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Isolation and partial characterization of surface membranes from Leishmania donovani promastigotes
Abstract:
Cell surface pellicular membranes (PM) were isolated from promastigote forms of Leishmania donovani by differential and discontinuous sucrose gradient centrifugation procedures. The PM had a density equivalent of approximately 1.19 g/cm3. As ascertained by electron microscopy, longitudinal parallel arrays of subpellicular microtubules (MT) remained attached to the isolated PM inner lamina, and this feature was used to assess membrane fraction purity. Gradient fractions having greater than or equal to 95% of all membranes combined with MT were obtained routinely. The attached MT imparted a structural asymmetry to the PM permitting uniequivocal identification of the membrane external and cytoplasmic surfaces. The supramolecular structure of attached MT was evident in negatively stained PM. In ultrathin sections, PM had a mean width of approximately 7.2 nm and attached MT a diameter of approximately 29 nm. The MT were apparently cross-bridged both to each other and to the PM via a flocculent filamentoid nexus. As determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, isolated PM contained approximately 40 peptide bands ranging in apparent molecular weight from less than or equal 1.2 x 10(4) to greater than or equal to 2.2 x 10(5) daltons. Of these, 19 were stained with periodic acid-Schiffs' reagent suggesting that most PM carbohydrate constituents were present as glycopeptides. A presumpative glycolipid!polysaccharide PM constituent was also identified in such gels.
Insights
Isolated cell surface pellicular membranes (PM) from Leishmania donovani revealed attached subpellicular microtubules (MT). This structural feature aids in identifying membrane surfaces and understanding parasite cell biology.
Area of Science:
- Parasitology
- Cell Biology
- Biochemistry
Background:
- Leishmania donovani is a protozoan parasite responsible for leishmaniasis.
- The cell surface pellicular membrane (PM) plays a crucial role in parasite structure and function.
- Understanding the composition and structure of the PM is key to developing anti-parasitic strategies.
Purpose of the Study:
- To isolate and characterize the cell surface pellicular membranes (PM) from Leishmania donovani.
- To investigate the structural association of subpellicular microtubules (MT) with the PM.
- To analyze the biochemical composition of the isolated PM.
Main Methods:
- Isolation of PM using differential and discontinuous sucrose gradient centrifugation.
- Electron microscopy for structural analysis and purity assessment.
- Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for biochemical composition analysis.
Main Results:
- Successfully isolated PM with attached subpellicular microtubules (MT), achieving >95% purity.
- Electron microscopy revealed structural asymmetry due to MT, aiding surface identification.
- SDS-PAGE identified approximately 40 peptide bands, with 19 periodic acid-Schiff positive, indicating glycopeptides and a glycolipid/polysaccharide component.
Conclusions:
- The study successfully isolated and characterized Leishmania donovani PM, highlighting the structural role of associated MT.
- The attached MT provide a unique feature for identifying membrane surfaces and assessing purity.
- Biochemical analysis revealed a complex composition of PM, rich in glycopeptides and potentially glycolipids/polysaccharides.