Μmol ADP Research Articles

Assessing seasonal and biological indices of juvenile Chinook Salmon for freshwater decision triggers that increase ocean survival

Decision triggers, used in adaptive management frameworks to decide when a specific management action will be implemented, are often informed by monitoring data. The identification and application of decision triggers is highly relevant to endangered fishes migrating through regulated rivers, as examined in the current study. The main goal was to determine whether seasonal patterns of behavioral, physical, and physiological indices of juveniles were related to subsequent smolt-to-adult return (SAR) survival and, if so, to determine whether these indices could be used to guide decisions related to the mitigation strategy of the juvenile fish transportation program in the Federal Columbia River Power System (Pacific Northwest, USA). Hatchery yearling Chinook Salmon (Oncorhynchus tshawytscha Walbaum in Artedi, 1792) were collected over the migration season at 3 dams in the hydrosystem and measured for fork length, wet mass, Fulton’s K (or condition factor), Na+/K+-ATPase (NKA) activity (or smoltification index), and % dry mass (or index of energetic reserves and smoltification). We estimated SAR survival from passive integrated transponder-tagged fish representative of our field samples and assessed its relationship to our fish indices, as well as indices of transported vs run-of-river passage and distance of sampling site to ocean. SAR survival was associated to interaction effects between juvenile fish transportation and % dry mass or NKA activity. Transported hatchery Chinook Salmon with dry mass <23% of whole fish wet mass and NKA activity >7 µmol ADP mg protein−1 h−1 showed greater SAR survival than their run-of-river counterparts. Fish with the highest predicted SAR survival had been transported and had fish indices consistent with smolts that were more developed (i.e., lower % dry mass and higher NKA activity). Furthermore, our results on % dry mass provided support for the hypothesis that greater lipid content increases fish buoyancy leading to greater susceptibility to predation. The buoyancy effect is expected to be greatest in hatchery fish. Overall, this study shows that decision triggers based on biological indices of migrating fish are potentially useful tools for in-season management.

Abiotic phosphorus recycling from adsorbed ribonucleotides on a ferrihydrite-type mineral: Probing solution and surface species

Iron (Fe) (oxyhydr)oxide minerals, which are amongst most reactive minerals in soils and sediments, are known to exhibit strong adsorption of inorganic phosphate (Pi) and organophosphate (Po) compounds. Beyond synthetic Po compounds, much still remains unknown about the reactivity of these minerals to transform naturally-occurring Po compounds to Pi, particularly with respect to solution versus surface speciation of Po hydrolysis. To investigate this reactivity with a ferrihydrite-type mineral and ribonucleotides, we employed high-resolution liquid chromatography-mass spectrometry (LC-MS), X-ray absorption near-edge structure (XANES), Fourier-transform infrared (FTIR) spectroscopy, and molecular modeling. Kinetic experiments were conducted with the mineral (1 g L−1) reacted with adenosine monophosphate, diphosphate, or triphosphate (respectively AMP, ADP, ATP; 50 µM). Analysis of solution organic species by LC-MS implied that only adsorption occurred with AMP and ADP but both adsorption and dephosphorylation of ATP were evident. Maximum adsorption capacities per gram of mineral were 40.6 ± 0.8 µmol AMP, 35.7 ± 1.6 µmol ADP, and 10.9 ± 1.0 µmol ATP; solution dephosphorylated by-products accounted for 15% of initial ATP. Subsequent XANES analysis of the surface species revealed that 16% of adsorbed AMP and 30% of adsorbed ATP were subjected to dephosphorylation, which was not fully quantifiable from the solution measurements. Molecular simulations predicted that ADP and ATP were complexed mainly via the phosphate groups whereas AMP binding also involved multiple hydrogen bonds with the adenosine moiety; our FTIR data confirmed these binding confirmations. Our findings thus imply that specific adsorption mechanisms dictate the recycling and subsequent trapping of Pi from ribonucleotide-like biomolecules reacted with Fe (oxyhydr)oxide minerals.

Influence of strain origin on osmoregulatory and endocrine parameters of two non-native strains of Atlantic salmon (Salmo salar L.)

Non-native strains of Atlantic salmon are used in reinstatement trials where populations are extinct. Environmental cues like photoperiod and temperature are known to influence the smolting process and there is evidence of strain-, stock- or population-specific differences associated with seaward migration or smoltification. The objective of this study was to compare morphological, osmoregulatory and endorcrine features between two strains, one originating from a cold and short river in Ireland (Cong) and another from a long and warm river in France (Loire-Allier), reared under Belgian conditions in order to highlight major differences in restocking adaptability. Comprehensive endocrine profiles, consistent with their interactive role of mediating changes associated with smolting, have been observed. Na+/K+ATPase activity (1.3–10.5 µmol ADP∗mg prot.−1∗h−1) and hormone plasma levels (e.g. 55–122 ng∗mL−1 of cortisol and 4.5–6.4 ng∗mL−1 of GH) were consistent with reported values. We observed strain-related differences of the influence of temperature and daylength on cortisol, GH and sodium plasma levels. These may be related to the respective environmental conditions prevailing in the river of origin, which have impacted the genetic background for smoltification. Using Na+/K+ATPase activity as an indicator, both strains smoltified successfully and simultaneously testifying a prevailing influence of environmental cues over genetic factors for smoltification.

Growth and Smoltification of Three Norwegian Strains of Atlantic Salmon Salmo salar Reared under Different Thermal Regimes

AbstractA comparative study was performed with juvenile Atlantic salmon Salmo salar from three stocks in Western Norway that differ in their natural conditions. One is from warm, lowland river conditions (Årdal), one is from cold glacial river conditions (Stryn) and the last one (Suldalslågen) is from a hydropower‐regulated river. The salmon parr were tagged and reared at 4, 5.5 and 7 °C and simulated natural water temperature (SNT) for river Suldalslågen. Size distribution was unimodal at 4 °C, with a change to a bimodal distribution, representing potential 1+ and 2+ smolts, at the other temperature regimes. The relative biomass of 1+ smolts varied between the stocks as Stryn (cold glacial river) stock had the highest number of smolts at 7 °C and the Suldalsågen stock (hydropower‐regulated river) displaying the highest number at the SNT regime. Overall, the Stryn stock, originating from cold river conditions, seemed to be well adapted to growth and smoltification at cold temperatures, whereas salmon parr from river Suldalslågen seem to be better adapted to the natural temperature regime (SNT) of this river than the other two stocks. This was reflected in the gill Na+,K+‐ATPase as the Suldalslågen stock showed increasing activity from 16 April (4.2 µmol ADP mg protein−1 h−1) to 10 May (9.2 µmol ADP mg protein−1 h−1), and at the end of the experiment, enzyme activity in Suldalslågen stock was significantly higher than both Stryn (5.7) and Årdal (5.9 µmol ADP mg protein−1 h−1) stock. In contrast, the warm lowland stock, Årdal, fish had low Na+,K+‐ATPase activity with no distinct peak at any of the sampled dates from March through May. Copyright © 2016 John Wiley &amp; Sons, Ltd.

Poor agreement between light transmission aggregometry, Verify Now P2Y12 and vasodilatator-stimulated phosphoprotein for clopidogrel low-response assessment: A potential explanation of negative results of recent randomized trials

Clopidogrel low response as assessed by several different biological tests correlates with poor prognosis after percutaneous coronary intervention (PCI). However, recent randomized clinical trials (RCT) testing the strategy of individual antiplatelet therapy tailoring based on one sole test have all shown negative results. Poor correlation between the different tests may explain the difficulties of patient selection and identification of “true poor responders” to clopidogrel. In this prospective study, clopidogrel response was assessed in 100 consecutive patients between 18 and 24 hours after a 600 mg clopidogrel loading dose using three different tests: light transmission aggregometry with 10 μmol ADP (LTA, results expressed as platelet aggregation percentage: PAP), Verify Now P2Y12 (VN, results expressed as P2Y12 reaction unit: PRU) and vasodilatator-stimulated phosphoprotein (VASP, results expressed as platelet reactivity index: PRI). Patients under chronic clopidogrel therapy were excluded. The mean PAP, PRU and PRI values were 38.6%, 176.1 PRU and 52.4%, respectively. When clopidogrel response was analyzed as continuous variable, there was a good correlation between the different tests: LTA/VN (R2 = 0.642, p < 0.001), LTA/VASP (R2 = 0.409, p < 0.001) and VN/VASP (R2 = 0.616, p < 0.001). However, when clopidogrel response was analyzed as pre-specified cut-off points to define patients as “poor or good responders” (according to the literature: 50% PAP for LTA, 235 PRU for VN and 50% PRI for VASP), only 47% of the patients were defined as “good” or “poor responders” by the three tests. Altogether, 33% of the patients were defined as “poor responders” by only one test, 20% by two tests and only 16% by the three tests. The correlation between the different tests is good when clopidogrel response is analyzed as continuous variable. Each individual is however rarely (less than 50%) defined as “poor or good responder” by all the three tests when pre-specified cut-off values are used. A sole test might not be sufficient to manage antiplatelet therapy in an individual patient and these results may explain the results of recent RCT showing the lack of benefit of systematic antiplatelet therapy monitoring strategy.

021: Clopidogrel low response and correlation between the different tests: light transmission aggregometry, VerifyNow-P2Y12 and V ASP

Clopidogrel low response correlates with poor prognosis after percutaneous coronary intervention (PCI). Many biological tests are currently available to test the clopidogrel response. However, the presence of any correlation between the different tests is today poorly reported. In this prospective study, clopidogrel response was assessed in 100 consecutive patients. All patients were tested between 18h and 24h after a 600mg clopidogrel loading dose using 3 different tests: light transmission aggregometry with 10 μmol ADP (LTA, results expressed as platelet inhibition percentage), VerifyNow-P2Y12 (VN, results expressed as PRU) and vasodilatator stimulated phosphoprotein (VASP, results expressed as IRP). Patients under chronic clopidogrel therapy were excluded. The mean platelet inhibition percentage, PRU value and IRP value were 38.5±13% by LTA, 178±89 PRU by VN and 52±21% by VASP. When results were analyzed as continuous variables, there was a good correlation between the different tests: LTA/VN (R2=0,642, p<0,001), LTA/VASP (R2=0,409, p<0,001) and VN/VASP (R2=0,616, p<0,001). However, when results were analyzed as pre-specified cut-off points to define patients as “low or good responders” (according to the literature: 50% for LTA, 235 PRU for VN and 50% IRP for VASP), only 47% of the patients were defined as “good” or “low responders” by the 3 tests. Altogether, 33% of the patients were defined as “low responders” by only 1 test, 20% by 2 tests and only 16% by the 3 tests. If the correlation between the different tests is good when results are analyzed as continuous variables, each individual is rarely (less than 50%) defined as “low or good responder” by all the 3 tests when recognized cut-off values are used. In that way, a sole test might not be sufficient to manage antiplatelet therapy in an individual patient.

Seawater tolerance and post‐smolt migration of wild Atlantic salmon Salmo salar× brown trout S. trutta hybrid smolts

High levels of hybridization between Atlantic salmon Salmo salar and brown trout Salmo trutta have been reported in the River Driva. This study presents the underlying mechanisms of development of seawater (SW) tolerance and marine migration pattern for S. salar×S. trutta hybrids. Migrating S. salar×S. trutta hybrid smolts caught in the River Driva, Norway (a river containing Gyrodactylus salaris), displayed freshwater (FW) gill Na(+), K(+) -ATPase (NKA) activity levels of 11·8 µmol ADP mg protein h(-1), which were equal to or higher than activity levels observed in S. salar and S. trutta smolts. Following 4 days of SW exposure (salinity 32·3), enzyme activity remained high and plasma ion levels were maintained within the normal physiological range observed in S. salar smolts, indicating no signs of ion perturbations in S. salar×S. trutta hybrids. SW exposure induced an increase in NKA α1b-subunit mRNA levels with a concurrent decrease in α1a levels. Salmo salar×S. trutta post-smolts migrated rapidly through the fjord system, with increasing speed with distance from the river, as is often seen in S. salar smolts. The present findings suggest that S. salar×S. trutta smolts, as judged by the activity and transcription of the NKA system, regulation of plasma ion levels and migration speed more closely resemble S. salar than S. trutta.

Growth and ionoregulatory ontogeny of wild and hatchery-raised juvenile pink salmon (Oncorhynchus gorbuscha)

Juvenile pink salmon ( Oncorhynchus gorbuscha (Walbaum, 1792)) enter seawater (SW) shortly following emergence. Little is known about growth and development during this life-history stage when sensitivity to sea louse exposure may be high, an issue that is of current concern in British Columbia. We tested the hypothesis that growth and ionoregulatory development were similar in hatchery-raised (Quinsam) and wild (Glendale and One’s Point) juvenile pink salmon (measured over 22 weeks) following SW entry. Fish body mass increased from 0.20 ± 0.01 to 6.47 ± 0.37 g, with mean specific growth rates of 2.74% to 3.05% body mass·day–1among the three groups. In all three groups, gill Na+–K+-ATPase (NKA) activity peaked at 12 µmol ADP·mg protein–1·h–1following 8 weeks post-transfer to SW. Whole body Na+and Cl–concentrations, which again did not differ among groups, were highest upon initial exposure to SW (~70 mmol·kg wet mass–1) and declined over time as gill NKA activity increased, indicating that the hypo-osmoregulatory capacity was not fully developed following emergence and initial entry into SW. Thus, consistent with our hypothesis, few differences were observed between hatchery-raised and wild juvenile pink salmon reared under laboratory conditions. These baseline data may be important for future studies in determining the effects of sea lice on wild juvenile pink salmon.

Dose Effect of Clopidogrel Reloading in Patients Already on 75-mg Maintenance Dose

Clopidogrel loading has mostly been studied in clopidogrel-naïve patients. Whether clopidogrel-treated patients readmitted for an acute coronary syndrome or percutaneous coronary intervention can benefit from a new load of clopidogrel and at what dose remain unknown. Our aim was to evaluate the impact of 3 different strategies of administration of a loading dose of 900 mg clopidogrel in patients already treated with a maintenance dose of 75 mg clopidogrel for at least 7 days on residual platelet aggregation. Patients treated long term by clopidogrel 75 mg/d were assigned to receive a first loading dose of 300, 600, or 900 mg clopidogrel and 4 hours later a second loading dose of 600, 300, or 0 mg, respectively, to achieve a total loading dose of 900 mg in all patients. Platelet aggregation was evaluated at baseline, at 4 hours after the initial load (and before second load), and at 24 hours using light transmission aggregometry with 20 micromol ADP and the point-of-care assay VerifyNow P2Y(12). The primary objective of the study was to evaluate the inhibition (relative change) of residual platelet aggregation (percentage of IRPA) between 600- and 900-mg first loading at 4 hours. IRPA at 24 hours also was evaluated as a secondary objective, as well as the rate of suboptimal response at 4 hours defined as IRPA <10%. We included 166 consecutive patients with acute coronary syndromes (n=80, 48%) or stable coronary artery disease (n=86, 52%). Baseline characteristics were similar in the 3 dose groups. A significant stepwise increase was found in percentage IRPA assessed at 4 hours in patients initially assigned to 300 versus 600 versus 900 mg (30.7% versus 40.3% versus 64.0%, respectively; P=0.0024). The difference in percentage IRPA at 4 hours was not significant between 300 and 600 mg but was significant between 600 and 900 mg and between 300 and 900 mg. Percentage IRPA assessed at 24 hours when all patients had received 900 mg did not differ between the 3 loading regimens. The rates of suboptimal response (IRPA <10% at 4 hours) were 23.6%, 20.4%, and 5.3% with 300, 600, and 900 mg, respectively (P=0.02 for all). In patients treated long term with 75 mg clopidogrel, a new loading dose of 900 mg improves IRPA and reduces poor and/or slow response to clopidogrel significantly more than that obtained with 300 or 600 mg.

The effects of daily ration on growth and smoltification in 0+ and 1+ Atlantic salmon (Salmo salar) parr

The effects of long-term variations in feed ration were studied during two experiments on Atlantic salmon parr. In the first experiment, three duplicate groups of approximately 500 salmon parr were fed at rates of 100%, 66% or 33% of the manufacturer's recommendation from shortly after first feeding. Each group was exposed to a photoperiod regime which was expected to result in smoltification 9 months after first feeding. In the second experiment, three duplicate groups of 550 fish were fed 100%, 66% or 33% of the manufacturer's recommendation from first feeding and exposed to a simulated natural photoperiod, which was expected to result in smoltification 13 months after first feeding.In both experiments fish size increased with ration, with recruitment to the upper modal group (UMG) of the population also related to ration (85–96%, 64–88% and 28–42% UMG fish for the full, two-thirds and one-third ration groups respectively, recorded at the conclusion of each experiment). Throughout each experiment the full and two-thirds ration fish maintained similar whole body lipid concentrations, although lipid concentrations in the one-third ration fish were generally lower. At the conclusion of experiment 1, gill Na+, K+-ATPase activity in UMG fish fed full rations reached 9.5 μmol ADP hydrolysed mg−1 protein−1 h−1, whereas ATPase activities were lower in the other ration groups. In experiment 2, all groups had similar gill Na+, K+-ATPase activities at the conclusion of the experiment (6.4–9.3 μmol ADP hydrolysed mg−1 protein−1 h−1). Following 24 h seawater challenges, conducted during the parr–smolt transformation, UMG fish from the full and two-thirds groups of experiment 1 displayed high survival rates (100%) and low serum osmolalities (335 mOsm kg−1), with lower survival rates (75%) and higher serum osmolalities (370 mOsm kg−1) recorded in the one-third ration fish. In experiment 2 similar survival rates (100%) and serum osmolalities (350 mOsm kg−1) were found in all ration groups.It is concluded that under accelerated production regimes, feed restriction may result in underyearling Atlantic salmon smolts developing a poor hypo-osmoregulatory ability. Variations in ration significantly influence growth, although it is believed that growth is dependant on the maintenance of a specific lipid level in the body.

Clopidogrel Resistance and Aspirin Resistance - a Cause of Coronary Artery Stent Thrombosis?.

Background: Occlusions of coronary artery stents are a seldom but often fatal complication after coronary artery intervention. With aggressive platelet inhibition therapy (combination of Aspirin (ASS) and clopidogrel) a reduction of stent thrombosis to less than 2% was achieved. The inhibition of platelet function has great interindividual variability and resistance against ASS and/or clopidogrel were previously described. Therefore we studied the relation between coronary artery stent thrombosis and insufficient inhibition of platelet function in patients after coronary artery intervention.Methods: Between 2000 and 2003 we examined 20 patients (pts) with coronary artery stent thrombosis within 4 weeks after coronary artery intervention. All patients received a clopidogrel loading dose of 300mg followed by 75mg/die and ASS 100mg/die. Platelet inhibition was studied by light transmittance aggregometry in platelet rich plasma (Aggregometer PAP 4, moelab inc.). Aggregation was recorded as the maximum percentage change in light transmittance from baseline using platelet poor plasma as a reference. We defined ASS resistance as platelet aggregation > 30% after induction with 0,5mg/ml arachidonic acid and clopidogrel resistance as platelet aggregation > 30% after induction with 20 μmol ADP. We compared these pts with 20 pts without stent thrombosis within 4 weeks after coronary artery intervention.Results: In the group with coronary artery stent thrombosis we found 13/20 patients with insufficient platelet inhibition. 2/20 pts in this group were resistent against both ASS and clopidogrel. In 2/20 pts we observed ASS resistance and in 9/20 pts clopidgrel resistance. In the group of patients without stent thrombosis we found only 1/20 pt with ASS resistance and 1/20 pt with clopidogrel resistance.Conclusions: Insufficient platelet inhibition could be one cause of early stent thrombosis after coronory artery intervention. Platelet function testing is able to detect insufficient platelet inhibition and early testing could be an instrument to prevent coronary artery stent thrombosis.

Future migratory behaviour predicted from premigratory levels of gill Na+/K+-ATPase activity in individual wild brown trout(Salmo trutta)

The relationship between premigratory gill Na+/K+-ATPase activity, determined at two dates during spring, and future migratory behaviour was investigated using non-lethal gill biopsies and PIT-tagging in wild brown trout (Salmo trutta) from two tributaries. No significant relationship between future migratory strategy (individuals eventually becoming migrants or residents) and gill Na+/K+-ATPase activity was found in late February-early March. By contrast, in mid-April, a highly significant logistic regression equation identifying the migratory strategy in 93% of the 75 individuals was obtained. The ability of this regression model from the tributaries to predict future migratory behaviour in an independent group of trout caught in early April in the mainstream was evaluated. A threshold probability of migration was used to predict the behaviour of the mainstream individuals as either future migrants or residents. The maximum percentage of correct predictions of future migratory behaviour in mainstream fish was observed at threshold probabilities between approximately 0.15 and 0.45 (corresponding to threshold gill Na+/K+-ATPase activities between 2.7 and 3.7 micromol ADP mg(-1) protein h(-1)), with an average of 91% of the predictions being correct. The present study shows that a non-lethal premigratory biochemical measurement can successfully select individual brown trout with high probability of migration.

Physiological characteristics of wild Atlantic salmon post‐smolts during estuarine and coastal migration

Changes were measured in some of the major physiological variables associated with seawater adaptability, growth and energetics in wild Atlantic salmon Salmo salar smolts and post‐smolts migrating from the river and through the estuary, fjord and coastal areas in the River Orkla and the Trondheimsfjord, Norway during late May to early June. Gill Na+,K+‐ATPase activity increased to levels of 12–16 µmol ADP mg protein−1 h−1 in post‐smolts caught in higher salinity zones, probably representing long‐term levels of Atlantic salmon post‐smolts in oceanic conditions. Muscle moisture was regulated within narrow limits (77·7–78·7%) in fish from all zones during both years, suggesting that post‐smolts adapt to marine conditions without any long‐term disturbance of hydro‐mineral balance. Lipid and glycogen content showed a general trend towards depletion from the river, through the fjord and into the ocean. There was, however, no significant change in protein content. The present results confirm that smolts are naturally ‘energy deficient’ during downstream migration, and suggest that post‐smolts also mobilize energy reserves during their early marine phase, while protein is allocated for somatic growth. Plasma growth hormone (GH) levels increased transiently during passage through the estuary and fjord, with lower levels observed in post‐smolts caught off‐shore, i.e. in fish which were feeding on marine prey and had adapted to the marine environment. These physiological changes may confer substantial selective advantages during the critical early marine phase of anadromous salmonids, and hence are adaptive for long‐term survival in sea water.

Inhibition of mitochondrial respiration and oxygen uptake in isolated rat renal tubular fragments by atractyloside

Atractyloside (ATR) is widely used as a specific inhibitor of mitochondrial adenine nucleotide translocase and it is also a potent nephrotoxin that selectively injures the proximal tubule in vivo. This regioselectivity has been attributed to the prominence of mitochondria in the proximal tubule cells, but there have been no investigations to confirm this. In order to better understand the molecular basis of ATR-induced renal injury, oxidative phosphorylation was studied in freshly isolated rat proximal tubular and glomeruli fragments, and in isolated rat renal cortical mitochondria. In isolated renal mitochondrial, ATR significantly inhibited state 3 respiration in a dose-dependent manner, with the maximum inhibition achieved at the highest ATR concentration. Low doses of ATR (53 μM) inhibited respiration by 50%, an effect which was reversed by 2.5 μmol ADP. 2,4-Dinitrophenol (5 mM), which stimulated respiration in control mitochondria, failed to do this in the presence of ATR. Basal oxygen consumption was significantly inhibited by ATR (> 50 μM) in proximal tubule previously incubated for 1 h at 37 °C. The concentration-dependent inhibition of oxygen uptake by the proximal tubule was maintained in the presence of 1 mM ouabain or 0.25 mg/ml nystatin. Glomeruli have active mitochondrial respiration (about half that of the proximal tubules), but were not affected by ATR at concentrations up to 500 μM. These data demonstrates that both purified renal mitochondria and freshly isolated fragments of the proximal tubule exposed to ATR in vitro exhibit similar alteration in respiratory parameters that demonstrate inhibition of state 3 mitochondrial respiration, but there was no significant effect on glomeruli cells. Thus, the inhibition of oxidative phosphorylation may be an early event in ATR-induced nephrotoxicity, where the prominence of mitochondria in the proximal tubule explain, in part, the localised injury. The resistance of the glomeruli suggest that preferential transport of ATR may also contribute to the sensitivity of the proximal tubule.

Purification and characterization of phosphoribulokinase from the N 2-fixing cyanobacterium Anabaena cylindrica

Phosphoribulokinase (PRuK, ATP:D-ribulose 5-phosphate 1-phosphotransferase, EC 2.7.1.19), a key enzyme of the reductive pentose phosphate cycle in autotrophs, was purified from the N 2-fixing heterocystous cyanobacterium Anabaena cylindrica by chromatographic methods, including blue-Sepharose affinity chromatography. The enzyme was purified 195-fold with a recovery of 10% and showed a specific activity of 1.95 μmol ADP formed (min·mg protein) −1. The enzyme was strongly activated by dithiothreitol (DTT)-reduced thioredoxin (TRX) and after activation the specific activity was 15.17 μmol ADP formed (min·mg protein) −1 with a recovery of 78% and a purification factor of 1517-fold. The apparent molecular mass of native PRuK was about 72 kDa as determined by chromatography on Sephacryl S-300. The optimum pH for activity depended on the method of assay used and was about 8.6 in the radioactive assay and 7.5 in the spectrophotometric assay. Mg 2+ appeared as a non-essential activator and at 5 mM increased the enzyme activity 5-fold but activity depended on the nature of the anion. The enzyme was more specific for the sugar phosphate substrate than for the nucleoside triphosphate. It showed classical Michaelis-Menten kinetics with apparent K m values of 0.29 mM and 1.42 mM for ribulose 5-phosphate (Ru5P) and ATP, respectively. The enzyme was not significantly affected in vitro by a number of typical effectors of PRuK purified from other sources. However, it could be partially activated in vitro by DTT and more efficiently by chemically-reduced homologous or heterologous TRXs. The presence of its substrate Ru5P accelerated the activation of the enzyme caused by reduced TRX.

AMP metabolism by the marine bacterium Vibrio (Beneckea) natriegens: purification and properties of adenylate kinase

Adenylate kinase (EC 2.7.4.3) has been purified 484-fold from extracts of Vibrio natriegens to a specific activity of 1350 μmol ADP formed∙min−1∙mg protein−1. The preparation was 97% pure as judged by gel electrophoresis and exhibited molecular weight values of 29 000 by gel filtration and 32 000 by SDS–gel electrophoresis. The isoelectric point was at pH 4.7. Only ATP (Km 0.067 mM), ADP (Km 0.45 mM), and AMP (Km 0.12 mM) exhibited high activity as substrates, though dATP or dAMP could serve as cosubstrates with AMP or ATP, respectively, at reduced rates. The equilibrium constant in the direction of ATP formation was 1.09, and the pH optimum in both directions was broad, from pH 7.2 to pH 7.6. Enzyme activity was sensitive to the thiolalkylating agents iodacetamide and p-chloromercuriphenyl sulfonate.

Physiological Levels and Properties of Polynucleotide Phosphorylase of Rhizobium meliloti

Summary: Polynucleotide phosphorylase activity was demonstrated in free-living and the symbiotic bacteroid forms of Rhizobium meliloti and in free-living Rhizobium japonicum. Crude extracts of R. meliloti f-28 catalysed the polymerization of 3.0 μmol ADP (mg protein)-1 h-1. The polynucleotide phosphorylase activity of symbiotic R. meliloti was about 50% of that of free-living R. meliloti and remained almost constant throughout the development of the nodules. Partially-purified fractions of free-living R. meliloti f-28 catalysed the polymerization of ribonucleoside diphosphates, the phosphorolysis of synthetic homopolymers and natural heteropolymers, and the β-phosphate exchange reaction. The enzyme had a pH optimum of 8.0 and had an obligatory requirement for Mg2+ for maximum activity. The phosphorylase was not highly primer-dependent, had low specificity for guanosine-containing substrates, and exhibited cooperative-type kinetics.

Sperrtonus und Nucleosidtriphosphate

The resting anterior byssal retractor muscle of Mytilus edulis (ABRM) contains 1 -1.2 μmol ATP and 0.6 - 0.8 μmol ADP per g wet weight, traces of AMP and 0.1 - 0.2 μmol GTP plus UTP. The ATP content and the ratio of ADP : ATP remains almost constant during contraction, catch and subsequent serotonin induced relaxation, IMP could not be detected, not even during thaw contracture. Consequently the catch (viscous tone, fused state) is a reversible rigor distinct from rigor in vertebrate skeletal muscle which is caused by ATP deficiency. ATP produces a very much stronger contraction in isolated actomyosin systems of ABRM than UTP, ITP and GTP.