Protein synthesis and oxygen consumption in fish cells

Oxygen consumption and protein synthesis were measured concurently in four fish cell types: BF-2 and TRG-2 cell lines, rainbow trout macrophages and scale cells. The fractional rates of protein synthesis (percentage of the protein mass synthesised per day) were ranked: BF-2 cells > macrophages > RTG-2 cells > scale cells. Oxygen consumption rates were ranked BF-2 cells = macrophages = RTG-2 cells > scale cells. Within three of the cell types (BF-2, RTG-2 and scale cells) oxygen consumption and protein synthesis were linearly correlated, whereas comparison between the four cell types gave rise to an exponential relationship between fractional rates of protein synthesis and oxygen consumption. Inhibition of protein synthesis with cycloheximide by 41–65% resulted in a 62–89% reduction in oxygen consumption depending on cell type. Calculations of the aerobic cost of protein synthesis using the cycloheximide-sensitive protein synthesis and oxygen consumption rates resulted in estimates ranging from 11 to 217 mol O₂·mg protein^-1 synthesised depending on the cell type. The lowest net protein synthesis costs, which are close to theoretical values for peptide bond formation, were associated with the most rapid rates of protein synthesis.Abbreviations ANOV A analysis of variance - BDH British Drug Housing - BOC British Oxygen Company - ADT A ethylenediaminetetra-acetic acid - FCS foetal calf serum - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethane-sulphonic acid] - LCD least square differnece - MEM munimum essential media - PBS phosphate-buffered saline - PCA perchloric acid - SIS spectral index of the sample - tSIE transformed spectral index of the external standard spectrum     

Interaction between aflatoxin B1 and oxytetracycline in isolated rat hepatocytes

Isolated rat hepatocytes were used as an in vitro model to investigate A possible interaction between oxytetracycline (OXT) and aflatoxin B₁ (AFB₁). LDH leakage, RNA and protein synthesis and glycogen accumulation were measured in the presence of both drugs, either separately or in combination. The evolution of LDH leakage during the incubation was identical in untreated and treated cells. AFB₁ inhibited RNA and protein synthesis at a concentration of 10^–7 M and 10^–6 M, respectively, and higher, whereas OXT did not influence RNA synthesis but inhibited protein synthesis at the highest tested concentration, 10^–3 M. As far as glycogen is concerned, rats were injected with glucagon before sacrifice in order to obtain a constant synthesis rate in isolated hepatocytes. AFB₁ inhibited the accumulation of glycogen from 10^–6 M upward. This effect was never observed before 90 min of incubation. OXT had no effect on glycogen synthesis. In the presence of both drugs, no interaction was demonstrated as far as RNA and protein synthesis were concerned, but OXT opposed the inhibition induced by AFB₁ on glycogen accumulation. If the in vivo protection, provided by OXT against AFBI-induced toxicity, is due to a direct interference in the toxic mechanisms of the mycotoxin, these results show that OXT does not influence the AFB₁-inhibition of RNA and protein synthesis. The latter are early and sensitive parameters inhibited by AFB₁. On the contrary, taking into consideration the results on glycogen accumulation, it seems more interesting to investigate further this metabolism.Abbreviations AFB₁ Aflatoxin B₁ - OXT Oxytetracycline - DMEM Dulbecco's Modified Eagle's Medium - LDH Lactate Dehydrogenase - DMSO Dimethyl Sulfoxide - BSA Bovine Serum Albumin     

Oxygen transport and cardiovascular responses in skipjack tuna (Katsuwonus pelamis) and yellowfin tuna (Thunnus albacares) exposed to acute hypoxia

Summary Responses to acute hypoxia were measured in skipjack tuna (Katsuwonus pelamis) and yellowfin tuna (Thunnus albacares) (1–3 kg body weight). Fish were prevented from making swimming movements by a spinal injection of lidocaine and were placed in front of a seawater delivery pipe to provide ram ventilation of the gills. Fish could set their own ventilation volumes by adjusting mouth gape. Heart rate, dorsal and ventral aortic blood pressures, and cardiac output were continuously monitored during normoxia (inhalant water (PO ₂>150 mmHg) and three levels of hypoxia (inhalant water PO ₂130, 90, and 50 mmHg). Water and blood samples were taken for oxygen measurements in fluids afferent and efferent to the gills. From these data, various measures of the effectiveness of oxygen transfer, and branchial and systemic vascular resistance were calculated. Despite high ventilation volumes (4–71·min^-1·kg^-1), tunas extract approximately 50% of the oxygen from the inhalant water, in part because high cardiac outputs (115–132 ml·min^-1·kg^-1) result in ventilation/perfusion conductance ratios (0.75–1.1) close to the theoretically ideal value of 1.0. Therefore, tunas have oxygen transfer factors (ml O₂·min^-1·mmHg^-1·kg^-1) that are 10–50 times greater than those of other fishes. The efficiency of oxygen transfer from water in tunas (65%) matches that measured in teleosts with ventilation volumes and order of magnitude lower. The high oxygen transfer factors of tunas are made possible, in part, by a large gill surface area; however, this appears to carry a considerable osmoregulatory cost as the metabolic rate of gills may account for up 70% of the total metabolism in spinally blocked (i.e., non-swimming) fish. During hypoxia, skipjack and yellowfin tunas show a decrease in heart rate and increase in ventilation volume, as do other teleosts. However, in tunas hypoxic bradycardia is not accompanied by equivalent increases, in stroke volume, and cardiac output falls as HR decreases. In both tuna species, oxygen consumption eventually must be maintained by drawing on substantial venous oxygen reserves. This occurs at a higher inhalant water PO₂ (between 130 and 90 mmHg) in skipjack tuna than in yellowfin tuna (between 90 and 50 mmHg). The need to draw on venous oxygen reserves would make it difficult to meet the oxygen demand of increasing swimming speed, which is a common response to hypoxia in both species. Because yellowfin tuna can maintain oxygen consumption at a seawater oxygen tension of 90 mmHg without drawing on venous oxygen reserves, they could probably survive for extended periods at this level of hypoxia.Abbreviations BP_da, BP_va dorsal, ventral aortic blood pressure - C _aO₂, C _vO₂ oxygen content of arterial, venous blood - DO₂ diffusion capacity - E_b, E_w effectiveness of O₂ uptake by blood, and from water, respectively - Hct hematocrit - HR heart rate - PCO₂ carbon dioxide tension - P _aCO₂, P _vCO₂ carbon dioxide tension of arterial and venous blood, respectively - PO₂ oxygen tension - P _aO₂, P _vO₂, P _iO₂, P _cO₂ oxygen tension of arterial blood, venous blood, and inspired and expired water, respectively - pHa, pHv pH of arterial and venous blood, respectively - P_w—b effective water to blood oxygen partial pressure difference - Pg partial pressure (tension) gradient - cardiac output - R vascular resistance - SV stroke volume - SEM standard error of mean - TO₂ transfer factor - U utilization - _g ventilation volume - O₂ oxygen consumption     

Myocardial oxygen consumption and mechanical efficiency of a perfused dogfish heart preparation

Summary Oxygen consumption of an in-pericardium heart preparation from the spiny dogfish (Squalus acanthias) was linearly related to cardiac power output. Basal oxygen consumption, predicted from the regression, was 0.127 l · s^-1 · g ventricle mass^-1 and increased by 0.189 l · s^-1 · g ventricle mass^-1 per milliwatt of power generated. From the relationship between cardiac power output and mechanical efficiency, mechanical efficiency was predicted to increase with cardiac power output to a maximum of 21 %. Mechanical efficiency was measured during volume loading and pressure loading at two power outputs (50% and 72% of maximum power output). At 50% of maximum power output, mechanical efficiency increased significantly by 2.87%, from 11.9±0.3% to 14.8±0.5% (n=7), when flow was halved and output pressure doubled to achieve the same power output. Similarly, at 72% of maximum power output, mechanical efficiency increased from 14.74±0.92% to 17.61±0.84% (n=6) when flow was halved and output pressure doubled to generate the same higher level of power output. The increased mechanical efficiency at higher output pressures is believed to result from cardiac myocytes working within a length range where they are able to generate the most tension during contraction and are most efficient. We speculate that the loss of mechanical efficiency associated with large changes in sarcomere length, when stroke volume is large, is a driving force behind the use of frequency as the principal means of increasing cardiac output as observed in more active fishes, birds and mammals.Abbreviations BM body mass - CO cardiac output - HR heart rate - P _i mean cardiac input pressure - P _o mean cardiac output pressure - PO partial pressure of oxygen - SV stroke volume of heart - VM ventricle mass     

Purification and characterization of a sialidase fromClostridium chauvoei NC08596

The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg^–1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn²⁺, Mg²⁺, and Ca²⁺ and bovine serum albumin have a positive effect on the sialidase activity, while Hg²⁺, Cu²⁺, and Zn²⁺, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.Abbreviations FPLC fast protein liquid chromatography - NCTC National Collection of Type Cultures - ATCC American Type Culture Collection - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - buffer A 0.02m piperazine, 0.01m CaCl₂, pH 5.5 - buffer B 0.02m piperazine, 0.01m CaCl₂, 1.0m NaCl, pH 5.5 - buffer C 0.1m sodium acetate, 0.01m CaCl₂, pH 5.5 - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - Neu5Ac N-acetylneuraminic acid - BSM bovine submandibular gland mucin - GD1a IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer - GM1 II³Neu5Ac-GgOse₄Cer - MU-Neu4,5Ac₂ 4-methylumbelliferyl--d-N-acetyl-4-O-acetylneuraminic acid - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol bis(2-aminoethyl-ethen)-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - IEF isoelectric focusing - IEP isoelectric point     

Ammonium uptake by cowpea Rhizobium strain MNF 2030 and Rhizobium trifolii MNF 1001

Free-living Rhizobium trifolii MNF 1001 and cowpea Rhizobium MNF 2030 grown in chemostat culture under nitrogen limitation had high activities of an ammonium permease. In phosphate-limited, nitrogen-excess conditions, strains MNF 1001 and MNF 2030 retained 20% and 50%, respectively, of the ammonium uptake activity found in nitrogen-limited cells. Uptake in both strains was sensitive to azide, cyanide, carbonyl cyanide m-chlorophenyl hydrazone and 2,4-dinitrophenol. A gradient of ammonium concentration greater than 150-fold developed across the membrane within 20 min in cells of strain MNF 1001 grown under ammonia limitation. The pH optimum for ammonium uptake by N-limited cells of both MNF 1001 and MNF 2030 was around pH 7. The apparent K _m values for the ammonium permease in strains MNF 2030 and MNF 1001 were 3.9±1.6 M and 2.0±1.6 M respectively, and the V _max was 47±2.6 nmol min^-1 (mg protein)^-1 for MNF 2030 and 101±5.1 nmol min^-1 (mg protein)^-1 for MNF 1001. Isolated snake bean bacteroids of strain MNF 2030 capable of transporting succinate and l-glutamate had no detectable ammonium uptake activity. It therefore appears that the ammonium permeases in cells of these two strains are not as tightly regulated as in R. leguminosarum MNF 3841.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrzone - HEPES N-Hydroxyethylpiperazine-N-2-ethanesulphonic acid     

On the use of Avena protoplasts to study chloroplast development

Different methods were tested to isolate protoplasts from etiolated, partially greened, and light-grown leaves of Avena sativa. Preparations with high yields and high photosynthetic capacities (time of illumination 4 h) were obtained when small transverse leaf segments were incubated for 2 h at 30°C in 2% cellulysin (Calbiochem), 0.6 M mannitol, and 0.5% bovine serum albumin (BSA) at pH 5.6, without shaking. As measured by light-dependent O₂ evolution or fixation of labeled bicarbonate, protoplasts exhibited rates of up to 124 mol per mg of chlorophyll per h at 20°C and saturating bicarbonate, which were nearly identical to those found with intact leaves. The assay conditions necessary for this activity were 0.6 M sorbitol, 50 mM N-2-hydroxy-ethylpiperazine-N-2-ethane sulfonic acid (pH 7.6), and 10 mM NaHCO₃. If plastids were isolated from these protoplasts, sorbitol was 0.45 M, including 10 mM ethylenediaminetetraacetate (EDTA). under these conditions, rates of photosynthesis were up to 125 (light-grown) and 71 (6 h illuminated) mol O₂ evolved or ¹⁴CO₂ fixed per mg of chlorophyll per h, compared to 3.5 mol·mg chl^-1·h^-1 obtained with mechanically isolated plastids. With this system, CO₂-dependent O₂ evolution was already detected after 3 h of illumination of etiolated tissue, but could only be observed at pH values between 7.6 and 8.6, in the presence of EDTA. At lower pH (7.3) or at pH 7.6 in the absence of EDTA, light-dependent O₂ evolution up to 24 h of greening was only measurable with 3-phosphoglycerate as the substrate. The possible effects of EDTA in this respect as well as the advantages of using protoplasts or plastids isolated from protoplasts for developmental studies are discussed.Abbreviations BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane-sulphonic acid - MES 2(N-morpholino) ethane sulphonic acid - PGA 3-phosphoglycerate     

Physiological energetics of a midge,Chironomus riparius Meigen (Insecta,Diptera): normoxic heat output over the whole life cycle and response of larva to hypoxia and anoxia

The rate of metabolism of laboratory reared Chironomus riparius was monitored by direct calorimetry over the entire life cycle from egg to adult stage. The metabolic response of the fourth instar larva to decreasing oxygen concentrations and anoxia was also measured. Normoxic measurements were carried out at 20°C and the hypoxic-anoxic experiments at 10°C. In larvae with body sizes ranging from 0.0028 to 0.645 mg ash-free dry mass (afdm), the rate of heat dissipation was related to body mass by a power function, with a mass exponent of 0.71±0.02 corresponding to an exponent of -0.29 for the relationship between mass-specific metabolic rate and body mass. However, the allometric equations applicable to larvae would not predict the metabolic rates of eggs, pupae and adults. Single egg batches used in the experiments consisted of 354±90 eggs, the individual egg with a mass of 0.99±0.01 g (mean±SD). The mass-specific rate of heat dissipation of the egg (13.7±1.8 W mg^-1 afdm) was considerably lower than that of the first and second instar larvae (44–53 W mg^-1) but equal to that of fourth instar larvae (13.1±3.9 W mg^-1). Heat dissipation by a pupa shortly before adult emergence was high (14.8±1.8 W mg^-1), probably due to high metabolism during metamorphosis. Emergence of the adult in the calorimeter was indicated by a short but intense burst of heat. The newly emerged imago had a ca. 20–35% higher metabolic rate than the pupa. In response to reduced O₂ partial pressure the fourth instar larva of C. riparius displayed metabolic regulation. In continuously declining oxygen partial pressure, the fourth instar larva maintained its aerobic energy metabolism (4.2 W mg^-1) with only a small decrease down to 0.8 kPa, corresponding to an oxygen concentration of 0.42 mg O₂l^-1 H₂O. Below this critical oxygen concentration (P_c), the rate of heat dissipation decreased rapidly down to the anoxic level which was only 14–17% of the normoxic level. The high relative reduction of metabolic rate under anoxia gives a wrong impression of short-term tolerance of C. riparius to anoxia. The absolute energetic costs of C. riparius associated with anaerobic energy metabolism (0.64±0.11 W mg^-1) are almost 6 times higher than those of more anoxia tolerant invertebrates such as sphaeriid bivalves.     

Hormonal regulation of temperature acclimation in catfish hepatocytes

Summary Protein synthesis (measured by ³H-leucine incorporation) by catfish hepatocytes in culture was enhanced when trace amounts of catfish serum were added. Serum from 15°C-acclimated fish was significantly more effective than serum from 25°C-acclimated fish.Total protein content of the cells was slightly diminished; DNA content was not altered.Added triiodothyronine (T₃) significantly reduced protein synthesis by cultured hepatocytes, more at 25°C than at 15°C culture. Threshold concentration of T₃ was 10^–9 M.Removal of T₃ from serum by exchange resin resulted in increased protein synthesis. Addition of T₃ to that preparation decreased protein synthesis.The concentration of T₃ in serum from 25°C-acclimated catfish is three times greater than the concentration in serum from 15°C-acclimated fish.Increase in protein synthesis after removal of T₃ suggests that there is a blood-borne stimulating factor, more active in cold- than in warm-acclimated fish. The stimulating substance was present after dialysis (2000 Da cutoff) and was partially inactivated by heat.Insulin stimulated protein synthesis; salmon insulin was more effective than bovine insulin. Insulin content did not differ in serum from 15°C- and 25°C-fish.The effects of growth hormone and prolactin were equivocal or negative.The inhibitory effect of T₃ may explain the reduction in metabolism during warm-acclimation. The nature of a stimulating hormone in cold acclimation is unknown.Abbreviations DNA desoxyribonucleic acid - DPM desintegrations per minute - GH growth hormone - HPLC high performance liquid chromatography - LDH lactate dehydrogenase - MEM minimal essential medium - PBS phosphate buffered saline - POPOP 1,4-bis [5-phenyl-2-oxazolyl]benzene 2,2-p-phenylene-bis[5-phenyloxazole] - PPO 2,5-diphenyloxazole - RIA radioimmunoassay - TCA trichloroacetic acid - T ₃ 3,5,5-triiodothyronine - T ₄ thyroxine - VO ₂ oxygen consumption     

Formal kinetics of poly-β-hydroxybutyric acid (PHB) production in Alcaligenes eutrophus H 16 and Mycoplana rubra R 14 with respect to the dissolved oxygen tension in ammonium-limited batch cultures

Summary Under chemolithoautotrophic growth conditions with the organism Alcaligenes eutrophus H16 the exponential growth phase is characterized by two different growth rates, each associated with different specific rates of ammonium consumption. On the basis of the analytical determination of Poly--hydroxybutyric acid (PHB), it can be conclusively shown that PHB is synthesized even during the exponential growth phase at a specific rate proportional to the specific growth rates of total biomass. After complete consumption of ammonium, the increase of biomass is exclusively due to PHB synthesis, whereas protein and rest biomass (cell dry weight minus PHB) remain constant. After an extended period of fermentation, the PHB content reaches a saturation value. The transient phase between the growth and the storage phase is very short in comparison to the duration of the whole fermentation. In the case of Alcaligenes eutrophus, strain H 16, high concentrations of dissolved oxygen strongly influence growth as well as PHB synthesis.Abbrevations ^cO₂,L concentration of oxygen in the liquid phase (dissolved oxygen tension: d.o.t) - ^cH₂,L concentration of hydrogen in the liquid phase - ^cCO₂,L concentration of carbon dioxide in the liquid phase - S limiting substrate, concentration of - X total biomass, concentration of; total cell dry weight - P product; PHB, concentration of - R rest biomass: X-P, concentration of - ^rX dX/dt growth rate - ^rP dP/dt rate of PHB synthesis - ^rR dR/dt rate of rest biomass production - ^r0 ^dcO₂,L/dt rate of oxygen consumption - X dX/dt·1/X=r_X·1/X specific growth rate - P dP/dt·1/P=r_P·1/P specific rate of product formation - R dR/dt·1/R=r_R·1/R specific rate of rest biomass formation - r₀/R specific respiration rate     

Kinetics of mitochondrial phosphate transport and rates of respiration and phosphorylation during greening of etiolated Avena leaves

Using the technique of silicone oil filtration of organelles and the inhibitor stop method, the kinetics of transport of inorganic phosphate across the inner mitochondrial membrane were tested in relation to different stages of greening (0 to 24 h) of etiolated laminae of Avena sativa L., and compared to the rates of oxygen consumption and ATP formation. The results demonstrate that there is a pronounced increase in phosphate transport after 3 h of greening, reaching values for V_max (about 17 mol mg protein^-1 h^-1) that are three times as high as those measured with mitochondria from etiolated tissue. This is also mirrored by the rates of respiration and oxidative phosphorylation. After 24 h of light treatment (4 Klx), respiration and ATP formation, as well as V decreased again to levels below those of the etiolated stage. In contrast to V, there was no change in the affinity between inorganic phosphate and the binding sites of the transporting systems involved, as indicated by a rather constant K_m (0.23 mM) for phosphate transport. Of the inhibitors of phosphate transport tested, mersalyl and methyl mercuric iodide were most efficient with identical characteristics of inhibition; but compared to animal mitochondria, the concentrations needed to result in similar amounts of inhibition, were more than ten times higher. The results are discussed with respect to plastid development.Abbreviations BSA bovine serum albumine - CH₃HgJ methyl mercuric iodide - Cyt cytochrome - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - MDH malate dehydrogenase - NEM N-ethylmaleimide     

The effect of short-term fasting and a single meal on protein synthesis and oxygen consumption in cod,Gadus morhua

Summary Rates of protein synthesis and oxygen consumption ( O₂) in cod were compared in both fasted and refed animals. During a 14-day fast both protein synthesis and respiration rates fell to stable values after 6 days. When a meal of whole sandeel at 6% body weight was fed to fish fasted for 6 days, protein synthesis and ( O₂) increased to a maximum at between 12 and 18 h after feeding. Peak ( O₂) was about twice the pre-feeding values, while whole animal protein synthesis increased four-fold. There were differences between tissues in the timing of maximum protein synthesis; the liver and stomach responded faster than the remainder of the body. Maximum protein synthesis rates in the liver and stomach occurred at 6 h after feeding, at which time their calculated contribution to total ( O₂) was 11%. Similar calculations suggested that the integrated increment in whole animal protein synthesis contributed between 23% and 44% of the post-prandial increase in ( O₂). It was concluded that protein synthesis is an important contributor to increased ( O₂) after feeding in cod.Abbreviations A _s absolute rate of protein synthesis - ASDA apparent specific dynamic action - ATP adenosine triphosphate - k _s fractional rate of protein synthesis - k _s/RNA amount of protein synthesized per unit RNA - ( O₂) oxygen consumption - PCA perchloric acid - RNA ribonucleic acid     

Interaction between new neoglycoproteins and thed-Man/l-fuc receptor of rabbit alveolar macrophages

New types of neoglycoproteins, -caseins coupled with ovalbumin-derived asparagine oligosaccharides (AO), aspartate aminotransferase-phosphopyridoxylated AO complex (AAT-PG), and streptavidin-biotinylated AO complex (SA-BAO), were tested for their inhibitory effect on binding of bovine serum albumin derivatized with thiomannoside, Man-AI-BSA [Lee YC, Stowell CP, Krantz MJ (1976) Biochemistry 15:3956–63] by rabbit alveolar macrophages. The -casein derivatives and the AAT-PG complex increased binding affinity as the number of oligosaccharide chains attached was increased. Their inhibitory potencies were closely related to those of the Man-Al-BSA derivatives [Hoppe CA, Lee YC (1983) J Biol Chem 258:14193–99] on the basis of terminal mannose density. The SA-BAO complex containing three AO chains gave stronger inhibitory potency than the -casein derivative with three AO residues, suggesting that proper orientation of the oligosaccharides on the protein can affect the receptor-ligand interaction.Abbreviations BSA bovine serum albumin - Man₄₃-BSA BSA derivative containing on average 43 residues of Man linked through an amidino-linkage [7] - AO asparagine oligosaccharide (Man₅-GlcNAc₂-Asn) from ovalbumin - AAT aspartate aminotransferase - PG phosphopyridoxylated AO - SA streptavidin - BAO biotinylated AO     

Effects induced by rotenone during aerobic growth ofParacoccus denitrificans in continuous culture

Paracoccus denitrificans was grown aerobically in chemostat culture in the presence of rotenone. After 6 to 10 generation times, cells showed an oxygen uptake which was completely rotenone-insensitive after removal of rotenone by washing with bovine serum albumin containing medium.The H⁺/O ratio of these cells for endogenous substrates decreased from about 7.50 to 3.95. The latter ratio was similar to the value obtained for starved cells oxidizing exogenous succinate, indicating that site I phosphorylation was absent in these rotenone-insensitive cells.Membrane particles prepared from these cells showed an 80% decrease in activity of reduced nicotinamide adenine dinucleotide oxidase and reduced nicotinamide adenine dinucleotide-ferricyanide oxidoreductase, while also the kinetic behaviour of the reduced nicotinamide adenine dinucleotide dehydrogenase in the reduced nicotinamide adenine dinucleotide-ferricyanide oxidoreductase assay was changed. Moreover the reduced nicotinamide adenine dinucleotide oxidase activity was practically rotenone-insensitive.Electron paramagnetic resonance spectroscopy on membrane particles from rotenone-insensitive cells at 15 K revealed that the resonance lines atg _z 2.05 andg _y g _x 1.92 arising from iron-sulfur center 2 were undetectable. The intensities of the other electron paramagnetic resonance signals originating from reduced nicotinamide adenine dinucleotide dehydrogenase linked iron-sulfur centers were only slightly diminished.These observations confirm our previous suggestion that site I phosphorylation, rotenone sensitivity and the presence of iron-sulfur center 2 are correlated.Abbreviations EPR electron paramagnetic resonance - BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophenylhydrazone - NAD nicotinamide adenine dinucleotide - NADP nicotinamide adenine dinucleotide phosphate - ATP adenosine triphosphate     

Isolation and characterization of the receptor for vitellogenin from follicles of the rainbow trout,Oncorhynchus mykiss

The isolation and characterization of the receptor for vitellogenin from follicle membranes of the rainbow trout, Oncorhynchus mykiss, is described. Follicle membrane proteins subjected to SDS-polyacrylamide gel electrophoresis and subsequently to either protein staining or ligand blotting with radiolabelled vitellogenin (¹²⁵iodine-vitellogenin) demonstrated that the vitellogenin receptor has an apparent molecular mass of 200 kD (probably comprising of two 100-kD subunits) under non-reducing conditions. The vitellogenin binding sites were identified as specific receptors: binding was saturable and the binding sites were both tissue specific to follicle membranes and exhibited ligand specificity. Scatchard analyses of specific binding data revealed a single class of binding sites with a high affinity for rainbow trout vitellogenin (K _d=8.2·10^-9 mol·1^-1). Both brown trout, Salmo trutta, vitellogenin and carp, Cyprinus carpio, vitellogenin were able to displace the radiolabelled rainbow trout vitellogenin from its receptor, although they were less effective than rainbow trout vitellogenin.Abbreviations B _max maximum number of binding sites available - BSA bovine serum albumin - bt-VTG brown trout vitellogenin - c-VTG earp vitellogenin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - K _d dissociatian constant - NCM nitrocellulose membranes - PMSF phenylmethylsulphonylfluoride - rt-VTG rainbow trout vitellogenin - VTG vitellogenin     

Glutamate dehydrogenase from Pisum sativum L.

A 2–8-fold increase in the activity of glutamate dehydrogenase (GDH), accompanied by an alteration of the GDH isoenzyme pattern, was observed in detached pea shoots floated on tap water (preincubated shoots). Sugars supressed the process, whereas NH ₊ ⁴ and various metabolites as well as inhibitors of energy metabolism and protein synthesis were ineffective. The subcellular distribution pattern revealed evidence that the GDH isoenzymes are exclusively located in the mitochondrial matrix. The alterations in GDH activity occurring in preincubated shoots are restricted to the mitochondria.An experimental device suitable for studying the GDH function in isolated intact mitochondria has been established. Using [¹⁴C] citrate as the carbon source and hydrogen donor, the mitochondria synthesized considerable amounts of glutamate upon addition of NH ₊ ⁴ . The rates of glutamate formation in dependency of increasing NH ₊ ⁴ levels follow simple Michaelis-Menten kinetics. Half-saturation concentrations of NH ₊ ⁴ of 3.6±1.2 mM; 1.9±0.06 mM and 1.6±0.1 mM were calculated for the mitochondria isolated from pea shoots, roots, and preincubated shoots, respectively. The results are discussed in relation to the possible role of GDH in NH_+/4 assimilation at elevated intracellular NH_+/4 levels.Abbreviations GDH Glutamate dehydrogenase - MDH malate dehydrogenase - GOT aspartate aminotransferase - SDH succinate dehydrogenase - HEPES 4-(2-hydroxyethyl)-1-piperazineethan-sulfonic acid - BSA bovine serum albumin - TPP thiamine pyrophosphate - DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCPIP 2,6-dichlorophenolindophenol Dedicated to Professor Dr. Maximilian Steiner on the occasion of his 75th birthday     

Dependence of oxygen uptake on ambient PO 2 in isolated perfused frog skin

Summary Rates of O₂ uptake across isolated perfused skin of bullfrogs (Rana catesbeiana) were measured in relation to blood flow at three levels of ambient O₂ tension: normoxia (O₂ tension=152 torr), hypoxia (12% O₂, 87 torr) and hyperoxia (42% O₂, 306 torr). At bulk perfusion rates ranging from 3.4 to 10.1 l·cm^-2·min^-1, O₂ uptake was positively correlated with hemoglobin delivery rate in both normoxia and hyperoxia, but was independent of delivery rate in hypoxia. Mean O₂ uptake in normoxia was 3.8 nmol O₂·cm^-2·min^-1 at a delivery rate of 9.8 nmol·cm^-2·min^-1 and 6.5 nmol O₂·cm^-2·min^-1 at a delivery rate of 28.3 nmol·cm^-2·min^-1. At any given bulk perfusion rate, oxygen uptake averaged about 49% lower in hypoxia than in normoxia, decreasing in proportion to the reduction of O₂ tension difference between medium and blood. In hyperoxia, O₂ uptake did not increase proportionally with the difference in O₂ tension between blood and medium, averaging only 50% higher at a 2.4-fold greater O₂ tension difference. Cutaneous diffusing capacity for O₂ averaged 0.041 nmol O₂·cm^-2·torr^-1·min^-1 during the first hour of perfusion in normoxia, and was not affected by reduction of ambient O₂ tension. The results indicate that cutaneous O₂ uptake in hypoxia is highly diffusion limited, and consequently, increases in cutaneous perfusion can not effectively compensate for reduction of ambient O₂ tension. In hyperoxia, O₂ uptake may be substantially perfusion limited because of reduced blood O₂ capacitance at high O₂ saturations.Abbreviations O₂ capacitance - C _Hb hemoglobin concentration - D diffusing capacity - PO₂ medium-blood PO₂ difference - Hb flow, hemoglobin delivery rate - Hepes N-[2-Hydroxyethyl]piperacine-N-[2 ethanesulfonic acid] - L _diff extent of diffusion limitation - MO₂ oxygen uptake rate - PO₂ oxygen tension - S O₂ saturation     

Partial purification and characterization of a soluble protein phosphatase fromLeishmania donovani promastigotes

A soluble protein phosphatase from the promastigote form of the parasitic protozoanLeishmania donovani was partially purified using Sephadex G-100, DEAE-cellulose and again Sephadex G-100 columns. The partially purified enzyme showed a native molecular weight of about 42, 000 in both Sephadex G-100 and sucrose density gradient centrifugation. The sedimentation constant, stokes radius and frictional ratio were found to be 3.43S, 2.8 nm and 1.20 respectively. The enzyme preferentially utilized phosphohistone as the best exogenous substrate. Mg²⁺ ions were essential for enzyme activity; among other metal ions Mn²⁺ can replace Mg²⁺ to a certain extent whereas Ca²⁺, Co²⁺ and Zn²⁺ could not substitute for Mg²⁺. The pH optimum of the enzyme was 6.5–7.5 and the temperature optimum 37°C. The apparent Km for phosphohistone was 7.14 M. ATP, ADP, inorganic phosphate and pyrophosphate had inhibitory effect on the enzyme activity whereas no inhibition was observed with sodium tartrate and okadaic acid. These results suggest thatL. donovani promastigotes possess a protein phosphatase which has similar characteristics with the mammalian protein phosphatase 2C.Abbreviations PMSF phenylmethylsulfonyl fluoride - DTT dithiothreitol - TCA trichloroacetic acid - BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - EGTA Ethyleneglycol-bis-(-aminoethyl ether) N,N,N,N-tetraacetic acid     

Effect of palmitoylcarnitine on mitochondrial activities

Mitochondria from pea (Pisum sativum L.) cotyledons and potato (Solanum tuberosum L.) tubers exhibited a palmitoyl carnitine-dependent, KCN-sensitive stimulation of the oxygen uptake measured in the presence of 0.2mmol·^–1 malate (sparker malate), provided a certain concentration range of palmitoylcarnitine was observed. Above this concentration range, which was dependent on the bovine serum albumin (BSA) concentration of the reaction mixture, the mitochondrial oxygen uptake was inhibited by palmitoylcarnitine. Palmitoylcarnitine (racemate) and palmitoyl-l-carnitine were equally effective in stimulating/inhibiting mitochondrial oxygen uptake in the presence of sparker malate. The mitochondrial membrane potential generated in the presence of sparker malate was partially dissipated by palmitoyl-lcarnitine concentrations stimulating the mitochondrial oxygen uptake. The formation of acid-soluble radioactivity in reaction mixtures provided with [1-¹⁴C]palmitoyll-carnitine was considerably lower than that expected minimally if the palmitoyl-l-carnitine-stimulated oxygen uptake resulted from palmitoyl-l-carnitine oxidation sparked by malate. Palmitoylcarnitine concentrations resulting in stimulation of the mitochondrial oxygen uptake in the presence of sparker malate also led to a stimulation of succinate-cytochrome c reductase activity, as well as to an increase in the measurable activities of mitochondrial matrix enzymes, indicating loss of both mitochondrial integrity and mitochondrial enzyme latency in the presence of palmitoylcarnitine. Correspondingly, malate-dependent NADH formation was stimulated by palmitoylcarnitine. Neither NAD reduction nor oxygen uptake were observed when the mitochondria were provided with palmitoylcarnitine only. The oxygen uptake due to glycine oxidation by mitochondria from green sunflower (Helianthus annuus L.) cotyledons was affected by palmitoylcarnitine in a similar manner to the oxygen uptake of pea cotyledon and potato tuber mitochondria in the presence of sparker malate. The results lead to the conclusion that the palmitoylcarnitine-dependent stimulation of mitochondrial oxygen uptake observed in the presence of sparker malate results substantially from an enhanced malate oxidation due to the detergent effect of palmitoylcarnitine on the mitochondrial membranes, rather than from palmitoylcarnitine -oxidation.Abbreviations BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophyenylhydrazone The work was supported by the Deutsche Forschungsgemeinschaft.     

Competition for limiting amounts of oxygen between Nitrosomonas europaea and Nitrobacter winogradskyi grown in mixed continuous cultures

Chemolithotrophic nitrifying bacteria are dependent on the presence of oxygen for the oxidation of ammonium via nitrite to nitrate. The success of nitrification in oxygen-limited environments such as waterlogged soils, will largely depend on the oxygen sequestering abilities of both ammonium- and nitrite-oxidizing bacteria. In this paper the oxygen consumption kinetics of Nitrosomonas europaea and Nitrobacter winogradskyi serotype agilis were determined with cells grown in mixed culture in chemostats at different growth rates and oxygen tensions.Reduction of oxygen tension in the culture repressed the oxidation of nitrite before the oxidation of ammonium was affected and hence nitrite accumulated. K _m values found were within the range of 1–15 and 22–166 M O₂ for the ammonium- and nitrite-oxidizing cells, respectively, always with the lowest values for the N. europaea cells. Reduction of the oxygen tension in the culture lowered the half saturation constant K _m for oxygen of both species. On the other hand, the maximal oxygen consumption rates were reduced at lower oxygen levels especially at 0 kPa. The specific affinity for oxygen indicated by the V _max/K _m ratio, was higher for cells of N. europaea than for N. winogradskyi under all conditions studied. Possible consequences of the observed differences in specific affinities for oxygen of ammonium-and nitrite-oxidizing bacteria are discussed with respect to the behaviour of these organisms in oxygen-limited environments.