Isolation and Partial Characterization of Surface Membranes From Leishmania Donovani Promastigotes*

Abstract

Cell surface pellicular membranes (PM) were isolated from promastigote forms of L. donovani by differential and discontinuous sucrose gradient centrifugation procedures. The PM had a density equivalent of .apprx. 1.19 g/cm3. As ascertained by EM, 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 .gtoreq. 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 .apprx. 7.2 nm and attached MT a diameter of .apprx. 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 .apprx. 40 peptide bands ranging in apparent MW from .ltoreq. 1.2 .times. 104 to .gtoreq. 2.2 .times. 105 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 identified in such gels.