Renaturation of Leishmania donovani 3'-nucleotidase following sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
1. Renaturation of a 3'-nucleotidase from the surface membrane of Leishmania donovani promastigotes was achieved following polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). 2. Enzyme activity was detected in situ in gels, following SDS removal, by incubating the gels in reaction mixtures containing 3'-AMP or 3'-UMP as substrate followed by staining for the inorganic phosphate (Pi) reaction product with malachite green-molybic acid solution. 3. Conditions for the removal of SDS by diffusion and for the renaturation of enzyme activity are described including evidence for the detergent requirement, which is best satisfied by 3[(3-cholamidopropyl)-dimethylammonio]2-hydroxy-1-propane sulfonate (CHAPSO). 4. Results indicate that the 3'-nucleotidase migrates under these conditions as a polypeptide with an Mr of 43,000.
D000818:Animals; D002793:Cholic Acids; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D007893:Leishmania donovani / Q000201:enzymology*; D008970:Molecular Weight; D009708:Nucleotidases / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity
D002793:Cholic Acids; D003902:Detergents; D012967:Sodium Dodecyl Sulfate; C048531:chapso; D009708:Nucleotidases; C040470:3'-nucleotidase