2 CYTOSOLIC PUROMYCIN-SENSITIVE AMINOPEPTIDASE ISOZYMES IN CHICKEN BRAIN - MOLECULAR HOMOLOGY TO BRAIN-SPECIFIC 14-3-3 PROTEIN

Two puromycin-sensitive aminopeptidase isozymes (PSA-I and PSA-II) wer e isolated from chicken brain cytosol by ammonium sulfate fractionatio n followed by column chromatography on Cellex D and AH-Sepharose 4B an d separated on Bio-Gel HTP. Each was purified to homogeneity on Sephad ex G-200, Arg-Tyr-AH-Sepharose, Bio-Gel HTP, and preparative gel elect rophoresis. On sodium dodecyl sulfate-polyacrylamide gel electrophores is, PSA-I appeared to be a monomer with a molecular mass of 105 kDa, a nd PSA-II to be composed of two subunits of 25 kDa and 100 kDa. The tr yptic maps of 100 kDa and 105 kDa protein in HPLC are different in pea k frequency, height, and composition. The internal peptide sequence of PSA-I has a considerable homology to PSA-II. Both isozymes have repea ted copies of common peptide segments and have no significant sequence homology to other peptidases and proteinases. These thio and Co2+-act ivated isozymes have a neutral pH optimum and are inhibited by puromyc in and bestatin. PSA-II is more sensitive to trypsin and heat treatmen t, has a lower K(m) to Met-enkephalin, and is more active on Arg BNA a nd Pro BNA. Our results suggest that PSA-I and PSA-II derive from tran slation of two RNAs of a new gene family related to the brain-specific 14-3-3 protein.