Enrichment of right-side-out Trypanosoma cruzi plasma membrane vesicles.
A simple method to prepare a high yield of Trypanosoma cruzi plasma membrane vesicles (PMV) from epimastigotes and metacyclic trypomastigotes is described. The method may be applicable to other protozoa. Solid-phase immunoassay to bind surface T. cruzi epitopes showed that this preparation was enriched with 80-82% PMV and that most of these were right-side out (81-92%). The method was based on the extraction of extrinsic proteins and subpellicular tubules with mild high and low ionic strength buffers without detergents (pH 7.4) and on the differential centrifugation of PMV based on their specific density (1.049 g/ml, 4 degrees C). Transmission electron microscopy of PMV pellets showed a heterogeneous population of vesicles without other significant cytoskeletal contaminants. T. cruzi PMV were also enriched with an ouabain- and oligomycin-insensitive magnesium-ATPase and contained an adenylyl cyclase, preserved for at least 3 months at -70 degrees C in storage buffer. Measurements of the [14C]-dextran and the 3H2O space indicated that T. cruzi PMV were not sealed, explaining why Lubrol PX and NaF failed to stimulate the adenylyl cyclase activity further and why T. cruzi PMV were unable to concentrate 86Rb in flow dialysis assays. No detectable DNA and RNA was found. The preparation was not capable of removing 51Cr or [3H]glucosamine from live L6 myoblast surfaces in physiologic conditions and acid phosphatase was extracted by this method. The contaminating fraction (18-20% by immunoassay) consisted of endoplasmic reticulum membranes with NADH oxidase activity and of kinetoplast membranes with cytochrome c oxidase and oligomycin sensitive magnesium-ATPase activity. The biologically active T. cruzi PMV retained the ability of living forms to trigger the alternate pathway of complement by releasing the Bb activation fragment from human Factor B.
Von Kreuter
B F
BF
Department of Pathology, Cornell University Medical College, New York, New York 10021.
McWilliam
J R
JR
Firpo
A
A
Santos-Buch
C A
CA
D000818:Animals; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism / Q000648:ultrastructure; D002463:Cell Membrane Permeability; D003167:Complement Activation; D003170:Complement Pathway, Alternative; D003911:Dextrans / Q000378:metabolism; D004798:Enzymes / Q000032:analysis; D007839:Functional Laterality; D006652:Histological Techniques; D008854:Microscopy, Electron; D009711:Nucleotides / Q000032:analysis; D012413:Rubidium / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000648:ultrastructure*
D003911:Dextrans; D004798:Enzymes; D009711:Nucleotides; D012413:Rubidium