The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells

Automated Edman degradation was performed using a Beckman Instru-ments Inc. (Futlerton, CA) 890C spinning cup sequencer (41) . GH3 cells were radiolabeled with [35S]methionine and the medium or cells treated with antihemoglobin antisera as outlined above. The protein A-Sepharose beads were incubated with 50 #1 of 2 M acetic acid and 8 M urea for 30 rain at 500C. The supernatant was removed and the pellet washed twice with 50 #1 of the same solution. A SEP-PAK (Waters Associates, Inc., Milford, MA) was prepared by washing with 5 ml of 80% CH3CN in 0.1% ,trifluoroacetic acid (TFA) followed by 5 ml of 0.1% TFA. To maximize binding, the combined supematants were loaded on the SEP-PAK three times which was then washed with 5 ml of 0.1% TFA. Bound material was eluted by three washes with 1 ml of 60% CH3CN in 0.1% TFA and three washes with 1 ml of 80% CH3CN in 0.1% TFA. 25 #1 of each fraction was counted. The first two 60% CH3CN washes containing 90% of the radioactivity were combined and loaded directly into the sequencer.