The O2-dependence of respiration and H2O2 production in the parasitic nematode Ascaridia galli.

1. Respiration in the parasitic nematode worm Ascaridia galli was inhibited at O2 concentrations in excess of 255 microM, and an apparent Km,O2 of 174 microM was determined. 2. Mitochondria-enriched fractions isolated from the tissues of A. galli have much lower apparent Km,O2 values (approx. 5 microM). They produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 3. Antimycin A inhibited respiration in muscle tissue mitochondria by 10%, but had no effect on respiration in gut + reproductive tissue mitochondria; the major portion of respiration in both types of mitochondria could be attributed to an alternative electron-transport pathway. 4. o-Hydroxydiphenyl, an inhibitor of alternative electron-transport pathways, inhibits respiration by 98% and completely inhibits the production of H2O2 in gut-plus-reproductive-tissue mitochondria; respiration and H2O2 production in muscle tissue mitochondria were inhibited by 90 and 86% respectively. 5. Another inhibitor of alternative electron transport, salicylhydroxamic acid, had the same effect as o-hydroxydiphenyl on H2O2 production and respiration in gut-plus-reproductive-tissue mitochondria. However, its effect on muscle tissue mitochondria was complex; a low concentration (0.35 mM) stimulated H2O2 production, whereas 3 mM inhibited respiration by 87% and prevented H2O2 production completely. 6. The similarities between the apparent Km,O2 values for H2O2 production and respiration in muscle mitochondria and in gut-plus-reproductive-tissue mitochondria suggests that the site of H2O2 production on the alternative electron-transport chain is cytochrome 'o'. 7. These results are discussed in relation to potential O2 toxicity in A. galli.     

Respiration in the filarial nematode Brugia pahangi

Glucose-supported O2 uptake in the filarial nematode Brugia pahangi was partially inhibited by antimycin A (30-40%), with the remaining activity being sensitive to o-hydroxydiphenyl or salicylhydroxamic acid (SHAM). The production of CO2 by B. pahangi in the presence of D-glucose was stimulated by O2; the stimulation of CO2; the stimulation of CO2 production was sensitive to antimycin A. The O2 dependencies of respiration showed that the apparent O2 affinity for B. pahangi was diminished in the presence of antimycin A; O2 thresholds for inhibition of respiration were observed which showed that the alternative electron transport pathway was less sensitive to inhibition at elevated O2 concentrations. H2O2 production and its excretion could be detected in whole B. pahangi; higher rates were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The effects of inhibitors on H2O2 production suggest two sites of H2O2 production, one associated with the classical antimycin A-sensitive pathway, the other with the alternative respiratory pathway. The similarity in the O2 dependencies of H2O2 production and respiration may indicate that H2O2 production is involved in O2-mediated toxicity. Succinate and malate respiring sub-mitochondrial particles of B. pahangi produced O2.- radicals at a site on the antimycin A-sensitive respiratory pathway. Inhibition of the alternative electron pathway by SHAM was unusual; sub-millimolar concentrations markedly stimulated respiration, H2O2 production and O2.- production by 30, 20 and 25%, respectively, whereas higher concentrations (greater than 2.5 mM) inhibited respiration by 75% and H2O2 and O2.- production by up to 85%.     

Oxygen consumption by ruminal microorganisms: protozoal and bacterial contributions

The relative contributions to O2 consumption made by the protozoal and bacterial populations present within the rumen were determined by using an open-type oxygen electrode system. Measurements indicated that two separate microbial populations contributed approximately equally to ruminal O2 consumption over the O2 concentration range experienced in situ (0.25 to 1.0 microM). The populations were observed to consume O2 under liquid-phase O2 concentrations of up to 7 microM, above which point rapid inactivation of O2 utilization was observed. Km values for the mixed population of bacteria and protozoa were 0.36 +/- 0.17 and 3.2 +/- 0.4 microM at concentrations of less than 1.6 and greater than 1.6 microM, respectively. O2 affinity values obtained for both the protozoal and bacterial populations were similar. O2 affinities of the isolated entodiniomorphid ciliates Polyplastron multivesiculatum and Eudiplodinium maggii showed O2 inhibition thresholds of 10 and 5, respectively, and apparent half-saturation constants (Km values) of 1.7 and 5.2 microM O2, respectively. Corresponding Vmax values were 7.8 microM O2 per min per 10(5) organisms for P. multivesiculatum and 3.6 microM O2 per min per 10(5) organisms for E. maggii. Mass spectroscopic analysis detected average rates of H2 production of 12.0 and 3.7 microM H2 per min per 10(5) organisms for P. multivesiculatum and E. maggii, respectively. Trace levels of dissolved O2 (less than 0.25 microM) stimulated the H2 production rate of E. maggii eightfold but inhibited that of P. multivesiculatum by 18%.     

Oxygen consumption by ruminal microorganisms: protozoal and bacterial contributions.

The relative contributions to O2 consumption made by the protozoal and bacterial populations present within the rumen were determined by using an open-type oxygen electrode system. Measurements indicated that two separate microbial populations contributed approximately equally to ruminal O2 consumption over the O2 concentration range experienced in situ (0.25 to 1.0 microM). The populations were observed to consume O2 under liquid-phase O2 concentrations of up to 7 microM, above which point rapid inactivation of O2 utilization was observed. Km values for the mixed population of bacteria and protozoa were 0.36 +/- 0.17 and 3.2 +/- 0.4 microM at concentrations of less than 1.6 and greater than 1.6 microM, respectively. O2 affinity values obtained for both the protozoal and bacterial populations were similar. O2 affinities of the isolated entodiniomorphid ciliates Polyplastron multivesiculatum and Eudiplodinium maggii showed O2 inhibition thresholds of 10 and 5, respectively, and apparent half-saturation constants (Km values) of 1.7 and 5.2 microM O2, respectively. Corresponding Vmax values were 7.8 microM O2 per min per 10(5) organisms for P. multivesiculatum and 3.6 microM O2 per min per 10(5) organisms for E. maggii. Mass spectroscopic analysis detected average rates of H2 production of 12.0 and 3.7 microM H2 per min per 10(5) organisms for P. multivesiculatum and E. maggii, respectively. Trace levels of dissolved O2 (less than 0.25 microM) stimulated the H2 production rate of E. maggii eightfold but inhibited that of P. multivesiculatum by 18%.     

Transport of C5 dicarboxylate compounds by Pseudomonas putida.

Induced glutarate and 2-oxoglutarate uptake and transport by Pseudomonas putida were investigated in whole cells and membrane vesicles, respectively. Uptake of 2-oxoglutarate, but not glutarate, was against a concentration gradient to 1.7-fold greater than the initial extracellular concentration. Membrane vesicles transported 2-oxoglutarate and glutarate against gradients to intramembrane concentrations fivefold greater than the initial extravesicle concentrations. The rates of transport of both compounds were greatest in the presence of the artificial electron donor system phenazine methosulfate-ascorbate. Malate and D-lactate were the only naturally occurring compounds that served as electron donors. Uptake and transport were inhibited by KCN, NaN3, and 2,2-dinitrophenol. Kinetic parameters of transport were: glutarate, apparent Km--1.22 mM, Vmax--400 nmol/min per mg of membrane protein; 2-oxoglutarate, apparent Km--131 microM, Vmax--255 nmol/min per mg of membrane protein. Studies of competitive inhibition indicated a common system for transport of five C5 dicarboxylate compounds. The apparent Km and Ki values with 2-oxoglutarate as a substrate placed the substrate affinity for transport in the order 2-oxoglutarate greater than glutarate greater than D-2-hydroxyglutarate and L-2-hydroxyglutarate greater than glutaconate.     

Glycolysis and respiration in yeasts. The Pasteur effect studied by mass spectrometry.

Simultaneous and continuous measurements of changes in CO2 and O2 concentrations in glucose-metabolizing yeast suspensions by mass spectrometry enabled a study of the Pasteur effect (aerobic inhibition of glycolysis) in Saccharomyces uvarum and Schizosaccharomyces pombe. A different control mechanism operates in Candida utilis to give a damped oscillation after the anaerobic-aerobic transition. The apparent Km values for respiration of the three yeasts were in the range 1.3-1.8 microM-O2. The apparent Km values for O2 of the Pasteur effect were 5 and 13 microM for catabolite-repressed and derepressed S. uvarum respectively and 7 microM for Sch. pombe. These results are discussed with respect to currently accepted mechanisms for the control of glycolysis.     

Hydrogen production by rumen holotrich protozoa: effects of oxygen and implications for metabolic control by in situ conditions

Experiments with washed suspensions of holotrich protozoa (Isotricha spp. and Dasytricha ruminantium) showed that both organisms have an efficient O2-scavenging capability (apparent Km values 2.3 and 0.3 microM, respectively). Reversible inhibition of H2 production increased almost linearly with increasing O2 up to 1.5 microM; higher levels of O2 gave irreversible inhibition. In situ determinations of H2, CH4, O2 and CO2 in ovine rumen liquor, using a membrane inlet mass spectrometer probe, indicated that O2 was present before feeding at 1-1.5 microM and decreased to undetectable levels (less than 0.25 microM) within 25 min after feeding. A transient increase in O2 concentration after feeding occurred only in defaunated animals and resulted in suppression of CH4 and CO2 production. The presence of washed holotrich protozoa decreases the O2 sensitivity of CH4 production by suspensions of a cultured methanogenic bacterium Methanosarcina barkeri. It is concluded that holotrich protozoa play a role in ruminal O2 utilization as well as in the production of fermentation end products (especially short-chain volatile fatty acids) utilized by the ruminant and H2 utilized by methanogenic bacteria. These hydrogenosome-containing protozoa thus both control patterns of fermentation by influencing O2 levels, and are themselves regulated by the low ambient O2 concentrations they experience in the rumen.     

The effect of inhibitors on the oxygen kinetics of terminal oxidases of Acanthamoeba castellanii.

1. Respiration of growing cultures of Acanthamoeba castellanii is inhibited less than 60% by azide (35 mM); the respiration of early-exponential-phase cultures differs from that of late-exponential-phase cultures in being stimulated by up to 120% by low concentrations (less than 1 mM) of this inhibitor. Azide (0.5 mM) plus 1 mM-salicylhydroxamic acid gives 80% inhibition of respiration in early- or late-exponential-phase cultures. 2. Lineweaver-Burk plots of 1/v against 1/[O2] for growing and stationary-phase cultures give values of less than 1 muM for the apparent Km for oxygen. 3. These values are not significantly altered when determined in the presence of 1 mM-salicylhydroxamic acid. 4. Higher values (greater than 7 muM) for apparent Km values for oxygen were obtained in the presence of azide, which gives non-linear Lineweaver-Burk plots. 5. Competitive inhibition of respiration by CO occurs with Ki 2.4 muM. 6. The results are discussed in terms of the presence of three terminal oxidases in this organism, namely two oxidases with high affinities for oxygen (cytochrome c oxidase of the main phosphorylating electron-transport chain and the salicylhydroxamic acid-sensitive oxidase) and a third oxidase with a low affinity for oxygen, sensitive to inhibition by cyanide but not by azide or salicylhydroxamic acid. The relative contributions to oxygen utilization by these oxidases change during the growth of a batch culture.     

Cyanide-resistant respiration in Taenia crassiceps metacestode (cysticerci) is explained by the H2O2-producing side-reaction of respiratory complex I with O2

The nature of the cyanide-resistant respiration of Taenia crassiceps metacestode was studied. Mitochondrial respiration with NADH as substrate was partially inhibited by rotenone, cyanide and antimycin in decreasing order of effectiveness. In contrast, respiration with succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was more sensitive to antimycin and cyanide. The saturation kinetics for O2 with NADH as substrate showed two components, which exhibited different oxygen affinities. The high-O2-affinity system (Km app=1.5 microM) was abolished by low cyanide concentration; it corresponded to cytochrome aa3. The low-O2-affinity system (Km app=120 microM) was resistant to cyanide. Similar O2 saturation kinetics, using succinate or ascorbate-TMPD as electron donor, showed only the high-O2-affinity cyanide-sensitive component. Horse cytochrome c increased 2-3 times the rate of electron flow across the cyanide-sensitive pathway and the contribution of the cyanide-resistant route became negligible. Mitochondrial NADH respiration produced significant amounts of H2O2 (at least 10% of the total O2 uptake). Bovine catalase and horse heart cytochrome c prevented the production and/or accumulation of H2O2. Production of H2O2 by endogenous respiration was detected in whole cysticerci using rhodamine as fluorescent sensor. Thus, the CN-resistant and low-O2-affinity respiration results mainly from a spurious reaction of the respiratory complex I with O2, producing H2O2. The meaning of this reaction in the microaerobic habitat of the parasite is discussed.     

Effects of fatty acids on activity of cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

Effects of fatty acids, prostaglandins, and phospholipids on the activity of purified cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver were investigated. Prostaglandins A2, E1, E2, F1 alpha, and F2 alpha, thromboxane B2, and most phospholipids were without effect; lysophosphatidylcholine was a potent inhibitor. Several saturated fatty acids (carbon chain length 14-24), at concentrations up to 1 mM, had little or no effect on hydrolysis of 0.5 microM [3H]cGMP or 0.5 microM [3H]cAMP with or without 1 microM cGMP. In general, unsaturated fatty acids were inhibitory, except for myristoleic and palmitoleic acids which increased hydrolysis of 0.5 microM [3H]cAMP. The extent of inhibition by cis-isomers correlated with the number of double bonds. Increasing concentrations of palmitoleic acid from 10 to 100 microM increased hydrolysis of [3H]cAMP with maximal activation (60%) at 100 microM; higher concentrations were inhibitory. Palmitoleic acid inhibited cGMP hydrolysis and cGMP-stimulated cAMP hydrolysis with IC50 values of 110 and 75 microM, respectively. Inhibitory effects of palmitoleic acid were completely or partially prevented by equimolar alpha-tocopherol. Palmitelaidic acid, the trans isomer, had only slightly inhibitory effects. The effects of palmitoleic acid (100 microM) were dependent on substrate concentration. Activation was maximal with 1 microM [3H]cAMP and was reduced with increasing substrate; with greater than 10 microM cAMP, palmitoleic had no effect. Inhibition of cGMP hydrolysis was maximal at 2.5 microM cGMP and was reduced with increasing cGMP; at greater than 100 microM cGMP palmitoleic acid increased hydrolysis slightly. Palmitoleic acid did not affect apparent Km or Vmax for cAMP hydrolysis, but increased the apparent Km (from 17 to 60 microM) and Vmax for cGMP hydrolysis with little or no effect on the Hill coefficient for either substrate. These results suggest that certain hydrophobic domains play an important role in modifying the catalytic specificity of the cGMP-stimulated phosphodiesterase for cAMP and cGMP.     

Properties of tyrosine hydroxylation in living mouse neuroblastoma clone N1E-115

Tyrosine hydroxylation was studied in intact cells of mouse neuroblastoma clone N1E-115 which have high levels of tyrosine 3-monooxygenase (EC 1.14.16.2) and which have been fully characterized for tyrosine transport. Measurement of [3H]OH formed from L-[3,5(-3)H]tyrosine in the medium was the method of assay and [3H]OH formed was stoichiometric with the formation of L-[3H]3,4-dihydroxyphenylalanine. Tyrosine hydroxylation was dependent on time of incubation, cell number, and the concentration of [3H]tyrosine in the medium. From velocity vs. [3H]tyrosine concentration experiments, two apparent Km values were obtained: Km1 = 10 +/- 2 microM; Km2 = 140 +/- 10 microM. Substrate inhibition occurred with tyrosine concentrations between 20 and 50 microM. The reaction was twice as fast at pH 5.5 as at pH 7.4. alpha,alpha'-Dipyridyl (1 mM) caused major inhibition (75%) when [3H]tyrosine concentration was 10 microM. L-3-Iodotyrosine was a competitive inhibitor with Ki = 0.3 microM. Dopamine was a non-competitive inhibitor with Ki = 500 microM. 1-Norepinephrine had no effect. These results show that the hydroxylation of tyrosine by living N1E-115 cells has many of the properties of the reaction catalyzed by purified tyrosine 3-monooxygenase from normal tissue.     

Activation by ATP of calcium-dependent NADPH-oxidase generating hydrogen peroxide in thyroid plasma membranes

An H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H2O2 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0.1 microM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hexokinase together with glucose to remove ATP. The Km value for NADPH was 35 microM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H2O2 production. The stoichiometry between the oxidation and the H2O2 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2 production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H2O2 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H2O2. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H2O2. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H2O2, a substrate for peroxidase.     

Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin B, the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a Km of 45 +/- 3 microM. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (IC50) observed at concentrations less than 30 microM. Of the pyrimidine nucleosides examined, only uridine (IC50, 41 +/- 9 microM) was a good inhibitor. Thymidine and cytidine were poor inhibitors with IC50 values greater than 300 microM. Direct measurements of [3H]thymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (IC50 values less than 25 microM), but not by formycin B, inosine, or guanosine (IC50 values greater than 600 microM). These data indicate that there are two sodium-dependent mechanisms for nucleoside transport in mouse intestinal epithelial cells, and that formycin B and thymidine may serve as model substrates to distinguish between these transporters. Neither of these sodium-dependent transport mechanisms was inhibited by nitrobenzylmercaptopurine riboside (10 microM), a potent inhibitor of one of the equilibrative (facilitated diffusion) nucleoside transporters found in many cells.     

Hydrogenase activity in Azospirillum brasilense is inhibited by nitrite, nitric oxide, carbon monoxide, and acetylene.

Nitrite, NO, CO, and C2H2 inhibited O2-dependent H2 uptake (H3H oxidation) in denitrifying Azospirillum brasilense Sp7 grown anaerobically on N2O or NO3-. The apparent Ki values for inhibition of O2-dependent H2 uptake were 20 microM for NO2-, 0.4 microM for NO, 28 microM for CO, and 88 microM for C2H2. These inhibitors also affected methylene blue-dependent H2 uptake, presumably by acting directly on the hydrogenase. Nitrite and NO inhibited H2 uptake irreversibly, whereas inhibition due to CO was easily reversed by repeatedly evacuating and backfilling with N2. The C2H2 inhibition was not readily reversed, partly due to difficulty in removing the last traces of this gas from solution. The NO2- inhibition of malate-dependent respiration was readily reversed by repeatedly washing the cells, in contrast to the effect of NO2- on H2-dependent respiration. These results suggest that the low hydrogenase activities observed in NO3(-)-grown cultures of A. brasilense may be due to the irreversible inhibition of hydrogenase by NO2- and NO produced by NO3- reduction.     

Reverse electron flow-induced ROS production is attenuated by activation of mitochondrial Ca2+-sensitive K+ channels

Mitochondria generate reactive oxygen species (ROS) dependent on substrate conditions, O(2) concentration, redox state, and activity of the mitochondrial complexes. It is well known that the FADH(2)-linked substrate succinate induces reverse electron flow to complex I of the electron transport chain and that this process generates superoxide (O(2)(*-)); these effects are blocked by the complex I blocker rotenone. We demonstrated recently that succinate + rotenone-dependent H(2)O(2) production in isolated mitochondria increased mildly on activation of the putative big mitochondrial Ca(2+)-sensitive K(+) channel (mtBK(Ca)) by low concentrations of 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619). In the present study we examined effects of NS-1619 on mitochondrial O(2) consumption, membrane potential (DeltaPsi(m)), H(2)O(2) release rates, and redox state in isolated guinea pig heart mitochondria respiring on succinate but without rotenone. NS-1619 (30 microM) increased state 2 and state 4 respiration by 26 +/- 4% and 14 +/- 4%, respectively; this increase was abolished by the BK(Ca) channel blocker paxilline (5 microM). Paxilline alone had no effect on respiration. NS-1619 did not alter DeltaPsi(m) or redox state but decreased H(2)O(2) production by 73% vs. control; this effect was incompletely inhibited by paxilline. We conclude that under substrate conditions that allow reverse electron flow, matrix K(+) influx through mtBK(Ca) channels reduces mitochondrial H(2)O(2) production by accelerating forward electron flow. Our prior study showed that NS-1619 induced an increase in H(2)O(2) production with blocked reverse electron flow. The present results suggest that NS-1619-induced matrix K(+) influx increases forward electron flow despite the high reverse electron flow, and emphasize the importance of substrate conditions on interpretation of effects on mitochondrial bioenergetics.     

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids.

The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate. A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Metabolism of unsaturated fatty acids by RBL-1 5-lipoxygenase: influence of substrate solubility and product inactivation

Several alternative fatty acid substrates have been employed to characterise the kinetics of rat basophilic leukaemia cell (RBL-1) 5-lipoxygenase. Using arachidonic acid (AA) as substrate, enzymes rates declined at high substrate concentrations (greater than 25 microM) and were associated with pronounced lag phases. The concentrations of AA at which apparent substrate inhibition and lag phases were observed were comparable with those at which AA induced emulsion formation in aqueous media. No evidence for substrate inhibition or lag phases was observed using eicosapentaenoic acid (EPA), a more soluble substrate which did not induce emulsion formation at concentrations up to 100 microM. Reactions catalysed by RBL-1 5-lipoxygenase terminated before exhaustion of substrate. AA and EPA induced time-dependent enzyme inactivation at concentrations 100-fold lower than their apparent Km values for the enzyme. The ability of several fatty acids to induce time-dependent inactivation was directly proportional to their substrate potency. We conclude that apparent substrate inhibition is a consequence of a change from monomeric to micellar substrate which has a lower affinity for the enzyme and that premature termination of the enzyme reactions is a consequence of product-induced enzyme inactivation.     

Bioconjugation of zirconium uridine monophosphate: application to myoglobin direct electrochemistry

Porous nano-granule of zirconium uridine monophosphate, Zr(UMP)2.H2O is, for the first time, synthesized under mild experimental conditions and applied to the bioconjugation of myoglobin (Mb) to realize its direct electron transfer. UV-vis and resonance Raman spectroscopies prove that Mb in the Zr(UMP)2.H2O film maintains its secondary structure similar to the native state. The conjugation film of the Mb-Zr(UMP)2.H2O on the glassy carbon (GC) electrode gives a well-defined and quasi-reversible cyclic voltammogram, which reflects the direct electron transfer of the heme Fe III/Fe II couple of Mb. On the basis of the satisfying bioelectrocatalysis of the nano-conjugation of Mb and genetic substrate, a kind of mediator-free biosensor for H2O2 is developed. The linear range for H2O2 detection is estimated to be 3.92-180.14 microM. The apparent Michaelis-Menten constant (Km) and the detection limit based on the signal-to-noise ratio of 3 are found to be 196.1 microM and 1.52 microM, respectively. Both the apparent Michaelis-Menten constant and the detection limit herein are much lower than currently reported values from other Mb films. This kind of sensor possesses excellent stability, long-term life (more than 20 days) and good reproducibility.     

The NADPH:O2 oxidoreductase of human neutrophils. Stoichiometry of univalent and divalent reduction of O2

The ratio of superoxide production to oxidation of NADPH affected by the NADPH:O2 oxidoreductase of human neutrophils is strongly influenced by pH, NADPH substrate concentration, aging of the enzyme, or its exposure to excess deoxycholate. Freshly prepared enzyme exhibited a Km for NADPH of 52 microM as determined by assaying NADPH oxidase activity, or approximately 33 microM by measurement of superoxide formation. In the range of 100-150 microM NADPH at pH 7.6 and in the presence of 0.06% deoxycholate, the univalent flux of electron equivalents given up by NADPH to O2 was 99%. Following storage of the oxidoreductase overnight on ice, its Km for NADPH rose to 125 microM as determined by monitoring oxidation of NADPH but was unaltered when measured in terms of superoxide production. Concomitantly, its capacity to affect univalent reduction of O2 fell approximately 20-30% under the same assay conditions. Univalent flux rates of less than 40% were observed with exposure of the enzyme to concentrations of deoxycholate in excess of 0.1% or to pH values below 6.0 or above 8.0. The capacity of the enzyme to carry out univalent reduction fell with increasing NADPH concentrations in a manner resembling that previously reported with increasing concentrations of xanthine in the case of xanthine oxidase (Fridovich, I. (1970) J. Biol. Chem. 245, 4053-4057). The reduced form of the neutrophil oxidoreductase, like xanthine oxidase, thus appears to be capable of conducting both 1- and 2-electron transfer steps in reducing O2. Its capacity to affect univalent reduction of O2 depends upon the concentration of electron donor (NADPH) supplied as well as conditions of storage and assay of the native enzyme.