Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8.

SDS, C12E8, 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 C12E8 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 4 degrees C after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C12E8/ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4 degrees C. 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 (K0.5 = 4.0 microM) and low (K0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 microg/ml) and sodium vanadate (3 microM) inhibited the ATPase activity of C12E8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5%, respectively. We have shown that Na,K-ATPase solubilized only with C12E8 can be purified and retains its activity. The activity is consistent with the form of (alphabeta)2 association.

Solubilization of Na, K-ATPase from rabbit kidney outer medulla using only C12E8

SDS, C12E8, 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 C12E8 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 C12E8/ml, yielding 98 percent 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 (K0.5 = 4.0 æM) and low (K0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 æg/ml) and sodium vanadate (3 æM) inhibited the ATPase activity of C12E8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5 percent, respectively. We have shown that Na,K-ATPase solubilized only with C12E8 can be purified and retains its activity. The activity is consistent with the form of (alphaß)2 association

[Stereotactic implantation of depth electrodes by magnetic resonance image in epilepsy surgery]. / Implantação estereotáxica de eletrodos profundos por ressonância magnética para cirurgia de epilepsia.

We present the case of a 40-year-old woman with refractory epilepsy since aged 18, who was submitted to video-EEG monitoring with intracerebral depth electrodes. The clinical history and examination, magnetic resonance image (MRI), video-EEG and neuropsychological study did not allow the determination of the cerebral onset of epileptic seizures. Depth electrodes inserted by MRI-guided stereotaxis allowed the recording of the epileptic activity and thus showed quite accurately the area of the brain to be surgically resected. She underwent a right anterior temporal lobectomy with amygdalohippocampectomy. The immediate postoperative period was uneventful and she is without epileptic seizures after three months of follow-up. The average pre-operative free-seizure period was two weeks. To our knowledge, this is the first stereotactic surgery for insertion of depth intracerebral electrodes in epilepsy in Brazil.