Alterations in Leishmania donovani 3'-nucleotidase/nuclease activity banding pattern in polyacrylamide gels.
1. In the absence of protease inhibitors, partial purification of the 3'-nucleotidase/nuclease (3'-N'ase) from detergent extracts of Leishmania donovani leads to the appearance of bands, at 42,000 and 38,000 Da, of enzyme activity which, in SDS-PAGE, migrate faster than 43,000 Da, the apparent weight of the intact polypeptide. 2. The generation of these faster migrating species is a consequence of detergent extraction and is prevented by the addition of the protease inhibitors PMSF and leupeptin, suggesting that degradation by an endogenous protease is responsible. 3. Treatment of leishmanial membranes with exogenous proteases yields activity at 38,000 Da which is membrane-associated. Protease treatment has no effect on living cells. 4. The observed changes in enzyme migration are discussed in terms of enzyme stability and regulation. 5. The advantages and limitations of the in situ gel activity assay are considered.
Campbell
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TA
Department of Immunology and Infectious Diseases, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205.
Mackow
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MC
Gottlieb
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D000818:Animals; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides; D007893:Leishmania donovani / Q000201:enzymology*; D008274:Magnesium; D008970:Molecular Weight; D009708:Nucleotidases / Q000302:isolation & purification*; D011480:Protease Inhibitors; D012967:Sodium Dodecyl Sulfate
D005960:Glucosides; D011480:Protease Inhibitors; C018619:octyl-beta-D-glucoside; D012967:Sodium Dodecyl Sulfate; D004492:Edetic Acid; D009708:Nucleotidases; C040470:3'-nucleotidase; D008274:Magnesium