylcarnitine by pea mitochondria

Acetylcarnitine was rapidly oxidised by pea mitochondria. (-)-carnitine was an essential addition for the oxidation of acetate or acetyl CoA. When acetate was sole substrate, ATP and Mg(2+) were also essential additives for maximum oxidation. CoASH additions inhibited the oxidation of acetate, acetyl CoA and acetylcarnitine. It was shown that CoASH was acting as a competitive inhibitor of the carnitine stimulated O2 uptake. It is suggested that acetylcarnitine and carnitine passed through the mitochondrial membrane barrier with ease but acetyl CoA and CoA did not. Carnitine may also buffer the extra- and intra-mitochondrial pools of CoA. The presence of carnitine acetyltransferase (EC 2.3.1.7) on the pea mitochondria is inferred.

Enhancement of the mitochondrial Ca2+ uptake rate by phenethylbiguanide and other organic cations with hypoglycemic activity.

At concentrations below 50 micronM, phenethylbiguanide enhanced the initial rate of enertized Ca2+ uptake into energized guinea pig liver mitochondria by as much as 45 per cent; Ca2+-stimulated O2 uptake increased in parallel. The biguanide concentration that enhanced Ca2+ uptake maximally was at least 15 times lower than that required for 50 per cent inhibition of respiration. Kinetic studies indicated that the enhanced rate of Ca2+ transport resulted from an increase in Vmax, while Km for Ca2+ was unaffected by the biguanide. Several other organic cations known to lower blood sugar in intact animals or to block the hepatic gluconeogenic response to glucagon also enhanced the mitochondrial Ca2+ uptake rate; three of these compounds did not inhibit respiration even at high concentrations. One organic cation, triethyltin, which is a potent respiratory inhibitor that does not affect blood sugar, had no effect on Ca2+ uptake. We concluded that enhancement of the mitochondrial Ca2+ uptake rate is related to the mechanism of therapeutic blood sugar lowering by these drugs, probably by impairing the gluconeogenic response of the liver to glucagon.

Respiration and ATP level in BHK21/13S cells during the earlist stages of rubella virus replication.

This study is concerned with the effect of virus infection on the energy balance of the cell and the possible involvement of mitochondria in virus replication. The process of rubella virus adsorption and penetration is immediately associated with a stimulation of O2 uptake. Shortly afterwards, ATP levels decline, possible because of increased ATP utilization. Virus infection appears to protect mitochondria against the uncoupling effect of dinitrophenol.

LIGHT PRODUCTION IN ALKALINE MIXTURES OF REDUCING AGENTS AND DIMETHYLBIACRIDYLIUM NITRATE*

Abstract— Kinetic studies were made of light production and 02 absorption elicited by treatment of dimethylbiacridylium hydroxide [D(OH)2] with reducing agents in alkaline aqueous solutions. D(OH)2 addition stimulated O2 uptake which proceeded with zero‐order kinetics until nearly all of the O2 or of the D(OH)2 was converted to end products. At the termination of the reactions with fructose as reductant the D(OH)2 was converted to methylacridone and to dimethylbiacridene each compound accounting for approximately one‐half the D(OH)2 consumed. O2 was reduced to H2O2. With hydroxylamine as the reducing agent the emitted light intensity was related to the first power of the D(OH)2 concentration and the rate of O2 uptake to the square root of the D(OH)2. The disappearance of D(OH)2 in these circumstances was by a first order or pseudo‐first order rate. Fructose as a reducing agent by contrast resulted in an O2 uptake nearly independent of D(OH)2 concentration over a range from 1 × 10‐5M‐1 × 10‐4M, while the light intensity and disappearance of D(OH)2 followed a one‐half order rate. O2 uptake was by zero order kinetics and the oxidation of fructose proceeded at the same rate as was found with ferricyanide as oxidant. The kinetics, quantum yields and temperature dependence of the fructose reactions were compared with similar reactions employing H2O2 as the light eliciting reagent. The results are interpreted as indicating that D(OH)2 acts as a chain initiator in a manner analogous to better known, radical producing compounds found to accelerate hydrocarbon autooxidations.

The action of gamma-hydroxybutyric acid on cerebral glucose metabolism.

Abstract— Experiments have been performed to study the effect of gamma‐hydroxybutyric acid (GHB) on glucose metabolism in vivo and in vitro. Administration of GHB (500 mg/kg) is followed by sleep in rats and mice. GHB is shown to increase the 1‐14C/6‐14C ratio in expired CO2 in mice in vivo by 300 per cent. The same effect is obtained with slices of cerebral cortical grey matter from GHB‐treated rats, where the 1‐14C/6‐14C ratio is increased from 1.72 to 3.63, but not with homogenates of cerebral cortex, nor with slices of kidney or diaphragm. GHB (500 mg/kg, intrapentoneally) specifically increases the activity of glucosed‐6‐phosphate dehydrogenase in vivo in rat and mouse whole brain by 27 per cent. The time course of this effect correlates with the sleeping time in both species. The activity of glucose‐6‐phosphate dehydrogenase is not altered in vitro by high concentrations of GHB. GHB stimulates O2 uptake by slices of cerebral cortical grey matter by 24 per cent, but it is not itself able to support respiration by the tissue. It is proposed that GHB specifically increases the activity of the pentose phosphate pathway in brain, and that this effect is mediated by an increase in glucose‐6‐phosphate dehydrogenase activity.

Effects of Freeze‐Drying on Permeability and Respiration of Germinating Lily Pollen

AbstractThe germination of lily pollen (Lilium longiflorum cv. Ace) was impaired by freeze‐drying. This loss of viability was associated with a modified pattern of respiration and an increased leakage of soluble carbohydrates, phosphate, and ninhydrin‐positive material into the culture medium. 2,4‐Dinitro‐phenol, (DNP), an uncoupler of oxidative phosphorylation showed a decreased ability to stimulate O2 uptake in freeze‐dried pollen. The altered viability, respiration, and permeability resulted from drying under vacuum and not the initial freezing of the pollen. Mature lily pollen contained approximately 0.3 “Ai phosphate, of which 15 % was inorganic phosphate and about 50 %> was acid soluble organic phosphate of unknown identity.

A magnesium-responsive defect of respiration and oxidative phosphorylation in skeletal muscle mitochondria of dystrophic hamsters.

Skeletal muscle mitochondria were isolated in the presence and absence of the proteinase Nagarse from dystrophic hamsters of the BIO 14.6 strain, aged 45–196 days, and from normal hamsters. Mitochondria from the dystrophic animals prepared by glass-on-glass homogenization without Nagarse in 0.25 M sucrose – 1 mM EDTA, pH 7.4, did not differ from normal in their respiratory rate or capacity for oxidative phosphorylation. However, these functions were subnormal in mitochondria isolated with Nagarse from the same animals, both in the presence and absence of albumin. Respiration measured with an O2 electrode was reduced by 50–70% and the stimulation of O2 uptake normally seen after ADP addition was minimal or absent. This was most marked in mitochondria from young hamsters about 65 days old with muscle necrosis. The defect was ameliorated by addition to the Polarographie test system of an ATP trap or of Mg2+, one of the trap constituents. This ion, when added to the defective mitochondria prior to ADP and substrate, restored respiration and oxidative phosphorylation to values that did not differ significantly from those found with skeletal muscle mitochondria of normal hamsters.

The low maximum temperature for growth of the psychrophile Sclerotinia borealis; evidence for the uncoupling of growth from respiration

The optimum temperatures for growth and respiration of cells of Sclerotinia borealis grown at 0 °C are, respectively, 0 °C and 25 °C while the maximum temperature for growth is approximately 15 °C. It is postulated that at temperatures above 15 °C growth becomes uncoupled from respiration. This is supported by the following evidence. With glucose as substrate, stimulation of O2-uptake by the uncoupling agents 2,4-dinitrophenol and dicoumarol was relatively greater at 5 than at 25 °C. The degree of stimulation at 25 °C varied inversely with the duration of exposure to that temperature while that at 5 °C remained constant. In the absence of glucose, 2,4-dinitrophenol inhibited O2-uptake strongly at both temperatures. Recovery on subsequent addition of glucose was slow. Prolonged exposure to 25 °C in the absence of glucose resulted in a gradual loss of ability to respire or take up glucose. When the distribution of 14C after utilization of glucose-u-14C was determined it was found that relatively only half as much 14C was incorporated into cell material at 25 °C as at 5 °C.

Effects of Various Concentrations of Carbon Dioxide on Respiration and Potassium Uptake in Barley Roots

AbstractBarley roots contain a CO2 sensitive respiratory fraction which is inhibited in 50 per cent CO2 and is partially restored upon subsequent exposure to air. The residual O2 consumption occurring at CO2 concentrations between 50 per cent and 95 per cent amounts to about 40 per cent of the O2 uptake in air and can support K+ uptake for a limited time at a rate equal to or higher than occurs in air. Above 95 per cent CO2 both O2 and K+ uptakes decrease rapidly. 2,4‐dinitrophenol (DNP), in the range of 10−6 to 10−5M, stimulates O2 uptake by the roots in air. The stimulation is absent when roots are treated with DNP in 80 per cent CO2, presumably because of the reduced demand for inorganic phosphate and phosphate acceptor at the lower respiratory level in high CO2. In either air or CO2, K+ uptake is strongly inhibited by DNP. A comparison of the respiratory and K+ uptake data indicates that O2 consumption is a necessary requirement for the uptake process in high CO2.Protoplasmic streaming in the root cells is rapidly stopped by high CO2 although K+ uptake and O2 consumption continue. The cation uptake mechanism in high CO2 appears to be limited to the stationary cytoplasm. It is also possible that a similar mechanism may be involved in cation uptake in air.