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

Translation in vitro was carried out for 1.5 h at 30°C in a 25,ul reaction volume containing all amino acids except methionine (1 mM each), 40 U RNasin, 25 pCi of a mixture of [35S]L-cysteine and [35 S]L-methionine (Tran 35 S-label, ICN Radiochemicals, 1,024 Ci/mmol), the RNA synthesized above, and a rabbit reticulocyte lysate preparation from Promega. Incubations containing dog pancreas microsomal membranes were carried out by addition of 1 .8 icl of microsomal membranes (Promega)/25-p1 reaction either before or following the translation reaction . Posttranslational addition of microsomal membranes was followed by a 45-min incubation at 30°C . Samples were either denatured directly in SDS sample buffer or processed for proteolysis by the addition of trypsin (Sigma Chemical Co.) to 100 jig/ml, t 0.1% Triton X-100, followed by incubation at 0°C for 1 h. Proteolysis was terminated by addition of 30 KIU of aprotinin and boiling in SDS sample buffer. SDS-PAGE and autoradiography were carried out as described (26) .