Immobilization of Na, K-ATPase isolated from rat brain synaptic plasma membranes (original) (raw)
Related papers
Influence of transition and heavy metal ions on ATP-ases activity in rat synaptic plasma membranes
Journal of the Serbian Chemical Society, 2004
The influence of transition metal (Cu 2+ , Zn 2+ , Fe 2+ and Co 2+ ) and heavy metal ions (Hg 2+ , Pb 2+ and Cd 2+ ) on the activities of Na + /K + -ATPase and Mg 2+ -ATPase isolated from rat synaptic plasma membranes (SPM) was investigated. The aim of the study was to elucidate the inhibition of both ATPase activities by exposure to the considered metal ions as a function of their affinity to bind to the -SH containing ligand L-cysteine, as a model system. The half-maximum inhibitory activities (IC 50 ) of the enzymes were determined as parameters of rectangular hyperbolas and correlated with the stability constant (K s ) of the respective metal-ion-L-cysteine complex. The linear Dixon plots indicate equilibrium binding of the investigated ions to both enzymes.
ATPases as Multi-Response Sensing System for Various Organic and Inorganic Analytes
2004
The possibility of using synaptic plasma membrane (SPM) enzymes Na þ =K þ -ATPase and Mg 2þ -ATPase, isolated from rat brain, as a biological component of multi-response sensing system for detection of different compounds (alkaline and heavy metal salts, organic compounds) was studied. The method is based on the spectrophotometric determination of inorganic ortho-phosphate (P i ) that serves as a measure of the enzymatic activity in the presence of various analytes. The concentration of P i , liberated by enzyme catalysed hydrolysis of adenosinetriphosphate (ATP), was followed spectrophotometrically, by single exposure to analytes or in the mixture. P i was dose dependent on the analyte concentration. Alkaline elements (Na, K, Mg), heavy metals (Pb, Cd, Hg, Cu, Fe, Co, Zn), toxic organic compounds (pyridine, urea, chlorpyrifos), and some drugs (digoxin, gitoxin) showed diverse effects, inducing the inhibition or stimulation of the enzymes activity. Development of simple test method for simultaneous detection of the investigated analytes based on the variation of medium assay composition was discussed. metals and organic compounds are required . Because these pollutants are known to influence the activity of many enzymes, the development of respective detection systems becomes increasingly interesting . They usually use enzymes immobilized on solid support or simply enzyme assays in the water solution .
Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8
Brazilian Journal of Medical and Biological Research, 2002
SDS, C 12 E 8 , CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C 12 E 8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,K-ATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C 12 E 8 /ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K 0.5 = 4.0 µM) and low (K 0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 µg/ml) and sodium vanadate (3 µM) inhibited the ATPase activity of C 12 E 8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5%, respectively. We have shown that Na,K-ATPase solubilized only with C 12 E 8 can be purified and retains its activity. The activity is consistent with the form of (aß) 2 association.
Na + /K + ATPase: Activity and inhibition
Russian Journal of Physical Chemistry, 2009
The aim of the study was to give an overview of the mechanism of inhibition of Na+/K+-ATPase activity induced by some specific and non specific inhibitors. For this purpose, the effects of some ouabain like compounds (digoxin, gitoxin), noble metals complexes ([PtCl2DMSO2], [AuCl4]−, [PdCl4]2−, [PdCl(dien)]+, [PdCl(Me4dien)]+), transition metal ions (Cu2+, Zn2+, Fe2+, Co2+), and heavy metal ions (Hg2+, Pb2+, Cd2+) on the activity of Na+/K+-ATPase from rat synaptic plasma membranes (SPM), porcine cerebral cortex and human erythrocytes were discussed.
Purification of the synaptosomal plasma membrane (Ca2+ + Mg2+)-ATPase from pig brain
Biochemical Journal, 1996
The Ca2+-ATPase from the synaptosomal plasma membrane has been purified nearly to homogeneity from pig brain by a new procedure involving the calmodulin-affinity-chromatography technique. This is a convenient alternative to the standard methods for the purification of the plasma membrane Ca2+-ATPase from different sources that were unsuitable to purify the enzyme from pig brain. The main feature of this procedure is the use of 15% (v/v) glycerol as stabilizing agent, instead of acidic phospholipid. By using this protocol the enzyme was purified 36-fold with respect to the plasma membrane vesicle fraction, showing a specific activity of 2.3 i.u. in the presence of acidic phospholipid. In SDS/PAGE, it appears as a single protein band around Mr 140000 that can be phosphorylated by [γ-32P]ATP in the presence of La3+ and recognized by specific antibodies against the plasma membrane Ca2+-ATPase from pig antral smooth muscle. Calmodulin activates the enzyme 1.5–1.8-fold in the presence of ...
Na+,K+ATPase as the Target Enzyme for Organic and Inorganic Compounds
Sensors, 2008
This paper gives an overview of the literature data concerning specific and non specific inhibitors of Na + ,K + -ATPase receptor. The immobilization approaches developed to improve the rather low time and temperature stability of Na + ,K + -ATPase, as well to preserve the enzyme properties were overviewed. The functional immobilization of Na + ,K + -ATPase receptor as the target, with preservation of the full functional protein activity and access of various substances to an optimum number of binding sites under controlled conditions in the combination with high sensitive technology for the detection of enzyme activity is the basis for application of this enzyme in medical, pharmaceutical and environmental research.
Biochimica Et Biophysica Acta - Biomembranes, 1978
Effects of commonly used purification procedures on the yield and specific activity of (Na ÷ + K*)-ATPase (Mg2÷-dependent, Na* + K÷-activated ATP phosphohydrolase, EC 3.6.1.3), the turnover number of the enzyme, and the kinetic parameters for the ATP<tependent ouabain-enzyme interaction were compared in canine brain, heart and kidney. Kinetic parameters were estimated using a graphical analysis of non-steady state kinetics. The protein recovery and the degree of increase in specific activity of (Na*+ K*)-ATPase and the ratio between (Na*+ K*)-ATPase and Mg2÷-ATPase activities during the successive treatments with deoxycholate, sodium iodide and glycerol were dependent on the source of the enzyme. A method which yields highly active (Na'+ K*)-ATPase preparations from the cardiac tissue was not suitable for obtaining highly active enzyme preparations from other tissues. Apparent turnover numbers of the brain (Na*+ K*)-ATPase preparations were not significantly affected by the sodium iodide treatment, but markedly decreased by deoxycholate or glycerol treatments. Similar glycerol treatment, however, failed to affect the apparent turnover number of cardiac enzyme preparations. Cerebral and cardiac enzyme preparations obtained by deoxycholate, sodium iodide and glycerol treatments had lower affinity for ouabain than renal enzyme preparations, primarily due to higher dissociation rate constants for the ouabain • enzyme complex. This tissue-dependent difference in ouabain sensitivity seems to be an artifact of the purification procedure, since less purified cerebral or cardiac preparations had lower dissociation rate constants. Changes in apparent association rate constants were minimal during the purification procedure. These results indicate that the presently used purification procedures may alter * To whom reprint requests should be sent. the properties of membrane (Na*+ K÷)-ATPase and affect the interaction between cardiac glycosides and the enzyme. The effect of a given treatment depends on the source of the enzyme. For the in vitro studies involving purified (Na ÷ + K÷)-ATPase preparations, the influence of the methods used to obtain the enzyme preparation should be carefully evaluated.
Toxicology Letters, 1999
Enzymatic activities of Na + /K + -ATPase and Mg 2 + -ATPase from rat brain synaptic plasma membrane were studied in the absence and presence of EDTA. The aim of the study was to examine the ability of this strong chelator to prevent and recover the CuSO 4 -induced inhibition. The influence of experimentally added CuSO 4 and EDTA on MgATP 2 − complex and 'free' Cu 2 + concentrations in the reaction mixture was calculated and discussed. CuSO 4 induced dose-dependent inhibition of both enzymes in the absence and presence of 1 mM EDTA. In the absence of EDTA, the IC 50 values of Cu 2 + , as calculated from the experimental curves, were 5.9 × 10 − 7 M for Na + /K + -ATPase and 3.6× 10 − 6 M for Mg 2 + -ATPase. One millimolar EDTA prevented the enzyme inhibition induced by CuSO 4 , but also reversed the inhibited activity, in a concentration-dependent manner, following exposure of the enzymes to the metal ion, by lowering 'free' Cu 2 + concentration. Kinetic analysis showed that CuSO 4 inhibits both the Na + /K + -ATPase and Mg 2 + -ATPase, by reducing their maximum enzymatic velocities (V max ), rather than apparent affinity for substrate MgATP 2 − (K 0.5 ), implying the noncompetitive nature of enzyme inhibition induced by the metal. The kinetic analysis also confirmed two distinct Mg 2 + -ATPase subtypes activated in the presence of low and high MgATP 2 − concentrations. K 0.5 and V max were calculated using a computer-based program. The results of calculation showed that MgATP 2 − concentration in the kinetic experiments exceeded three times the apparent K 0.5 value for the enzyme activation.
The Na,K-ATPase (NKA) System is thé receptor for thé cardioactive steroids of plant or animal origin. It is not yet known whether passive ion f luxes traverse thé înactivated receptor and thereby contribute to thé hormonal, pharmacological or toxic actions of thèse compounds. To look for putative passive ionfluxes across thé ouabain-NKA complex, we incorporated it into thé artificial membrane of liposomes. Since this synthetic membrane is virtually imperméable to Na and K ions, thé hypothetical ion-fluxes mediated by thé NKA molécule can be determined. E2-forms and E2-ouabain-forms of purified NKA were incorporated, in parallel, into separate liposome préparations and thé permeability of thé resulting E2-liposomes and E2-ouabainliposomes to K, Na and Ça ions was compared. The E2-liposomes expressed a typical K-permeabilty which was not observed in thé E2-ouabain-liposomes; thé latter showed a slightly higher Na-permeability and a similar Ca-permeability as compared to thé former. Thus, ouabain does not induce leaks for K or Ça ions in thé NKA molécule.