Dependent Adenosine Triphosphatase Research Articles

The Requirement and Protective Effects of Dietary Protein against Chronic Ammonia Exposure in Juvenile Genetically Improved Farmed Tilapia (Oreochromis niloticus).

Ammonia is a key risk factor in intensive aquaculture systems. This experiment is aimed at investigating the influence of dietary protein levels on genetically improved farmed tilapia (GIFT, Oreochromis niloticus) under chronic ammonia stress. GIFT juveniles of 4.00 ± 0.55 g were exposed to high ammonia level at 0.88 mg/L and fed with six diets comprising graded protein levels at 22.64%, 27.26%, 31.04%, 35.63%, 38.47%, and 42.66% for 8 weeks. The fish in negative control was fed the diet with 31.04% protein in normal water (0.02 mg ammonia/L water). Our results showed that high ammonia exposure (0.88 mg/L) caused significant decrease in fish growth performance, hematological parameters, liver antioxidant enzymes (catalase and glutathione peroxidase), and gill Na+- and K+-dependent adenosine triphosphatase (Na+/K+-ATP) activity. When fish were under high ammonia exposure, the weight gain rate, special growth rate, feed efficiency, and survival rate elevated significantly with dietary protein supplementation increase to 35.63%, whereas protein efficiency ratio, hepatosomatic index, and viscerosomatic index showed a decreased tendency. Dietary protein administration significantly enhanced crude protein but reduced crude lipid contents in the whole fish. Fish fed diets with 35.63%-42.66% protein had higher red blood cell counts and hematocrit percentage than fish fed 22.64% protein diet. The values of serum biochemical indices (lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase), hepatic antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase), and gill Na+/K+-ATP activity were all elevated with the increment of dietary protein. Moreover, histological analysis indicated that dietary protein administration could prevent the ammonia-induced damages in fish gill, kidney, and liver tissues. Based on weight gain rate as a response criterion, the optimal dietary protein requirement for GIFT juveniles under chronic ammonia stress was 37.9%.

Effect of thyroxine on Na+/K+-ATPase and SDH activities in gills and kidney during osmotic adjustments of Heteropneustes fossilis at higher salinity

Aim: The aim of the present study was to investigate the facilitatory role of thyroxine during adaptation process of catfish, Heteropneustes fossilis in higher salinity following its exogenous administration by monitoring changes in plasma osmolality, branchial and renal sodium-potassium dependent adenosine triphosphatase and succinic dehydrogenase enzyme activity profiles and study the effect of direct transfer of fish to higher salinities on plasma thyroxine levels. Methodology: Catfish were directly transferred from tap water to 30 and 35% sea water and plasma thyroxine profiles were analysed. Fish were injected with thyroxine at a dose of 2 and 5 µg g-1 b.wt. daily for five days and then transferred to tap water and 30% sea water. Fish were sampled after day 3 and day 6, post-transfer and plasma osmolality was measured, and the enzyme activities were determined in gills and kidney. Results: Higher levels of plasma thyroxine were observed after transfer of fish from tap water to higher salinities. Treatment of fish with thyroxine at higher dose (5 µg g-1 b.wt.) showed a significant increase in plasma osmolality in tap water (p<0.05) while the enzymes in gills were found to be significantly higher both in tap water (p<0.05; p<0.01for Na+/K+-ATPase and p<0.001 for SDH) and 30% sea water (p<0.001for Na+/K+-ATPase and p<0.01; p<0.001 for SDH). No significant changes were observed in any of the parameters analysed after exogenous administration of thyroxine at lower dose (2 µg g-1 b.wt.). Interpretation: Thyroxine affects the osmotic adjustment of fish following transfer to higher salinities and its exogenous administration at at a dose of 5 µg g-1 b.wt. may improve the hypoosmoregulatory ability. Key words: Hormonal control, Osmoregulation, Teleost, Thyroid hormones

Recruitment of Muscle Genes as an Effect of Brown Adipose Tissue Ablation in Cold-Acclimated Brandt’s Voles (Lasiopodomys brandtii)

Skeletal muscle-based nonshivering thermogenesis (NST) plays an important role in the regulation and maintenance of body temperature in birds and large mammals, which do not contain brown adipose tissue (BAT). However, the relative contribution of muscle-based NST to thermoregulation is not clearly elucidated in wild small mammals, which have evolved an obligate thermogenic organ of BAT. In this study, we investigated whether muscle would become an important site of NST when BAT function is conditionally minimized in Brandt's voles (Lasiopodomys brandtii). We surgically removed interscapular BAT (iBAT, which constitutes 52%~56% of total BAT) and exposed the voles to prolonged cold (4 °C) for 28 days. The iBAT-ablated voles were able to maintain the same levels of NST and body temperature (~37.9 °C) during the entire period of cold acclimation as sham voles. The expression of uncoupling protein 1 (UCP1) and its transcriptional regulators at both protein and mRNA levels in the iBAT of cold-acclimated voles was higher than that in the warm group. However, no difference was observed in the protein or mRNA levels of these thermogenesis-related markers except for PGC-1α in other sites of BAT (including infrascapular region, neck, and axilla) between warm and cold groups either in sham or iBAT-ablated voles. The iBAT-ablated voles showed higher UCP1 expression in white adipose tissue (WAT) than sham voles during cold acclimation. The expression of sarcolipin (SLN) and sarcoplasmic endoplasmic reticulum Ca2+-dependent adenosine triphosphatase (SERCA) in skeletal muscles was higher in cold than in warm, but no alteration in phospholamban (PLB) and phosphorylated-PLB (P-PLB) was observed. Additionally, there was increased in iBAT-ablated voles compared to that in the sham group in cold. Moreover, these iBAT-ablated voles underwent extensive remodeling of mitochondria and genes of key components related with mitochondrial metabolism. These data collectively indicate that recruitment of skeletal muscle-based thermogenesis may compensate for BAT impairment and suggest a functional interaction between the two forms of thermogenic processes of iBAT and skeletal muscle in wild small mammals for coping cold stress.

Altering membrane potential for cancer

Cancer Polymorphisms in the G protein–coupled receptor GPR35 are associated with increased risk for certain inflammatory diseases that can progress to cancer. Schneditz et al. found that GPR35 promoted the activity of Na+- and K+-dependent adenosine triphosphatase (Na+,K+-dependent ATPase), a transmembrane pump that sets the membrane potential in cells. This effect was enhanced by a disease-associated GPR35 variant. Stimulation of Na+,K+-ATPase activity by GPR35 increased glycolysis and proliferation in intestinal epithelial cells. Na+,K+-ATPase deficiency or treatment with a pepducin targeting GPR35 decreased tumor burden in mouse models of intestinal cancer. Sci. Signal. 12 , eaau9048 (2019).

Fluorescent Ca2+ indicators directly inhibit the Na,K-ATPase and disrupt cellular functions.

Fluorescent Ca2+ indicators have been essential for the analysis of Ca2+ signaling events in various cell types. We showed that chemical Ca2+ indicators, but not a genetically encoded Ca2+ indicator, potently suppressed the activity of Na+- and K+-dependent adenosine triphosphatase (Na,K-ATPase), independently of their Ca2+ chelating activity. Loading of commonly used Ca2+ indicators, including Fluo-4 acetoxymethyl (AM), Rhod-2 AM, and Fura-2 AM, and of the Ca2+ chelator BAPTA AM into cultured mouse or human neurons, astrocytes, cardiomyocytes, or kidney proximal tubule epithelial cells suppressed Na,K-ATPase activity by 30 to 80%. Ca2+ indicators also suppressed the agonist-induced activation of the Na,K-ATPase, altered metabolic status, and caused a dose-dependent loss of cell viability. Loading of Ca2+ indicators into mice, which is carried out for two-photon imaging, markedly altered brain extracellular concentrations of K+ and ATP. These results suggest that a critical review of data obtained with chemical Ca2+ indicators may be necessary.

Preventive Effect of Phytic Acid on Isoproterenol-Induced Cardiotoxicity in Wistar Rats

This study was aimed to evaluate the preventive role of phytic acid on membrane bound enzymes such as sodium potassium- dependent adenosine triphosphatase (Na+ /K+ ATPase), calcium-dependent adenosine triphosphatase (Ca2+ ATPase) and magnesium- dependent adenosine triphosphatase (Mg2+ ATPase) and glycoproteins such as hexose, hexosamine, fucose and sialic acid in isoproterenol (ISO)-induced myocardial infarction (MI) in rats. Male albino Wistar rats were pretreated with phytic acid (25 and 50 mg/kg, respectively) for a period of 56 days. After the treatment period, ISO (85 mg/kg) was subcutaneously injected to rats at an interval of 24 h for 2 days. ISO-induced rats showed a significant decrease in the activity of Na+ /K+ ATPase and increase in the activities of Ca2+ and Mg2+ ATPase in the heart and a significant (P<0.05) increase in the levels of glycoproteins in serum and the heart were also observed in ISO-induced rats. Pretreatment with phytic acid for a period of 56 days exhibited a significant (P<0.05) effect and altered these biochemical parameters positively in ISO-induced rats. Thus, our study shows that phytic acid has cardioprotective role in ISO-induced MI in rats.

Effect of dietary consumption of sheep meat on thyroid hormone levels and energy expenditure of Sprague - Dawley rats

The purpose of the present study was to investigate the effects of a sheep meat-based diet on the thyroid hormone concentrations and energy expenditure of Sprague–Dawley rats. The diets, a sheep meat diet (SMD) or a casein control diet (CD), each based on a standard formulation (AIN-93G), contained 18.3% protein, 7.4% fat and 60.0% carbohydrate, and were isocaloric (15.9kJ/g dry matter). Serum tri-iodothryonine (T3), thyroxine (T4), free tri-iodothyronine (FT3) and free thyroxine (FT4) concentrations, oxygen consumption rate (OCR), bodyweight, bodyweight gain and activities of liver sodium–potassium-adenosinetriphosphatase (Na,K-ATPase) and Ca2+-dependent adenosine triphosphatase (Ca-ATPase) were measured. Compared with the CD, the SMD increased the serum FT3 concentration on Day 7 (P = 0.004) and Day 14 (P < 0.001). Serum albumin concentrations, which may influence the amount of thyroid hormones bound, were significantly lower in the SMD group on Day 7 (P < 0.001) and Day 14 (P = 0.02) than those in the CD group. Both liver Na,K-ATPase and Ca-ATPase activities were significantly higher in rats consuming the SMD on Day 7 (P < 0.001, P = 0.001) and Day 14 (P < 0.001, P < 0.001) respectively. OCRs were significantly higher in rats consuming the SMD on Day 6 (P = 0.023) and Day 13 (P = 0.014). Both bodyweight and bodyweight gains were significantly lower in the SMD group. We concluded that sheep meat consumption decreased the serum albumin concentrations, which increased the serum FT3 concentrations. This lead to FT3 promoting the OCRs and the activities of Na,K-ATPase and Ca-ATPase, resulting in reduced bodyweight and bodyweight gain, presumably through greater energy expenditure. Thus, energy expenditure appears to have been enhanced in rats fed the SMD.

Characterization of a response regulator protein that binds to Anabaena sp. strain L-31 kdp-promoter region

The potassium dependent adenosine triphosphatase (Kdp-ATPase), encoded by the kdp operon, is a potassium uptake system found in several prokaryotes. The cyanobacterium Anabaena sp. strain L-31 shows the presence of two kdp operons (kdp1 and kdp2) of which the kdp2 is predominantly induced in response to potassium limitation or desiccation stress. Two ORFs, encoding a sensor kinase and a response regulator, respectively, were identified upstream of the kdp2 operon in Anabaena sp. strain L-31. The response regulator protein, tagged with 6 additional C-terminal histidine residues, was over-expressed in Escherichia coli and purified by affinity chromatography. Employing the protein-specific antiserum, the response regulator protein was detected in Anabaena L-31 cytosolic fractions. The response regulator protein bound to the kdp2 promoter region with higher affinity than kdp1 promoter region. Addition of acetyl phosphate increased the ability of the protein to bind to kdp2 promoter region by several fold, suggesting a phosphorylation-dependent binding of response regulator to the promoter. These data implicate the response regulator protein in regulation of kdp2 expression in Anabaena sp. strain L-31.

Subcellular redistribution of trimeric G-proteins – potential mechanism of desensitization of hormone response; internalisation, solubilization, down-regulation

Agonist-induced subcellular redistribution of G-protein coupled receptors (GPCR) and of trimeric guanine-nucleotide binding regulatory proteins (G-proteins) represent mechanisms of desensitization of hormone response, which have been studied in our laboratory since 1989. This review brings a short summary of these results and also presents information about related literature data covering at least small part of research carried out in this area. We have also mentioned sodium plus potassium dependent adenosine triphosphatase (Na, K-ATPase) and 3H-ouabain binding as useful reference standard of plasma membrane purity in the brain.

Preventive effect of rutin on lipids, lipoproteins, and ATPases in normal and isoproterenol‐induced myocardial infarction in rats

The present study was designed to evaluate the preventive role of rutin on lipids, lipoproteins, and ATPases in normal and isoproterenol (ISO)-induced myocardial infarction in rats. Rutin (40 and 80 mg/kg) was orally administered to rats for a period of 42 days. After that period, isoproterenol (150 mg/kg) was injected subcutaneously to male wistar rats at an interval of 24 h for 2 days. The weight of heart and the concentrations of total cholesterol, triglycerides, and free fatty acids were increased significantly (p < 0.05), and the concentration of phospholipids was decreased significantly (p < 0.05) in the heart of ISO-treated rats. ISO-treated rats also showed a significant increase (p < 0.05) in the levels of total cholesterol, triglycerides, phospholipids, low-density lipoprotein cholesterol (LDL-C), and very low-density lipoprotein cholesterol (VLDL-C) with a significant decrease (p < 0.05) in high-density lipoprotein cholesterol (HDL-C) level in serum. The activities of sodium potassium dependent adenosine triphosphatase (Na(+)/K(+) ATPase) and magnesium-dependent adenosine triphosphatase (Mg(2+) ATPase) were decreased significantly (p < 0.05), and the activity of calcium-dependent adenosine triphosphatase (Ca(2+)ATPase) was increased significantly (p < 0.05) in the heart in ISO-treated rats. Pretreatment with rutin at doses of 40 or 80 mg/kg to ISO-treated rats showed a significant (p < 0.05) effect in all the parameters studied. Oral administration of rutin to normal rats did not show any significant effect. Thus, the results of our study show that pretreatment with rutin maintained the levels of lipids, lipoproteins, and ATPases in ISO-induced myocardial infarcted rats. The observed effects might be due to the antioxidant potential of rutin.

Preventive effect of Aegle marmelos leaf extract on isoprenaline-induced myocardial infarction in rats: biochemical evidence

We have evaluated the preventive effects of an aqueous Aegle marmelos leaf extract (AMLEt) in isoprenaline (isoproterenol)-induced myocardial infarction in rats. Rats were pretreated with AMLEt (50, 100 or 200 mg kg(-1)) for 35 days. After the treatment period, isoprenaline (200 mg kg(-1)) was administered subcutaneously to rats at an interval of 24 h for two days. The activity of creatine kinase (CK) and lactate dehydrogenase (LDH) was significantly increased in serum and significantly decreased in heart of isoprenaline-treated rats. Pretreatment with AMLEt decreased the activity of CK and LDH in serum and increased them in the heart. The activity of sodium-potassium dependent adenosine triphosphatase (Na(+)K(+)ATPase) was significantly decreased while the activity of calcium dependent adenosine triphosphatase (Ca(2+)ATPase) was simultaneously increased in the heart and aorta. AMLEt pretreatment increased the activity of Na(+)K(+) ATPase and decreased the activity of Ca(2+)ATPase in the heart and aorta simultaneously. The levels of cholesterol and triglycerides increased, while the levels of phospholipids decreased in the heart and aorta of isoprenaline-treated rats. In AMLEt-pretreated rats the levels of cholesterol and triglycerides decreased whereas phospholipids increased in heart and aorta. All the deranged biochemical parameters were restored with 200 mg kg(-1) AMLEt. Similarly alpha-tocopherol (60 mg kg(-1))-pretreatment to isoprenaline-treated rats exhibited a significant effect on all the parameters studied. The results from this study may have clinical relevance.

Effect of the digitoxigenin derivative, INCICH-D7, on Na +, K +-ATPase

Compound 14β,17β-cycloketoester-3β-OH androstane (INCICH-D7) is a semisynthetic product of a structural modification of the digitoxigenin molecule. INCICH-D7 has a heterocyclic ketoester type fusion between positions C14 and C17 of the steroid nucleus, which confers this molecule stronger electronegativity than that of digitoxigenin. INCICH-D7 retained positive inotropic effect, with a greater safety margin, when compared to digitoxigenin and ouabain. In this study we have examinated the INCICH-D7 effect on Na +, K +-dependent adenosinetriphosphatase (Na +, K +-ATPase) and compared these results with the ones observed with digitoxigenin and ouabain. The inhibitory effect of INCICH-D7 on Na +, K +-ATPase was five times lower (IC 50 = 4 μM) than that of ouabain (IC 50 = 0.8 μM) and 70 times lower than that of digitoxigenin (IC 50 = 0.06 μM). The inhibitory effect of INCICH-D7 and ouabain on the enzyme was irreversible while digitoxigenin's one was reversible in up to an 80%. Our results indicate that inclusion of the heterocycle between positions C14 and C17 in the digitoxigenin molecule lowers significantly the inhibitory effect on Na +, K +-ATPase and renders the interaction between INCICH-D7 and enzyme irreversible under the studied reaction conditions.

The hepatoprotective action of ten herbal extracts in CCl4 intoxicated liver

The effect of ten phytotherapeutic products on CCl(4) intoxicated liver in albino male Wistar rats was investigated. Biochemical parameters, including serum transaminase activity (GPT and GOT), histoenzymological measurements (lactate dehydrogenase, LDH; succinate dehydrogenase, SDH, cytochromoxidase, CyOx; Mg(2+)-dependent adenosine triphosphatase, ATP-ase) and histochemical (Sudan black) and histological examinations (haematoxylin-eosin staining) of the liver were investigated. Some positive effects such as the reduction of hepatocytolysis and steatosis, and a return to normal values of the activity of some enzymes in the following plants: Chrysanthemum balsamita, Echinacea pallida, Calendula officinalis and Corylus avelana were obtained.

Turbellaria Phagocata sibirica: Some Enzymes of Carbohydrate and Energy Metabolisms

Activities and properties of some enzymes of carbohydrate and energy metabolisms in free-living turbellaria Phagocata sibirica are studied. The enzymes are studied in various subcellular fractions. A high activity of hexokinase is accompanied by high activity of glucose-6-phosphate dehydrogenase (G6PDG). The level of pyruvate kinase activity is sufficient to provide dissimilation of phosphoenolpyruvate with formation of pyruvate. P. sibirica has highly-active lactate dehydrogenase (LDH) and malate dehydrogenase (MDH); a predominance of MDH activity over LDH and a low activity of phosphoenolpyruvate carboxykinase is revealed. NADP-dependent isocitrate dehydrogenase is found, which is activated by Mn2+ and Mg2+ and inhibited by salts of heavy metals and p-chloromercuribenzoate. Activities and properties of α-ketoglutarate dehydrogenase, succinate dehydrogenase (SDH), and fumarate reductase are studied, and it is concluded that in P. sibirica there is the system of succinate oxidation, whereas the system of fumarate reduction into succinate is absent. Mitochondrial and microsomal fractions from P. sibirica had Mg2+- and Ca2+-dependent adenosine triphosphatases.

Changes in the structure and function of the kidney of rats chronically exposed to cadmium. II. Histoenzymatic studies.

Early effects of cadmium (Cd) on the structure and function of the kidney were studied in an experimental model using rats intoxicated with Cd at the levels of 5 and 50 mg Cd/l drinking water. The effect of Cd was evaluated histopathologically and biochemically. Damage to the cellular structures was assessed on the basis of histoenzymatic analyses of the activity and localization of indicator enzymes (succinate dehydrogenase, lactate dehydrogenase, glucose-6-phosphatase, Mg(2+)-dependent adenosine triphosphatase and acid phosphatase). The histochemical observations indicate that Cd causes damage to the organization and function of the nephron. Several structures, i.e. endoplasmic reticulum, mitochondrion, lysosome, cellular and intracellular membrane, as well as their biological functions, i.e. aerobic and anaerobic respiration, transport functions and biochemical processes taking place in the endoplasmic reticulum, were affected. The cytotoxic action of Cd occurs mainly in the tubules and partially also in the glomeruli. The results clearly indicate that Cd damages kidney structurally and functionally even at a relatively low level (5 mg/l) corresponding to human environmental exposure, and they confirm our previous hypothesis that the threshold for the kidney effects of Cd is less than 4.08+/-0.33 microg/g kidney wet weight and higher than 2.40+/-0.15 microg/g. The target for Cd action in the kidney is the tubules (proximal convoluted tubules and straight tubules), and disturbance in their function is the main toxic effect of Cd. Renal glomeruli are also injured, but only partially, whereas in other parts of the nephron the damage is slight. The results, together with observations reported in the first paper of the study, incline us to conclude that humans environmentally exposed to Cd are at risk of tubular damage.

Close Encounters Between the Na Pump and the IP 3 R

Miyakawa-Naito et al. uncovered an intriguing interaction between the Na,K-ATPase (Na + - and K + -dependent adenosine triphosphatase) and the inositol 1,4,5-trisphosphate receptor (IP 3 R), implicating a possible interaction of these two proteins in cell signaling. Ouabain, a cardiac glycoside initially identified as an arrow poison and long used experimentally as an inhibitor of the Na,K-ATPase, has more recently been proposed to function as a hormone. Partial inhibition of the Na,K-ATPase by ouabain elicits oscillations in intracellular calcium concentration ([Ca 2+ ] i ) and stimulates several transcription factors, suggesting that association of ouabain (a steroid) with the plasma membrane Na,K-ATPase activates a cell signaling pathway. Miyakawa-Naito et al. used pharmacological analysis to determine that ouabain-induced [Ca 2+ ] i oscillations in rat renal proximal tubule cells depended on IP 3 R-mediated release of calcium from endoplasmic reticulum (ER) stores. Fluorescence resonance energy transfer (FRET) analysis in COS-7 cells indicated close proximity of Na,K-ATPase to IP 3 R2 and IP 3 R3 and coimmunoprecipitation indicated that the Na,K-ATPase associated with IP 3 R3 in a complex. FRET and coimmunoprecipitation were increased by ouabain and abolished by disruption of the actin cytoskeleton, which also abolished ouabain-induced Ca 2+ oscillations, implicating the cytoskeleton in the association of plasma membrane Na,K-ATPase with ER IP 3 R. Oscillations persisted in cells treated with a phospholipase C inhibitor or transfected with a high-affinity IP 3 -binding protein. Partial truncation of the Na,K-ATPase α-1 subunit N-terminal indicated that the ability of ouabain-stimulated Na,K-ATPase to elicit Ca 2+ oscillations and activate nuclear factor-κB depended on a region that was not required for enzymatic activity. This study thus defines a functional microdomain linking the plasma membrane to ER Ca 2+ stores and adds weight to the possible role of the Na,K-ATPase as a hormone receptor and transducer of cell signaling. A. Miyakawa-Naito, P. Uhlén, M. Lal, O. Aizman, K. Mikoshiba, H. Brismar, S. Zelenin, A. Aperia, Cell signaling microdomain with Na,K-ATPase and inositol 1,4,5-trisphosphate receptor generates calcium oscillations. J. Biol. Chem . 278 , 50355-50361 (2003). [Abstract] [Full Text]

Mechanism of acute silver toxicity in Daphnia magna

Daphnids (Daphnia magna) were exposed to AgNO3 at 0.303 +/- 0.017 microg silver/L (46.9% as Ag+), in the absence of food, in moderately hard synthetic water under static conditions for up to 48 h. Results from accumulation experiments demonstrated that silver body burden was inversely related to body mass. Daphnids exposed to silver exhibited ionoregulatory disturbance, which was characterized by decreases in whole-body sodium concentration. This ionoregulatory disturbance was explained, at least in part, by a competitive inhibition of the whole-body sodium uptake (six- to sevenfold increase in the Michaelis constant with no change in maximal velocity), which was complete by 1 h of exposure, and resulted in approximately 40% inhibition of sodium influx from the water. A rapidly developing inhibition of whole-body Na+,K(+)-dependent adenosine triphosphatase (Na+,K(+)-ATPase) activity, significant by 2 h and complete at 90% blockade by 12 h, also was observed during exposure to AgNO3. Therefore, these findings clearly demonstrate that the key mechanism involved in acute Ag+ toxicity in D. magna, the most sensitive freshwater organism tested to date, resembles that described for freshwater fish--that is, inhibition of active sodium uptake by blockade of Na+,K(+)-ATPase. Furthermore, the results showed that Na+,K(+)-ATPase inhibition was directly related to silver accumulation in the whole body of D. magna. However, the nature of the sodium uptake inhibition (competitive vs noncompetitive in fish) and the fact that whole-body chloride concentration was not disturbed in daphnids was different from fish. With regard to the biotic ligand model (BLM) for silver, our results yielded a log K value of about 8.9. However, the current version of the BLM uses a rainbow trout log K value (7.3) but achieves the correct sensitivity of the model for daphnids by reducing the saturation of toxic sites needed to cause toxicity. An alternative way may be to use the log K value derived from the present results.

Interaction of cisplatin drug with Na,K-ATPase: drug binding mode and protein secondary structure.

cis-Pt(NH(3))(2)Cl(2) (cisplatin) is an antitumor drug with many severe toxic side effects including enzymatic changes associated with its mechanism of action. This study was designed to examine the interaction of cisplatin drug with the Na(+), K(+)-dependent adenosine triphosphatase (Na,K-ATPase) in H(2)O and D(2)O solutions at physiological pH, using drug concentrations of 0.1 microM to 1 mM. UV absorption spectra and Fourier transform infrared difference spectroscopy with its self-deconvolution, second derivative resolution enhancement and curve-fitting procedures were applied to characterize the drug binding mode, the drug binding constant and the protein secondary structure in the cisplatin-ATPase complexes. Spectroscopic evidence showed that at low drug concentration (0.1 microM), cisplatin binds mainly to the lipid portion of the enzyme, whereas at higher drug contents, the Pt cation interaction is through the polypeptide C==O and C-N groups with overall binding constant of K=1.93 x 10(4) M(-1). At high cisplatin concentration (1 mM), drug binding results in protein secondary structural changes from that of the alpha-helix 19.8%; beta-pleated 25.6%; turn 9.1%; beta-antiparallel 7.5% and random 38%, in the free Na,K-ATPase to that of the alpha-helix 22.2%; beta-pleated 23.2%; turn 9.4%; beta-antiparallel 2.2% and random 43%, in the cis-Pt-ATPase complexes.

The Effects of Anions on the Solution Structure of Na,K-ATPase

Anions interact with protein to induce structural changes at ligand binding sites. The effects of anion complexation include structural stabilization and promote cation-protein interaction. This study was designed to examine the interaction of aspirin and ascorbate anions with the Na+, K+-dependent adenosine triphosphatase (Na, K-ATPase) in H2O and D2O solutions at physiological pH, using anion concentrations of 0.1 μM to 1 mM with final protein concentration of 0.5 to 1 mg/ml. Absorption spectra and Fourier transform infrared (FTIR) difference spectroscopy with its self-deconvolution, second derivative resolution enhancement and curve-fitting procedures were applied to characterize the anion binding mode, binding constant, and the protein secondary structure in the anion-ATPase complexes. Spectroscopic evidence showed that the anion interaction is mainly through the polypeptide C=O and C-N groups with minor perturbation of the lipid moiety. Evidence for this came from major spectral changes (intensity variations) of the protein amide I and amide II vibrations at 1651 and 1550 cm−1, respectively. The anion-ATPase binding constants were K=6.45 × 103 M−1 for aspirin and K=1.04 × 104 M−1 for ascorbate complexes. The anion interaction resulted in major protein secondary structural changes from that of the αhelix 19.8%; β-pleated sheet 25.6%; turn 9.1%; β-antiparallel 7.5% and random 38% in the free Na, K- ATPase to that of the αhelix 24–26%; β-pleated 17–18%; turn 8%; β-antiparallel 5–3% and random 45. 0% in the anion-ATPase complexes.

THE DECOMPENSATED DETRUSOR III: IMPACT OF BLADDER OUTLET OBSTRUCTION ON SARCOPLASMIC ENDOPLASMIC RETICULUM PROTEIN AND GENE EXPRESSION

THE DECOMPENSATED DETRUSOR III: IMPACT OF BLADDER OUTLET OBSTRUCTION ON SARCOPLASMIC ENDOPLASMIC RETICULUM PROTEIN AND GENE EXPRESSION