Nmol O2 Min-1 Research Articles

Energy pathways associated with sustained spermatozoon motility in the endangered Siberian sturgeon Acipenser baerii.

Sturgeon spermatozoa are unique for their sustained motility. We investigated the relative importance of bioenergetic pathways in the energy supply of Siberian sturgeon Acipenser baerii spermatozoa during motile and immotile states. Spermatozoon motility and oxygen consumption rate (OCR) were analysed following exposure to inhibitors of oxidative phosphorylation (sodium azide, NaN3 ), glycolysis (2-deoxy-D-glucose, DOG) and β-oxidation of fatty acids (sodium fluoride, NaF), and to an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenyl hydrazine, CCCP). No significant difference in curvilinear velocity was observed after addition of these reagents to activation medium (AM) or nonactivation medium (NAM) for incubation. Incubation of spermatozoa in NAM containing CCCP or NaN3 resulted in significantly decreased motility duration compared to controls. The OCR of sturgeon spermatozoa in AM (11.9 ± 1.4 nmol O2 min-1 (109 spz)-1 ) was significantly higher than in NAM (8.2 ± 1.5 nmol O2 min-1 (109 spz)-1 ). The OCR significantly declined with addition of NaN3 to AM and NAM. No significant difference in motility parameters or OCR was observed with NaF or DOG. These results suggest active oxidative phosphorylation in both immotile and motile spermatozoa. Nevertheless, mitochondrial respiration occurring during motility is not sufficient to meet the high energy demands, and the energy required for sustained motility of Siberian sturgeon spermatozoa is derived from adenosine triphosphate accumulated during the quiescent state.

Effects of domestic effluent discharges on mangrove crab physiology: Integrated energetic, osmoregulatory and redox balances of a key engineer species

Mangroves are increasingly used as biofiltering systems of (pre-treated) domestic effluents. However, these wastewater discharges may affect local macrofauna. This laboratory study investigates the effects of wastewater exposure on the mangrove spider crab Neosarmatium meinerti, a key engineering species which is known to be affected by waste waters in effluent-impacted areas. These effects were quantified by monitoring biological markers of physiological state, namely oxygen consumption, the branchial cavity ventilation rate, gill physiology and morphology, and osmoregulatory and redox balance. Adults acclimated to clean seawater (SW, 32 ppt) and freshwater (FW, ∼0 ppt) were compared to crabs exposed to wastewater for 5 h (WW, ∼0 ppt). Spider crabs exposed to WW increased their ventilation and whole-animal respiration rates by 2- and 3-fold respectively, while isolated gill respiration increased in the animals exposed to FW (from 0.5 to 2.3 and 1.1 nmol O2 min−1 mg DW−1 for anterior and posterior gills, respectively) but was not modified in WW-exposed individuals. WW exposure also impaired crab osmoregulatory capacity; an 80 mOsm kg−1 decrease was observed compared to FW, likely due to decreased branchial NKA activity. ROS production (DCF fluorescence in hemolymph), antioxidant defenses (superoxide dismutase and catalase activities) and oxidative damage (malondialdehyde concentration) responses varied according to animal gender. Overall, this study demonstrates that specific physiological parameters must be considered when focusing on crabs with bimodal breathing capacities. We conclude that spider crabs exposed to WW face osmoregulatory imbalances due to functional and morphological gill remodeling, which must rapidly exhaust energy reserves. These physiological disruptions could explain the ecological changes observed in the field.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Phagocytosis of microbial symbionts balances the carbon and nitrogen budget for the deep‐water boreal sponge Geodia barretti

AbstractGeodia barretti is a massive nearly spherical sponge that forms dense assemblages on the continental shelf of the North Atlantic and the Norwegian Sea. We studied the metabolism of individual sponges collected using a remotely operated vehicle and maintained in large tanks with high volumes of unfiltered water brought from 160 m depth. We used direct methods (In‐Ex) to measure excurrent flow rates, oxygen removed, and carbon and nutrient flux through the sponges. G. barretti had very low specific filtration (0.26 mL min−1 mL−1 sponge tissue) and low respiration (5.34 ± 0.98 nmol O2 min−1 mL−1 sponge tissue; 8.44 ± 1.51 μmol O2 h−1 g C−1) rates in comparison to other sponges. A net release of nitrogen was detected as . Bacteria were removed from the water filtered with up to 99% efficiency, yet comprised only 5% of the sponges′ total carbon budget; the remainder consisted of dissolved organic carbon and detritus. High bacterial removal was aided by the presence of a tight gasket of cells that surrounds the collar of each choanocyte filter. A test for potential bypass canals showed removal of fluorescent microspheres until they were excreted 5–12 h after feeding. Electron micrographs showed active uptake of Escherichia coli “fed” to the sponge as well as phagocytosis of symbiont microbes by sponge cells in the mesohyl. These data provide the first comprehensive study of metabolism in a deep‐water high microbial abundance sponge.

Diversity and Ecophysiology of New Isolates of Extremely Acidophilic CS 2 -Converting Acidithiobacillus Strains

Biofiltration of industrial carbon disulfide (CS2)-contaminated waste air streams results in the acidification of biofilters and therefore reduced performance, high water use, and increased costs. To address these issues, we isolated 16 extremely acidophilic CS2-converting Acidithiobacillus thiooxidans strains that tolerated up to 6% (vol/vol) sulfuric acid. The ecophysiological properties of five selected strains (2Bp, Sts 4-3, S1p, G8, and BBW1) were compared. These five strains had pH optima between 1 (2Bp) and 2 (S1p). Their affinities for CS2 ranged between 80 (G8) and 130 (2Bp) μM. Strains S1p, G8, and BBW1 had more hydrophobic cell surfaces and produced less extracellular polymeric substance than did strains 2Bp and Sts 4-3. All five strains converted about 80% of the S added as CS2 to S(0) when CS2 was supplied in excess. The rate of S(0) consumption varied between 7 (Sts 4-3) and 63 (S1p) nmol O2 min(-1) ml culture(-1). Low S(0) consumption rates correlated partly with low levels of cell attachment to externally produced S(0) globules. During chemostat growth, the relative amount of CS2 hydrolase in the cell increased with decreasing growth rates. This resulted in more S(0) accumulation during CS2 overloads at low growth rates. Intermittent interruptions of the CS2 supply affected all five strains. Strains S1p, G8, and BBW1 recovered from 24 h of starvation within 4 h, and strains 2Bp and Sts 4-3 recovered within 24 h after CS2 was resupplied. We recommend the use of mixtures of Acidithiobacillus strains in industrial biofilters.

Liver mitochondrial and whole-animal level metabolic compensation in a catfish during seasonal acclimatization

Abstract To examine whether metabolic compensation during seasonal acclimatization at the liver mitochondrial level is consistent with that at the whole-animal level, respiration rates of liver mitochondria and resting metabolic rates in winter- and summer-acclimatized southern catfish (Silurus meridionalis Chen) were measured. At 12.5, 17.5, 22.5, 27.5 and 32.5°C, the mean values of state 3 respiration rates were 12.21, 13.84, 18.96, 24.78 and 32.01 nmol O2 min-1 mg-1 mitochondrial protein in the winter group, and 8.56, 9.20, 17.32, 22.74 and 26.32 nmol O2 min-1 mg-1 in the summer group, respectively. At the five assay temperatures the resting metabolic rates were 24.86, 42.68, 61.59, 84.10 and 125.65 mg O2 h-1 kg-1 body mass in the winter group, and 22.89, 40.59, 52.94, 75.13 and 109.35 mg O2 h-1 kg-1 in the summer group, respectively. Total mitochondrial respiration rates in the liver organ were estimated based on state 3 respiration rates, mitochondrial protein content and organ mass, and the mean values were 72.96, 71.87, 112.47, 167.35 and 183.27 nmol O2 min-1 in the winter group, and were 47.89, 47.39, 105.67, 138.18 and 132.29 nmol O2 min-1 in the summer group, respectively. Metabolic compensation caused by seasonal acclimatization occurred at the liver mitochondrial level and compensation at the liver organ level was found to be more efficient because of an increase in metabolic capacity of mitochondria and a boost in organ mass. Metabolic compensation at the whole-animal level was not detected. During seasonal acclimatization, the effect of metabolic compensation at liver mitochondrial level is inconsistent with that at the whole-animal level in the southern catfish. This may be due to different degrees of regulation of metabolic mechanisms among various tissues and organs in an acclimatized organism.

Altered Mitochondrial Energetics and Increased ROS Generation Act Synergistically to Dampen β-Adrenergic Stimulated Contractility in the Diabetic Heart

Background: Excitation-contraction coupling and β-adrenergic activation are altered in diabetic hearts, contributing to contractile dysfunction. We hypothesized that mitochondrial dysfunction in diabetic hearts contributes to altered β-adrenergic responses via increased oxidative stress and respiratory uncoupling. Methods: Basal and isoproterenol (ISO)-induced changes in sarcomere shortening and Ca2+ transients were assessed in cardiomyocytes from wild-type (WT) and db/db mice under euglycemia (5.5mM) or hyperglycemia (30mM). Reactive oxygen species (ROS; H2O2 and O2.-), NADH, and ΔΨm were monitored using two photon laser scanning fluorescence microscopy. Results: Basal fractional shortening (FS) and Ca2+ transients were not significantly different between WT and db/db myocytes, regardless of glucose concentration. Following ISO (10nM), FS increased by ≅150% and Ca2+ transients by ≅30%, in both WT and db/db myocytes under euglycemia. Under hyperglycemia, the WT ISO response was intact, but the increase in FS and Ca2+ transients was blunted in db/db cells (68±2%, and 12±3%, respectively, both p<0.01 vs euglycemia). Under euglycemia, db/db cardiomyocytes had H2O2 signals 31% higher than WT (p<0.001), but under hyperglycemia, they possessed higher H2O2 (+12%; p<0.01) and lower O2.- levels (−22%; p<0.05) vs WT. Isolated WT and db/db mitochondria showed impaired respiration for substrates of Complex I (16±5 and 23±8 nmol O2min−1mg−1, respectively), but normal activity for substrates of Complexes II or IV. Impaired energetics correlated with high levels of ROS generation from Complex I or II observed under similar conditions. State 3 mitochondrial respiration with succinate and total respiratory capacity were significantly lower in db/db cells compared to WT. Conclusions: The findings suggest that the reduced effectiveness of ISO in diabetic hearts under hyperglycemia is mediated by impaired mitochondrial energetics coupled to increased oxidative stress, leading to a deleterious synergistic effect on β-adrenergic response.

Oxygen consumption by a coral reef sponge

Oxygen consumption of the Red Sea coral reef sponge Negombata magnifica was measured using both incubation and steady-state methods. The latter method was found to be the more reliable because sponge activity remained stable over time. Oxygen consumption rate was measured during three levels of sponge activity: full activity, reduced activity and basal activity (starved). It was found that the active oxygen consumption rate of N. magnifica averaged 37.3+/-4.6 nmol O2 min(-1) g(-1) wet mass, which is within the upper range reported for other tropical marine sponges. Fully active N. magnifica individuals consumed an average of 41.8+/-3.2 nmol O2 min(-1) g(-1) wet mass. The mean basal respiration rate was 20.2+/-1.2 nmol O2 min(-1) g(-1) wet mass, which is 51.6+/-2.5% of the active respiration rate. Therefore, the oxygen used for water pumping was calculated to be at most 10.6+/-1.8 nmol O2 min(-1) g(-1) wet mass, which is 25.1+/-3.6% of the total respiration. Combined oxygen used for maintenance and water pumping activity was calculated to be 30.8 nmol O2 min(-1) g(-1) wet mass, which is approximately 74% of the sponge's total oxygen requirement. The remaining oxygen is directed to other physiological activities, mainly the energy requirement of growth. These findings suggest that only a relatively minor amount of energy is potentially available for growth, and thus might be a factor in controlling the growth rate of N. magnifica in oligotrophic coral reefs.

A sulphite respiration system in the chemoheterotrophic human pathogen Campylobacter jejuni

The ability to use sulphite as a respiratory electron donor is usually associated with free-living chemolithotrophic sulphur-oxidizing bacteria. However, this paper shows that the chemoheterotrophic human pathogen Campylobacter jejuni has the ability to respire sulphite, with oxygen uptake rates of 23 +/- 8 and 28 +/- 15 nmol O(2) min(-1) (mg cell protein)(-1) after the addition of 0.5 mM sodium sulphite or metabisulphite, respectively, to intact cells. The C. jejuni NCTC 11168 Cj0004c and Cj0005c genes encode a monohaem cytochrome c and molybdopterin oxidoreductase, respectively, homologous to the sulphite : cytochrome c oxidoreductase (SOR) of Starkeya novella. Western blots of C. jejuni periplasm probed with a SorA antibody demonstrated cross-reaction of a 45 kDa band, consistent with the size of Cj0005. The Cj0004c gene was inactivated by insertion of a kanamycin-resistance cassette. The resulting mutant showed wild-type rates of formate-dependent respiration but was unable to respire with sulphite or metabisulphite as electron donors. 2-Heptyl-4-hydroxyquinoline-N-oxide (HQNO), a cytochrome bc(1) complex inhibitor, did not affect sulphite respiration at concentrations up to 25 microM, whereas formate respiration (which occurs partly via a bc(1) dependent route) was inhibited 50%, thus suggesting that electrons from sulphite enter the respiratory chain after the bc(1) complex at the level of cytochrome c. Periplasmic extracts of wild-type C. jejuni 11168 showed a symmetrical absorption peak at 552 nm after the addition of sulphite, demonstrating the reduction of cytochrome c. No cytochrome c reduction was observed after addition of sulphite to periplasmic extracts of the Cj0004c mutant. A fractionation study confirmed that the majority of the SOR activity is located in the periplasm in C. jejuni, and this activity was partially purified by ion-exchange chromatography. The presence of a sulphite respiration system in C. jejuni is another example of the surprising diversity of the electron-transport chain in this small-genome pathogen. Sulphite respiration may be of importance for survival in environmental microaerobic niches and some foods, and may also provide a detoxification mechanism for this normally growth-inhibitory compound.

Respiration of steelhead trout sperm: sensitivity to pH and carbon dioxide

Steelhead trout Oncorhynchus mykiss sperm held in seminal plasma or sperm‐immobilizing buffer (pH 8·6) at 10° C consumed O2 at the rate of c. 2 nmol O2 min−1 10−9 sperm; the rate of O2 consumption was not different in sperm held for 4 or 24 h. Decreasing the extracellular pH from 8·5 to 7·5 either by diluting semen with buffer titrated with HCl or by increasing the partial pressure of CO2 in the incubation atmosphere resulted in c. a 40% decrease in the rate of sperm respiration. The data did not, however, support the hypothesis that the precipitous reduction in the capacity for sperm motility that occurs as external pH is reduced is a result of a decrease in cellular metabolism. The rate of O2 consumption of freshly collected semen from different males was not correlated to cellular ATP content or to the proportion of sperm that were motile upon activation; the initial ATP content and sperm motility were positively correlated. The rate of O2 consumption was not significantly increased following sperm activation or by the addition of an uncoupler of oxidative phosphorylation, carbonyl cyanide p‐trifluoromethoxyphenylhydrazone, suggesting that these sperm have little, if any, capacity for increased oxidative metabolism.

Stimulation of potato tuber respiration by cold stress is associated with an increased capacity of both plant uncoupling mitochondrial protein (PUMP) and alternative oxidase.

The CO2 evolution of intact potato tubers (Solanum tuberosum, L., var. "Bintje") was analyzed during a 10-day period of their warm (25 +/- 2 degrees C) or cold (5 +/- 1 degrees C) storage, to evaluate cold-stress effects on expression and activities of plant uncoupling mitochondrial protein (PUMP) and alternative oxidase (AOX). CO2 evolution rates were analyzed at 20 degrees C, to reflect their possible capacities. The 20 degrees C CO2 production declined from 13 to 8 mg kg(-1) h(-1) after 2 days of warm storage and then (after 3 to 7 days) decreased from 8 to 6.5 mg kg(-1) h(-1). In contrast, 20 degrees C CO2 evolution did not change after the first day of cold storage, increased up to 14.5 mg kg(-1) h(-1) after 2 days, and decreased to about 12 mg kg(-1) h(-1) after 3 to 7 days of cold storage. Cold storage increased PUMP expression as detected by Western blots and led to elevated capacities of both PUMP (44%) and CN-resistant AOX (10 times), but not the cytochrome pathway. Since we found that cold storage led to about the same mitochondrial respiration of 40 nmol O2 min(-1) mg(-1) attributable to each of the respective proteins, we conclude that both AOX and PUMP equally contribute to adaptation of potato tubers to cold.

Chemolithoautotrophy and mixotrophy in the thiophene-2-carboxylic acid-utilizing xanthobacter tagetidis

Xanthobacter tagetidis grew as a chemolithotrophic autotroph on thiosulfate and other inorganic sulfur compounds, as a heterotroph on thiophene-2-carboxylic acid, acetic acid and alpha-ketoglutaric acid, and as a mixotroph on thiosulfate in combination with thiophene-2-carboxylic acid and/or acetic acid. Autotrophic growth on one-carbon organosulfur compounds, and intermediates in their oxidation are also reported. Thiosulfate enhanced the growth yields in mixotrophic cultures, presumably by acting as a supplementary energy source, since ribulose bisphosphate carboxylase was only active in thiosulfate-grown cells and was not detected in mixotrophic cultures using thiosulfate with thiophene-2-carboxylic acid. Bacteria grown on thiophene-2-carboxylic acid also oxidized sulfide, thiosulfate and tetrathionate, indicating these as possible sulfur intermediates in thiophene-2-carboxylic acid degradation. Thiosulfate and tetrathionate were oxidized completely to sulfate and, consequently, did not accumulate as products of thiophene-2-carboxylic acid oxidation in growing cultures. Km and Vmax values for the oxidation of thiosulfate, tetrathionate or sulfide were 13 &mgr;M and 83 nmol O2 min-1 (mg dry wt.)-1, respectively; thiosulfate and tetrathionate became autoinhibitory at concentrations above 100 &mgr;M. The true growth yield (Ymax) on thiophene-2-carboxylic acid was estimated from chemostat cultures (at dilution rates of 0.034-0.094 h-1) to be 112.2 g mol-1, with a maintenance coefficient (m) of 0.3 mmol thiophene-2-carboxylic acid (g dry wt.)-1 h-1, and the maximum specific growth rate (&mgr;max) was 0.116 h-1. Growth in chemostat culture at a dilution rate of 0. 041 h-1 indicated growth yields [g dry wt. (mol substrate)-1] of 8.1 g (mol thiosulfate)-1, 60.9 g (mol thiophene-2-carboxylic acid)-1, and 17.5 g (mol acetic acid)-1, with additive yields for growth on mixtures of these substrates. At a dilution rate of 0.034 h-1, yields of 57.8 g (mol alpha-ketoglutaric acid)-1 and 60.7 g (mol thiophene-2-carboxylic acid)-1 indicated some additional energy conservation from oxidation of the thiophene-sulfur. SDS-PAGE of cell-free preparations indicated a polypeptide (Mr, 21.0 kDa) specific to growth on thiophene-2-carboxylic acid for which no function can yet be ascribed: no metabolism of thiophene-2-carboxylic acid by cell-free extracts was detected. It was shown that X. tagetidis exhibits a remarkable degree of metabolic versatility and is representative of facultatively methylotrophic and chemolithotrophic autotrophs that contribute significantly to the turnover of simple inorganic and organic sulfur compounds (including substituted thiophenes) in the natural environment.

The role of polyglucose in oxygen-dependent respiration by a new strain of Desulfovibrio salexigens

Desulfovibrio salexigens strain Mastl was isolated from the oxic/anoxic interface of a marine sediment. Growth under sulfate-reducing conditions was accompanied by polyglucose accumulation in the cell with every substrate tested. Highest polyglucose storage was found with glucose (0.8–1.0 g polyglucose (g protein)−1), but the growth rate with this substrate was very low (0.015 h−1). Anaerobically grown cells of strain Mastl exhibited immediate oxygen-dependent respiration. The endogenous oxygen reduction rate was proportional to the polyglucose content. The rate of aerobic respiration of pyruvate was also directly related to the polyglucose content indicating that this organism was only able to respire with oxygen as long as polyglucose was present. Maximum oxygen reduction rates were found at air saturating concentrations and were relatively low (3–50 nmol O2 min−1 (mg protein)−1). Catalase was constitutively present in anaerobically grown cells. When batch cultures were exposed to oxygen, growth ceased immediately and polyglucose was oxidized to acetate within 40–50 h. Like the oxygen reduction activity, the nitro blue tetrazolium (NBT)-reduction activity in these cells was proportional to the polyglucose content. Under anaerobic starvation conditions there was no correlation between the NBT-reduction activity and polyglucose concentration and polyglucose was degraded slowly within 240 h. The ecological significance of aerobic polyglucose consumption is discussed.

A common pathway of sulfide oxidation by sulfate-reducing bacteria

The pathway of sulfide oxidation with oxygen as electron acceptor was studied with five strains of freshwater, marine and alkaliphilic sulfate-reducing bacteria. Electrode measurements with washed cells indicated that all strains oxidized sulfide to elemental sulfur. In a second step, the elemental sulfur formed was disproportionated to sulfate and sulfide. During this phase, most of the disappeared sulfide was formed back. Since oxygen could be replaced by nitrate or nitrite as electron acceptor, the described biphasic reaction was independent of molecular oxygen. With Desulfobulbus propionicus and the alkaliphilic strain Z-7935, sulfide back-formation started after oxygen was consumed completely. By contrast, with the freshwater strains Desulfovibrio desulfuricans CSN (DSM 9104) and Essex 6 (DSM 2032) and the marine strain P1B, sulfide back-formation already started before oxygen was consumed. The addition of hydrogen as electron donor increased simultaneously the rate of aerobic respiration and sulfide back-formation. Both reactions stopped when the oxygen was consumed, indicating that the electron transport to oxygen and sulfur was coupled. Sulfide-oxidizing activity (84 nmol O2 min−1 (mg protein)−1) was found in the periplasmic fraction prepared by osmotic shock treatment of suspensions of D. desulfuricans CSN. This fraction oxidized sulfide with oxygen to elemental sulfur. It is concluded that in different sulfate-reducing bacteria sulfide oxidation proceeds via a common pathway with the formation of elemental sulfur as intermediate and its disproportionation to sulfate and sulfide. The process is independent of molecular oxygen.

Metabolic specialization of mitochondria from scallop phasic muscles

In fast, glycolytic muscles, oxidative phosphorylation presumably facilitates recuperation from exhaustive exercise and supports growth and maintenance metabolism. Given the shifts in pH with extensive glycolytic activity, the pH optima of mitochondrial processes should indicate whether mitochondria are adapted for recuperation from exercise or for growth and maintenance. We examined this question using mitochondria from the phasic adductor muscle of the scallop, Euvola (Pecten) ziczac, collected from the Golfo de Cariaco, Venezuela in 1992 and 1993. Scallop muscle mitochondria showed well coupled oxidation of glutamate and pyruvate at pH 7.0 and 6.4. The preferred substrates (glutamate, pyruvate and succinate) were oxidized at approximately 40 nmol O2 min-1 mg-1 mitochondrial protein at 25°C, while malate and glutamine were oxidized at ∼ 75% and proline at ∼ 30% of these rates. Neither palmitoyl carnitine nor aspartate were oxidized. Succinate oxidation was not coupled to ADP utilization at pH 7.0 but was somewhat coupled at pH 6.4. Generally, State 3 rates of oxygen uptake were similar at pH 7.0 and 6.4. Maximal rates of oxidation of glutamate and pyruvate showed broad pH optima. For both glutamate and pyruvate, the highest respiratory control ratio (RCR) values were found at pH 6.5. The saturation curves of scallop muscle mitochondria for pyruvate, glutamate and ADP were well described by the Michaelis-Menten equation. The affinity for pyruvate was greater at pH 6.4 (apparent Km, app=0.013 mM) than at pH 7.0 (Km, app=0.026 mM) while the affinity for ADP (Km, app=0.015 mM) and that for glutamate (Km, app=0.55 mM) changed little with pH. The ADP affinity was the same whether pyruvate or glutamate was the carbon substrate. The combination of maintenance of sensitivity to ADP with an enhanced affinity for pyruvate at acidic pH values should facilitate recuperation from bouts of glycolytic activity. Scallops harvested in September and those harvested in January differed in the maximal rates of glutamate and pyruvate oxidation.

Acetylene Reduction by Symbiosomes and Free Bacteroids from Broad Bean (Vicia faba L.) Nodules (Role of Oxalate).

We report the presence of oxalate in the organic acid fraction of broad bean (Vicia faba L.) nodule cytosol. Using both high-performance liquid chromatography and enzymic assays, high levels of oxalate were detected (70.4 [plus or minus] 2.4 mM). To study the potential role of oxalate as an energy-yielding substrate for nitrogenase activity, free bacteroids were isolated from nodules and found to oxidize oxalate in support of C2H2 reduction under O2 tensions that were lower than those required to oxidize succinate, another dicarboxylate commonly detected in legume nodules. Symbiosomes of broad bean, isolated for the first time from amide-producing nodules, were provided with [14C]oxalate and found to have uptake kinetics with a lower affinity [Km(oxalate) = 330 [mu]M] than that for free bacteroids [Km(oxalate) = 130 [mu]M]. In anaerobic preparations of symbiosomes supplied with purified oxyleghemoglobin, O2 consumption was stimulated by oxalate from 20.2 [plus or minus] 0.8 nmol O2 min-1mg-1 protein to 24.5 [plus or minus] 1.1 nmol O2 min-1 mg-1 protein but always remained lower than the rate of O2 consumption in free bacteroids (32.2 [plus or minus] 1.4 nmol O2 min-1 mg-1 protein). Under these conditions, C2H2 reduction activity was 9.7 [plus or minus] 0.8 and 15.1 [plus or minus] 0.9 nmol C2H4 min-1 mg-1 protein for symbiosomes and bacteroids, respectively. These data support the suggestion that oxalate may play a role as a carbon substrate in support of N2 fixation in broad bean nodules.

Supply of O2 regulates O2 demand during utilization of reserves of poly-beta-hydroxybutyrate in N2-fixing soybean bacteroids.

A liquid reaction medium containing dissolved air and oxyleghaemoglobin, but no energy-yielding substrate, was supplied to bacteroids confined in a stirred flow reaction chamber. The relative oxygenation of the leghaemoglobin in the chamber was determined automatically by spectrophotometry of the effluent solution, and the concentrations of free, dissolved O2 ([O2]) and rates of O2 consumption were calculated. Dissolved CO2 and NH3 from N2 fixation were determined in fractions of the effluent solution. Bacteroids utilized endogenous reserves of poly-beta-hydroxybutyrate (PHB), which were depleted by 9.2% during a typical 5 h-long experiment. Stepwise increases in flow rate (increasing supply of O2) initially produced a drop in O2 demand and resulted in a rise in [O2] and a decline in N2 fixation. Subsequently, O2 demand rose (presumably because of increased mobilization of substrate from PHB) and [O2] declined to a low level. N2 fixation was fully restored, or even enhanced, within 15-20 min of establishment of a new, steady [O2]. This pattern of regulation by O2 supply was completely eliminated by adding low concentrations (20-50 microM) of oxidizable substrate (succinate, malate, ethanol) to the reaction medium. During endogenous activity, rates of CO2 evolution were proportional to, but less than, rates of O2 consumption up to 5.4 nmol O2 min-1 mg-1, above which CO2 evolution exceeded O2 consumption. These and other features of endogenous activity are discussed in relation to sustaining N2 fixation by nodules in vivo.

Aerobic respiration in sulfate-reducing bacteria*

Cultures of Disulfovibrio desulfuricans strain CSN (incubated in a sulfide- and sulfate-free medium) reduced up to 5 mM O2 with H2 as electron donor. Aerobic respiration was not coupled with growth, but resulted in ATP formation. Washed cells incubated in H2-saturated phosphate buffer revealed respiration rates of up to 250 nmol O2 min−1 mg protein−1. The uncoupler carbonyl-cyanide m -chlorophenylhydrazone (CCCP) stimulated the respiration rate and abolished ATP formation. The terminal oxidase has not yet been identified. Respiration was microaerophilic, insensitive to cyanide and azide, but inhibited after heat treatment of the cells (80°C for 10 min). The ph optimum was at pH 6 with less than 50% activity at pH 4.5 and pH 9. Besides H2, organic eletron donors (formate, ethanol, lactate or pyruvate) and inorganic sulfur compounds (H2S, thiosulfate, sulfite) were used as electron donors for aerobic respiration. Sulfite and thiosulfate were oxidized completely to sulfate. The capability of aerobic respiration was also detected in Desulfovibrio vulgaris, D. sulfidismutans, Desulfobacterium autotrophim, Desulfobulbus propionicus , and Desulfococcus multivorans .

OXYGEN CONSUMPTION AND LUMINESCENCE OF ISOLATED Porichthys PHOTOPHORES IN RESPONSE TO ADRENERGIC STIMULATIONS

Abstract—Isolated photophores of Porichthys maintained in saline at 20oC, consume oxygen at a mean rate of 115 ± 5 ± 10 3 nmol O2 min1 (n= 165).In the presence of 0.1 and 1 mM adrenalin or noradrenaline the photophore produces a fast peak of light followed by a slow luminescence. A large increase in oxygen consumption occurs prior to the fast light response and it remains slightly above the resting level during the following slow light emission.When isolated photophores do not produce the fast peak of light(in response to isoprenalin or after treatment with phentolamine), there is only a slight transient increase of oxygen consumption. During the production of the slow luminescence the oxygen consumption remains close to the resting level.A relationship between the oxygen consumption and the light emission during the fast luminescence has been found: it is suggested that most of the oxygen consumed could be involved in a triggering mechanism of light production.

Coupling of active sodium transport to oxidative metabolism in the rabbit distal colon.

1. Active transepithelial Na+ transport (Ji) and O2 consumption (Jr) were measured simultaneously in rabbit distal colon, under standard (control) incubation conditions and after various manoeuvres, known to inhibit Na+ transport. 2. The determination of Jr was complicated by the presence of fluctuations of the PO2 in the incubation solution and by spontaneous variations of the tissue respiration, which usually declined slowly with time. 3. The control values of Ji and Jr after 2 h incubation were 55 +/- 4 nequiv min-1 cm-2 and 16 +/- 1 nmol O2 min-1 cm-2, respectively (n = 44). The electrical resistance was 386 +/- 23 omega cm2; it was stable over 6 h. 4. Ji was reduced to a very low level with either amiloride, ouabain or Na+ substitution with choline. In all instances, Jr decreased concomitantly by 15-30%. 5. A plot of the change in Jr versus the change in Ji gave a straight line for all situations, i.e. for the spontaneous decline of Na+ transport and respiration and for the effects of the inhibitors. 6. The linearity between Jr and Ji allows for the determination of a stoichiometric ratio. It is of similar magnitude, when calculated either with the data of spontaneous variations or with those obtained by the action of any inhibitor tested. It is 15-20 Na+ ions per O2 molecule, a value close to that reported previously for amphibian epithelia and also close to the maximum theoretical value of 18 Na+ ions per O2 molecule.