Figure 1.
Methodology and terminology used in this study. P1798 murine T lymphoma cells (109) were homogenized in TKM buffer containing 73% (wt/vol) sucrose. Aliquots of the cell homogenate (equivalent to 150 × 106 cells), treated with detergent or left untreated at 4°C for 20 min, were adjusted to 40% (wt/vol) sucrose and subjected to equilibrium density gradient centrifugation. One-milliliter fractions collected from the top and fractions 3–5 enriched for the GPI-anchored proteins were pooled, or the GPI-enriched floating membranes visible as a light-scattering band at the 5–36% sucrose interface were collected directly (∼3 ml) and stored at −20°C until use. (A) Untreated GPI-enriched membranes. (B) Detergent-treated GPI-enriched membranes. (C) Subcellular fractionation of the cell homogenate from 1–2 × 109 cells was carried out to obtain plasma membrane fractions with specific gravity of 1.034–1.09 g/ml (light membranes) and 1.09–1.136 g/ml (heavy membranes) as described previously (Hoessli and Rungger-Brändle, 1983; Arni et al., 1996). The most frequently used terms are boxed.