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.