Isolation of an enriched plasma membrame subpellicular microtubule fraction of Leishmania mexicana amazonensis

Abstract

A cell fractionation procedure previously developed for Trypanosoma cruzi was applied to isolated the plasma membrane of promastigotes of Leishania mexicana amazonensis. The cell, swollen in an hypotonic mediun, were disrupted in the presence of a nonionic detergent and the membrane fraction isolated by differencial centrifugation. Electron microscopy showed that the fraction consisted of pieces of the plasma membrane associated with subpellicular microtubules. It was also shown that this fraction is able to induce cell-mediated immune response in mice.

Cell fractionation of parasitic protozoa: a review

“…They found that the subpellicular microtubules remained attached to the plasma membrane, and could be used as a morphological criterion in assessing the purity of the fraction. This association of isolated membranes with microtubules has been confirmed for L. collosoma (Linder & Staehelin 1977), Trypanosoma cruzi (Meyer & De Souza 1976), Trypanosoma brucei (Voorheis et al 1979), Leishmania mexicana (Timm et al 1980), Leishmania donovani (Dwyer 1980), and Trypanosoma lewisi (Dywer & D'Alessandro 1980). In some cases, the microtubules did not remain associated with the plasma membrane (Pereira et al 1978, Franco da Silveira et al 1979, Zingales et al 1979, Rovis & Baekkeskov 1980.…”

“…The specific activities of Mg 2+ -dependent ATPase and adenyl cyclase in the membrane fraction were two and five times higher, respectively, than in the whole homogenate. Electron-microscopy showed slight contamination with ribosomes (Timm et al 1980).…”

“…Since different methods have been used to rupture the cells, we cannot exclude the possibility that, in some cases, the attachment of microtubules to the membrane depends on differences in the methods used. But even when the same method was used for the isolation of the membrane, it gave different results in T. cruzi (Pereira et al 1978) and in L. mexicana (Timm et al 1980). Three procedures have been used to obtain a purified plasma membrane fraction from epimastigotes of T. cruzi.…”

How to obtain the organelles of prokaryotic and microbial eukaryotic cells

“…To obtain a purified plasma membrane fraction from epimastigotes of T. cruzi, cells previously swollen by treatment with Triton X-100 were disrupted using a Dounce-type homogenizer followed by consecutive steps of differential centrifugation and its purity was evaluated by electron microscopy and by testing for enzyme markers. Electron-microscopy showed slight contamination with ribosomes of T. cruzi (Timm et al, 1980). In another approach, cells were ruptured by sonication and the membrane fraction was isolated by differential centrifugation, followed by equilibrium centrifugation on sucrose gradients.…”

Análise de imunogenicidade e proteção gerada pelos imunógenos de terceira geração pVAX1-FSD e pVAX1-SP contra a leishmaniose tegumentar americana experimental

“…Um dos primeiros trabalhos sobre vacinas experimentais de 2ª geração contra as espécies de Leishmania do Novo Mundo data da década de 80(TIMM et al 1980). Nesse trabalho, os autores purificaram frações antigênicas de membrana plasmática de L. (L.) amazonensis e avaliaram sua imunogenicidade em camundongos Swiss.…”