Figure 3.

Mammalian cytosol contains an N-glycanase. (A) Aliquots of ≈4 × 10⁴ cpm of unglycosylated tripeptide or 1.6 × 10⁴ cpm of glycotripeptide derived from a yeast export reaction were loaded onto SepPak C₁₈ reverse-phase cartridges and eluted in 1-ml steps with increasing concentrations of acetonitrile as indicated. FT, flowthrough. ¹²⁵I cpm in each fraction were detected by γ counting. (B and C) Glycotripeptide (1.6 × 10⁴ cpm) derived from a yeast export reaction were incubated with endo H (0.1 unit/ml), PNGase F (20 units/ml), or mammalian cytosol (10 mg/ml) for 1 hr at 37°C and subsequently analyzed by reverse-phase chromatography as above. (D) Purified glycotripeptide was incubated with water (lane 3), endo H (0.1 unit/ml, lane 4), PNGase F (20 units/ml, lane 5), or mammalian cytosol (7 mg/ml, lane 6) for 1 hr at 37°C and analyzed by thin-layer chromatography as described. Lane 1, unglycosylated peptide; lane 2, purified glycopeptide. (E) Aliquots of purified glycotripeptide were incubated with rat liver cytosol for 20 min at 32°C in various buffers as described in Material and Methods. At the end of the incubation, remaining glycotripeptide was quantified by Con A-Sepharose precipitation and γ counting, and activity expressed as percent glycopeptide degraded during the incubation.