FIG. 4.

GXM is present in purified vesicles. (A) Immunogold labeling of purified vesicles with MAb 18B7 revealed a preferential intravesicular distribution of GXM, as observed in different vesicular bodies (a to c). A higher magnification of the boxed area shown in panel c demonstrates the occurrence of bilayered membrane (d). Bars, 150 (a), 180 (b), 120 (c), and 30 (d) nm. (B) The presence of GXM inside the vesicles was confirmed by polysaccharide detection in purified 100,000 × g fractions from culture supernatants (white bar). Supernatants obtained from these suspensions were assayed as a control of vesicle integrity, and in fact, GXM was detected at low levels in these preparations (black bar), suggesting that some of these structures may be disrupted. (C) GXM content of vesicle suspensions and vesicle-depleted supernatants after serial passage through a column composed of Sepharose-bound MAb 18B7. Sequential passages of vesicle-depleted supernatant through this column result in a rapid loss of reactivity, while the GXM content of the vesicular preparation is relatively constant. (D) GXM-containing vesicles are not formed extracellularly, since the addition of the polysaccharide to culture supernatants during or after growth of Cap67 cells is not followed by its detection in purified 100,000 × g fractions.