Total ATPase Activity Research Articles

Thermal stability of myofibrillar protein from Indian major carps

AbstractThe characteristics and stability of natural actomyosin (NAM) from rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) were investigated. The total extractable actomyosin (AM) was higher (7.60 mg ml−1) in the case of rohu compared with that from catla and mrigal (5 mg ml−1). Although the specific AM ATPase activity was similar (0.43–0.5 µmol P min−1 mg P−1) among the three species, the total ATPase activity was lower in mrigal (25 µmol g−1 meat) compared with the other species (37 µmol g−1 meat). The inactivation rate constants (kd) of AM Ca ATPase activity showed differences in the stabilities of actomyosin among these fish, the actomyosin from catla being least stable. The NAM from these species was stable up to 20 °C at pH 7.0. Catla AM became unstable at 30 °C, while rohu and mrigal AM could withstand up to 45 °C. The thermal denaturation with respect to solubility, turbidity, ATPase activity, sulphhydryl group and surface hydrophobicity showed noticeable changes at around these temperatures. Copyright © 2004 Society of Chemical Industry

Normal expression and inflammation-induced changes of Na and Na/K ATPase activity in spinal dorsal horn of the rat

We tested whether that peripheral inflammation induces changes in the spinal dorsal horn ATPase activity. Adult Sprague–Dawley rats were anesthetized (thiobarbital), the left hind paw (inflammation group; n = 15) was immersed in water at 60 °C for 60 s, which induced a local inflammation. A control group ( n = 12) was tested with water at room temperature. After 60 min of peripheral inflammation left (LDH) or right lumbar dorsal horn (RDH) were processed for total, Na/K, Na and remanent ATPase activities (nM P i (mg protein) −1 min −1). In control animals isoenzymatic activities were: Na (31.2%); Na/K (20.6%) and remanent (48.2%) from total ATPase activity. No LDH-RDH asymmetry was found. The inflammation group presented an ipsilateral increase of total ATPase activity in LDH ( X ± S.E.M.; 4798.9 ± 601) over the RDH (3982.2 ± 451; Δ + 817; P < 0.05). This is due to an increase in Na ATPase activity (1609.3 ± 297) over RDH (1164.2 ± 166; Δ + 445; P < 0.05). ATPase activities were increased in LDH from inflamed over the control group as follows: total (4798.9 ± 601; Δ + 840; P < 0.05), Na/K (1298.1 ± 301; Δ + 483; P < 0.05) and Na (1609.3 ± 297; Δ + 373; P < 0.05). These increased ATPase activities, induced in a short time, can be considered a functional marker of nociceptive neuronal activity.

Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F 1F o ATP synthase

His-tagged cysteine-less F 1F o ATP synthase from Escherichia coli was purified using Ni-NTA affinity chromatography. During the purification procedure the loss of total ATPase activity did not exceed 50%, and the extent of purification was about 80-fold. The purified enzyme was essentially free of other proteins, was highly active in ATP hydrolysis (75 units/mg at pH 8 and 37 °C), and was sensitive to N, N′-dicyclohexylcarbodiimide (70%). Incorporation of F 1F o into soybean liposomes yielded well-coupled and highly active proteoliposomes. The entire procedure, from the disruption of cells by French press to the preparation of proteoliposomes, took only about 8 h. Some improvements in procedures for the estimation of rates of both ATP hydrolysis and ATP-dependent 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching are described.

Effects of Aluminium and Lead on ATPase Activity of Knockout GDNF+/– Mouse Cerebral Synaptosomes In Vitro

In this in vitro study, changes in the activity of the neural membrane integral protein, ATPase, were recorded after the exposure of isolated synaptosomes to different concentrations of aluminium and lead. Both total ATPase activity and Mg(2+)-ATPase activity were studied. A specific mouse strain, heterozygous for a glial cell line-derived neurotrophic factor (GDNF), and the corresponding wildtype mouse cerebral tissue, were used for the synaptosome isolations. The ATPase activities of the +/- mouse synaptosomes were compared with those of wild-type synaptosomes. The decrease in total ATPase activity was similar in both types of synaptosomes, but after exposure to aluminium, the decrease of Mg(2+)-ATPase activity in the GDNF+/- synaptosomes was smaller than that in the wild-type synaptosomes. After exposure to lead, the protective effect of GDNF was not so clear. The synaptosomal effects of lead were already found at concentrations lower than those where cell toxicity appeared in SH-SY5Y cell cultures. Thus, synaptosomal ATPase activity was considered to be a sensitive marker for the detection of lead-induced neurotoxicity.

Effect of low-intensity (3.75-25 J/cm2) near-infrared (810 nm) laser radiation on red blood cell ATPase activities and membrane structure.

The biostimulation and therapeutic effects of low-power laser radiation of different wavelengths and light doses are well known, but the exact mechanism of action of the laser radiation with living cells is not yet understood. The aim of the present work was to investigate the effect of laser radiation (810 nm, radiant exposure 3.75-25 J/cm(2)) on the structure of protein and lipid components of red blood cell membranes and it functional properties. The role of membrane ATPases as possible targets of laser irradiation was analyzed. A variety of studies both in vivo and in vitro showed significant influence of laser irradiation on cell functional state. At the same time another group of works found no detectable effects of light exposure. Some different explanations based on the light absorption by primary endogenous chromophores (mitochondrial enzymes, cytochromes, flavins, porphyrins) have been proposed to describe biological effects of laser light. It was suggested that optimization of the structural-functional organization of the erythrocyte membrane as a result of laser irradiation may be the basis for improving the cardiac function in patients under a course of laser therapy. Human red blood cells or isolated cell membranes were irradiated with low-intensity laser light (810 nm) at different radiant exposures (3.75-25 J/cm(2)) and light powers (fluence rate; 10-400 mW) at 37 degrees C. As the parameters characterizing the structural and functional changes of cell membranes the activities of Na(+)-, K(+)-, and Mg(2+)-ATPases, tryptophan fluorescence of membrane proteins and fluorescence of pyrene incorporated into membrane lipid bilayer were used. It was found that near-infrared low-intensity laser radiation changes the ATPase activities of the membrane ion pumps in the dose- and fluence rate-dependent manner. At the same time no changes of such integral parameters as cell stability, membrane lipid peroxidation level, intracellular reduced glutathione or oxyhaemoglobin level were observed. At laser power of 10 mW, an increase of the ATPase activity was observed with maximal effect at 12-15 J/cm(2) of light dose (18-26% for the total ATPase activity). At laser power of 400 mW (fluence rate significantly increased), inhibition of ATPases activities mainly due to the inhibition of Na(+)-, K(+)-ATPase was observed with maximal effect at the same light dose of 12-15 J/cm(2) (18-23% for the total ATPase activity). Fractionation of the light dose significantly changed the membrane response to laser radiation. Changes in tryptophan fluorescent parameters of erythrocyte membrane proteins and the increase in lipid bilayer fluidity measured by pyrene monomer/excimer fluorescence ratio were observed. Near-infrared laser light radiation (810 nm) induced long-term conformational transitions of red blood cell membrane which were related to the changes in the structural states of both erythrocyte membrane proteins and lipid bilayer and which manifested themselves as changes in fluorescent parameters of erythrocyte membranes and lipid bilayer fluidity. This resulted in the modulation of membrane functional properties: changes in the activity of membrane ion pumps and, thus, changes in membrane ion flows.

Haemato-biochemical and immuno-pathophysiological effects of chronic toxicity with synthetic pyrethroid, organophosphate and chlorinated pesticides in broiler chicks

Haemato- biochemical and immuno-pathophysiological changes following feeding of broiler chicks with 20 ppm fenvalerate (synthetic pyrethroid, SP), 2 ppm monocrotophos (organophosphate, OP) and 2 ppm endosulfan (chlorinated hydrocarbon, CH) were studied. Four groups of broiler birds (30 each) were fed poultry mash without (control) or mixed with pesticides for 8 weeks. Blood glucose, serum globulin and acetyl cholinesterase (AChE) activity level were decreased ( P<0.01) in all treated groups compared to control, but not the serum albumin and BUN. The total ATPase activity was enhanced ( P<0.01) in fenvalerate and monocrotophos than birds in control group. Body weight, total erythrocyte count, packed cell volume, haemoglobin, eosinophil and monocyte count did not show any changes. Total leucocytes and T-lymphocyte count was lower ( P<0.01) in all treated groups as compared to control group. B-cell count ( P<0.01), mean 2-4-dinitrofluorobenzene (DNFB) dermal sensitivity score and splenic indices from graft vs. host reaction ( P<0.05) were decreased in fenvalarate and endosulfan but the values for monocrotophos were intermediate between control and other treated groups. Pesticide intoxication reduced nitroblue tetrazolium (NBT) positive cells (active splenic macrophages) ( P<0.05) and spleen weight ( P<0.01). Whereas bursal weight was reduced only with endosulfan, thymic weight was reduced on endosulfan and fenvalerate-treated feed. Microscopic examination of these organs further revealed atrophy/hypoplasia, decrease in the size of follicles with depletion of lymphocytes and haemorrhages in thymus. The study concludes that the chronic exposure of chicks to small amount of SP, OP and CH pesticide leads to deleterious effects on metabolism and immune system of birds.

The V-type H(+)-ATPase in Malpighian tubules of Aedes aegypti: localization and activity.

The V-type H(+)-ATPase is thought to provide the driving force for transepithelial electrolyte and fluid secretion in Malpighian tubules. To confirm the presence of this proton pump in Malpighian tubules of the yellow fever mosquito Aedes aegypti, we used several antibodies raised against the V-type H(+)-ATPase of Manduca sexta. Western blot analysis confirmed the presence of the V-type H(+)-ATPase in Malpighian tubules of Aedes aegypti. In situ immunostaining identified the V-type H(+)-ATPase at the apical membrane of the mitochondrion-rich brush border of principal cells. The V-type H(+)-ATPase was not found in stellate cells. Measurements of ATPase activity revealed that bafilomycin-sensitive and NO(3)(-)-sensitive ATPase activity accounted for 50-60% of total ATPase activity in crude extracts of Malpighian tubules. No significant ouabain- or vanadate-sensitive Na(+)/K(+)-ATPase activity was detected. These results support the conclusion reached previously in electrophysiological studies that the mechanisms for transepithelial electrolyte secretion in the Aedes Malpighian tubules rely on the V-type H(+)-ATPase as the principal energizer of epithelial transport. Measures of transepithelial Na(+) and K(+) secretion and estimates of the H(+) flux mediated by the V-type H(+)-ATPase suggest a 1:1 stoichiometry for Na(+)/H(+) and K(+)/H(+) exchange transport across the apical membrane.

Eel ATPase activity as biomarker of thiobencarb exposure

European eels ( Anguilla anguilla) were exposed to a sublethal thiobencarb concentration of 0.22 mg/L in a flow-through system for 96 h. Mg 2+ and Na +–K + adenosine triphosphatase (ATPase) activities were evaluated in gill and muscle tissues at 2, 12, 24, 48, 72, and 96 h of thiobencarb exposure. Gill ATPase activities were rapidly inhibited from 2 h of contact onward. Highest inhibition was registered for Na +, K +-ATPase (85%) from 2 to 12 h. Both Mg 2+ and total ATPase were inhibited (>73%) during the first hours of toxicant exposure. At the end of the exposure period (96 h) ATPase activities were still different from those of the controls (>50%). Significant inhibition was detected in Na +, K +-ATPase activity (80%) in muscle tissue after 2 h and it was maintained over the entire exposure time. However, Mg 2+-ATPase and total ATPase showed only perturbations after 2 h of exposure. Eels were exposed to 0.22 mg/L of thiobencarb for 96 h and then a recovery period in herbicide-free water was allowed for 192 h. Gill and muscle samples were removed at 8, 24, 72, 96, 144, and 192 h and ATPase activity was evaluated. Following 144 h of recovery, Mg 2+- and Na +, K +-ATPase activities, as well as total ATPase activity, in gills of those animals previously exposed to 0.22 mg/L of thiobencarb were still significantly different compared to controls. Thiobencarb seems to act to alter the ionic profiles. Since ion-dependent ATPases are known to regulate the influx and efflux of ions across the membrane to maintain the physiological requirements of the cells, the inhibition of Na +, K +-ATPase probably induced osmoregulatory perturbations. On the other hand, thiobencarb exposure causes increases in the muscle water content of A. anguilla. The results indicated that water content increased significantly (>100% higher than the controls) during the first 24 h of exposure.

The synaptosomal membrane bound ATPase as a target for the neurotoxic effects of pyrethroids, permethrin and cypermethrin

Pyrethroids are used widely as insecticides both in agriculture and in households. A cellular target of pyrethroids is the sodium channel in the membrane. In the present study, the activity of the membrane bound integral protein ATPase was studied as a biomarker for the membrane effect of the pyrethroids permethrin and cypermethrin. Male Sprague–Dawley rats were used for cerebral synaptosome preparation. The isolation of synaptosomes was performed with the Percoll gradient method. Both total ATPase and Mg 2+ activated ATPase were studied by determining inorganic phosphate liberated from the substrate ATP. One hour exposure to permethrin (Biokill) and cypermethrin (Ripcord) insecticide products affected ATPase activities. The activity of Na +, K + ATPase decreased dose-dependently in 10–50 μM concentrations of permethrin, and Mg 2+ activated ATPase increased over twofold in the same concentrations of the active components. The effect of the cypermethrin compound Ripcord was not clearly dose-dependent. The activity of total ATPase was almost entirely lost in the concentrations of 100 μM of permethrin and cypermethrin. The results support the idea that membrane ATPases are one target of the neurotoxic effect of pyrethroid compounds.

The Contractile Potency of Adenosine Triphosphate and Ecto-adenosine Triphosphatase Activity in Guinea Pig Detrusor and Detrusor From Patients With a Stable, Unstable or Obstructed Bladder

We compared the potency of adenosine triphosphate (ATP) and its nonhydrolyzable analogue alpha,beta-methylene ATP for generating contractions in human detrusor smooth muscle from patients with a stable, unstable and obstructed bladders. The different ATP potencies were compared with the ecto-adenosine triphosphatase (ATPase) of these samples. Contractile experiments were done in vitro by superfusing samples with purines and dose-response curves were generated. Ecto-ATPase activity was measured from the rate of ATP hydrolysis sensitive to the ecto-ATPase inhibitor ARL 67156 with a luciferin-luciferase assay. ATP generated contractions with a mean EC50 of 933 microM. in tissue from stable bladders and was significantly more potent in tissue from unstable and obstructed bladders (EC50 141 and 172 microM., respectively). alpha,beta-methylene ATP was more potent in tissue from stable and unstable bladders (mean combined EC50 3 microM.). In guinea pig detrusor the mean EC50 for ATP and alpha,beta-methylene ATP was 138 and 5.5 microM., respectively. Mean total ATPase activity in unstable bladder biopsies plus or minus standard deviation was about 50% of that in stable bladder biopsies (2.54 +/- 1.50 versus 1.37 +/- 0.46 nmol. per second per mg. protein ). The ARL 67156 sensitive fraction was also significantly less in samples from unstable compared with stable bladders (mean 0.94 +/- 0.41 versus 0.36 +/- 0.26 nmol. per second mg. protein ). The greater potency of ATP for generating contractions in detrusor from unstable bladders may be due to reduced extracellular hydrolysis, allowing purine greater access to detrusor smooth muscle. This finding may explain atropine resistant purine based contractions in detrusor from unstable bladders.

Unaltered cerebral Na +,K +-ATPase activity after hypoxic/ischemic injury in piglets

We examined whether hypoxic/ischemic (H/I) stress decreased the cerebral Na +,K +-ATPase enzyme activity (NEA) of newborn pigs. The effects of global ischemia (10 min), asphyxia (10 min), and incomplete forebrain ischemia (45 min) were analyzed in ten different brain regions. The lengths of the reperfusion periods varied between 15 min and 3 h. NEA was determined as the ouabain-sensitive fraction of the total ATPase activity of the sample. Marked regional differences in NEA were observed in all experimental groups, whereas NEA was not significantly affected in any of the brain structures investigated. The present results suggest that damaged brain Na +,K +-ATPase may not be the cause of the neuronal–vascular impairment following H/I stress.

Neurotoxicologic sequelae of tributyltin intoxication in rats

Tributyltin oxide (TBTO) is a commonly used biocide. The purpose of this study is to correlate the toxicity of TBTO with the alterations of brain neurotransmitters and ATPases. TBTO was given by stomach tube to rats at either 37.5 or 75 mg kg −1for 3 consecutive days. Nervous signs appeared in treated animals and the mortality reached 12 and 30%, respectively. The levels of brain dopamine, norepinephrine and serotonin decreased in a dose-dependent manner. The activities of brain total ATPase, Mg 2+-ATPase and Na +/K +- ATPase were suppressed. The activity of Na +/K +- ATPase was more severely affected than that of Mg 2+-ATPase. Histopathological changes in brain included hyperaemia, focal haemorrhages in vacuolated myelinated fibres, chromatolysis, or complete necrosis of neurons, degenerative changes, or complete absence of purkinje cells in the cerebellum.

Attenuation of Ca 2+-activated ATPase and shortening velocity in hypertrophied fast twitch skeletal muscle from aged Japanese quail

The effect of aging on the in vitro contractile properties of the patagialis (PAT) muscle of 35 young adult (YA; 8 weeks of age) and 35 aged adult (AA, 110 weeks of age) Coturnix quails was examined after 0–30 days of stretch-overload. Overload was achieved by placing a weight equivalent to 12% of the birds' body weight on one wing. The contralateral wing served as the intra-animal control. Overload increased the weight of the PAT by 45.1±2.1% in YA, and 24.1±2.6% in AA. Twitch contraction time increased with loading from 43.2±1.2 to 67.3±2.2 ms in YA birds and 57.2±1.7 to 77.4±1.9 ms in AA birds. Unloaded shortening velocity (Vo) decreased by 40.1±2.2 and 38.8±3.2% in YA and aged birds, respectively. The decrease in fast myosin expression was greater in overloaded muscles of YA (20%) as compared to AA birds (12%). However, this was accompanied by a greater decrease in total muscle ATPase activity in aged birds (61%) compared to YA birds (40%). Myosin isozyme Ca 2+-ATPase activity was 26% lower in FM1 but not other fast myosins in YA birds, but it was ∼30% lower in all fast myosins in PAT muscles of aged birds. These data show that the reduction of Vo and the increase in twitch duration with aging may be due in part to reductions in ATPase activity in all myosin isoforms, as compared to myosin isoforms isolated from YA birds.

Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production.

Effect of isoproturon pretreatment on the biochemical toxicodynamics of anilofos in male rats

Anilofos and isoproturon are important herbicides of organophosphorus and substituted phenylurea groups, respectively. Isoproturon is an inducer of hepatic drug-metabolizing enzymes. Animals and humans have the potential to be exposed to the mixture of these intentionally introduced environmental xenobiotics, but toxicological interactions between these herbicides are not known. Effects of isoproturon pretreatment (675 mg/kg/day for 3 consecutive days) on the toxic actions of anilofos administered orally as a single dose (850 mg/kg) were evaluated by determining some biochemical attributes in blood (erythrocyte/plasma), brain and liver of rats. Anilofos or isoproturon alone or in combination failed to produce any noticeable signs of cholinergic hyperactivity and behavioural alterations. Isoproturon did not potentiate the anticholinesterase action of anilofos in blood and liver. Inhibition of brain acetylcholinesterase was significantly protected. No significant alteration in anilofos-mediated production of lipid peroxidation was observed in erythrocyte and brain of isoproturon-pretreated rats, but it was significantly increased in liver. Anilofos did not affect GSH and GST. The isoproturon-mediated increase in GSH levels of brain (threefold) and liver (3.6-fold) was also not affected following combined administration. GST activity was increased in liver of rats given isoproturon alone (fourfold) or in combination with anilofos (2.8-fold). Activities of total ATPase, Mg 2+-ATPase and Na +-K +-ATPase were not affected in rats given either anilofos alone or herbicides in sequence. With these treatments, there were no alterations in the protein content of plasma, brain and liver. Overall findings of the study indicate that isoproturon pretreatment does not alter the toxicity of anilofos, the GSH–GST metabolic pathway may not have a significant implication in the detoxification of anilofos and the production of a reactive oxygen species may be a factor in mediating anilofos toxicity.

ATP utilization for calcium uptake and force production in different types of human skeletal muscle fibres.

The contractile properties and ATPase activity of skinned human skeletal muscle fibres from vastus lateralis were examined. Fibre types were resolved from single fibre segments by SDS-polyacrylamide gel electrophoresis. ATPase activity was determined by enzymatic coupling of ATP resynthesis to the oxidation of NADH. The partitioning of ATPase activity into (a) calcium-activated activity due to actomyosin (AM) interaction, (b) calcium-activated activity of the sarcoplasmic reticular (SR) calcium pump, and (c) basal (calcium independent) activity was investigated by comparing ATP utilization before and after exposure of the preparations for 30 min to a solution containing 0.5 % Triton X-100, which effectively abolished the SR ATPase activity. Partitioning of ATPase activity was also determined by measuring ATP utilization and force at different concentrations of butanedione monoxime (BDM), which inhibits AM interaction. The results obtained with Triton X-100 and BDM were similar. At saturating Ca2+ concentrations and 20 degrees C, the AM, SR and basal ATPase activities per litre cell volume (+/- S.E.M.) amounted to 46 +/- 4, 51 +/- 4 and 19 +/- 2 muM s-1 in type I fibres (n = 21), 139 +/- 14, 69 +/- 8 and 30 +/- 3 muM s-1 in type IIA fibres (n = 25), 137 +/- 22, 175 +/- 28 and 26 +/- 8 muM s-1 in type IIA/B fibres (n = 4) and 108 +/- 13, 169 +/- 42 and 32 +/- 8 muM s-1 in type IIB fibres (n = 2). These results indicate that ATP utilization for SR Ca2+ pumping in fast fibres is considerably larger than in slow fibres. The SR ATPase activity in human muscle represents a considerable fraction of the total (AM + SR + basal) ATPase activity.

Simulated biological effects of microgravity on phospholipid and energy metabolism of chicken embryonic brain cells studied by31P-NMR spectroscopy

Levels of phosphomonoester (PME), phosphodiester (PDE), ATP and pH in brain cells of chicken embryos rotated for 24 h in a clinostat during the period of hatching the 13th day (E13) and 15th day (E15) embryos were investigated by using(31)P-NMR spectroscopy. Significant increases in the values of PME, ATP and pH were identified after E13 rotating for 24 h. With the same treatment, differences were obtained in the phospholipid and energy metabolism of E15, but no significant levels have been reached. The calorimetric assay (malachite green method) was used for measuring the activity of total ATPase. A dramatic decrease was evident in the activity of ATPase in brain cells of rotated E13 and E15. The former is more sensitive than the latter. All the levels mentioned above could restore in 24 h after the rotation stopped, except that the level of ATP was still higher than the control.

Characterization of (Na +, K +)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na +, K +)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na +, K +)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na +, K +)-ATPase activity. Western blotting showed that M. olfersii gill contains a single α-subunit isoform of about 110 kDa. The (Na +, K +)-ATPase hydrolyzed ATP with Michaelis–Menten kinetics with K 0.5=165±5 μM and V max=686.1±24.7 U mg −1. Stimulation by potassium ( K 0.5=2.4±0.1 mM) and magnesium ions ( K 0.5=0.76±0.03 mM) also obeyed Michaelis–Menten kinetics, while that by sodium ions ( K 0.5=6.0±0.2 mM) exhibited site–site interactions ( n=1.6). Ouabain ( K 0.5=61.6±2.8 μM) and vanadate ( K 0.5=3.2±0.1 μM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A 1. These data suggest that the (Na +, K +)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F 0F 1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na +, K +)-ATPase activity may be regulated by the Na + concentration of the external medium.

Activity of Na +, K +-activated adenosine triphosphatase in healthy Nigerian children

The activity of Na +, K + - ATPase, a cell membrane enzyme which maintains trans — membrane ionic concentration gradient, was determined in 40 healthy young Nigerian Children (22 males and 18 females) using the erythrocyte membrane. The mean ATPase activity is 0.230 + 0.090 μmol/h/mg protein at 37° C. This constituted 70 percent of total ATPase activity. This study shows that in absolute terms, both total and ouabain sensitive ATPase activities differ from values obtained in similar age range in other regions and from older age groups in same locality. The study was carried out as a preliminary part of a more detailed study of the status of the sodium pump in severely malnourished Nigerian children.

Piperonyl butoxide potentiates the synaptosome ATPase inhibiting effect of pyrethrin

Pyrethrins are widely used insecticides in both agriculture and households. In many commercial formulations piperonyl butoxide (PBO) is used with pyrethrins. PBO is a well-known synergist of pyrethrins, used to intensify their effects. One of the cellular targets of pyrethrins is the sodium channel in the membrane. In the present study, the activity of the membrane-bound integral protein ATPase was studied as a biomarker for the membrane effects of pyrethrin and PBO. Cerebral synaptosomes of rat brain were used in the study. The isolation of synaptosomes was performed with the Percoll gradient method. Both total ATPase and Mg 2+ activated ATPase were studied by determining inorganic phosphate. Exposure to 0.1–1000 μM of pyrethrin and to 0.4–4000 μM of PBO decreased ATPase activity dose-dependently. The most efficient mixture was the one consisting of one part of pyrethrin and four parts of PBO. The activity of total ATPase decreased 15% in concentrations of 0.1–10 μM pyrethrin, and a 50% decrease was found at 100 μM pyrethrin. The mixture of pyrethrin and PBO caused a 15–60% decrease in the total ATPase activity at 0.1–10 μM pyrethrin and 0.4–40 μM PBO. A 85% decrease was found after exposure to the mixture of 100 μM pyrethrin and 400 μM PBO. PBO alone had no effect at 0.4–40 μM concentrations, but a marked effect was seen at over 40 μM concentrations. The results indicate that PBO is an effective synergist of pyrethrin and that it is very toxic in high concentrations. The results also confirm that neuronal sodium homeostasis is one target of the neurotoxic effect of pyrethroid compounds.