Isolation and Partial Biochemical Characterization of the Brush Border Plasma Membrane from the Cestode, Hymenolepis diminuta

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Abstract
A procedure was devised to remove the tegument from the cestode H. diminuta [isolated from rats] using 0.2% Triton X-100. The brush border fragments were collected separately from cytoplasmic vesicles by differential centrifugation, and the brush border plasma membrane was further enriched by disruption in high osmolarity Tris-HCl buffer. The distribution of plasma membrane was quantified by comparing the specific activity of 3H-concanavalin A in all fractions following incubation of intact organisms with the radioactive lectin. SDS[sodium dodecyl sulfate]-polyacrylamide gels stained with Coomassie Blue reveal no qualitative differences between plasma membranes and vesicles. When gels are stained with Stains-all, 7 proteins of the plasma membrane stain blue, whereas the vesicular counterparts stain red. Treatment of plasma membrane with calf intestinal alkaline phosphatase results in a decrease in the intensity of 5 of the blue-staining bands, indicating that these proteins are phosphoproteins. Membrane-bound alkaline phosphatase is resistant to dodecyl sulfate plus dithiothreitol and was detected histochemically in a region corresponding to 104,000 daltons following SDS polyacrylamide gel electrophoresis. Lactoperoxidase catalyzes the iodination of 7 externally oriented plasma membrane macromolecules, 6 of which stain as glycoproteins with PAS [periodic acid Schiff]. The incorporation of 125I-iodide increases approximately 10-fold when the organisms are preincubated in buffer for 30 min. This suggests that either adsorbed proteins are being removed or that, in the absence of host proteases, these external macromolecules are accumulating in the plasma membrane with time.