Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans

Biosynthetic Labeling of NIH-373 Cells and COS Cells NIH-3T3 cells were grown in 100-mm culture dishes in DME containing 10% calf serum. For biosynthetic labeling 3T3 or COS cells in 100-mm plates were washed twice with Met-free DME and incubated in Met-free DME for 30 min . Cells were then labeled for 60 min at 37°C with 4 ml Met-free DME containing 120 ttCi/nil [35 S]-labeled mixture of methionine and cysteine (Trap 35 S-label, 1,024 Ci/mmol ; ICN Radiochemicals) . Cell monolayers were washed with DME/10% FCS and chased for 60 min with Moremen and Robbins cDNA Cloning and Expression of Golgi a-Mannosidase 111523 the unlabeled medium . Cells were collected by trypsinization, washed twice in PBS, and vigorously resuspended in lysis buffer containing 1% Triton X-100, 0.5 M NaCl, 20 mM Tris HCl, pH 7.5 . The extract was clarified by centrifugation at 200,000 g for 30 min in a centrifuge (TL-100; Beckman Instruments, Inc., Palo Alto, CA) using a TLA 100.3 rotor at 2°C. The clarified extracts were preadsorbed with 100 Wl of a 50% (vol/vol) slurry of protein ASepharose beads (Phamtacia Fine Chemicals, Piscataway, NJ) for 1 h at 4°C. After the removal ofthe beads by centrifugation, 5 p1 of anti-Man II antiserum (27) was added and incubated at 4°C for 4 h with constant mixing. Protein ASepharose was added (50 Wl of the 50% slurry) and the mixture was incubated for an additional 2 h at 4°C with mixing. The immunoprecipitates were washed, eluted, and resolved by SDS-PAGE as previously described (27) . Samples digested with N-glycanase (Genzyme) were processed as described (27) .