title: Association of alpha- and beta-subunits during the biosynthesis of beta-hexosaminidase in cultured human fibroblasts.
abstract: Subunit association of beta-hexosaminidase was studied in intact fibroblasts using antisera that discriminate between free and associated alpha-chains. These were anti-beta-hexosaminidase A (anti-alpha beta), which precipitated all alpha-chains, free or associated; anti-beta-hexosaminidase B (anti-beta beta), which precipitated those alpha-chains that were associated with beta; and anti-alpha-chains, which recognized only monomeric alpha-chains. After biosynthetic labeling, beta-hexosaminidase or its free alpha-subunit were immuno-precipitated from extracts of cells and medium with the aid of protein A-bearing Staphylococcus aureus, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by fluorography. Pulse-chase labeling showed that the alpha-chains existed predominantly in the monomeric precursor form during the first 5 h, and then began to accumulate in the mature (lysosomal) associated alpha beta form. Precursor alpha beta complexes were secreted, along with some precursor alpha monomers; the latter were catalytically inert. Both alpha- and beta-chains were phosphorylated (a Golgi modification) prior to association. Thus alpha-beta association probably occurred in the Golgi area before transfer to lysosomes and before secretion. Cycloheximide inhibited the association and subsequent maturation of preformed alpha-chains, perhaps by causing a depletion of a pool of beta-chain precursor upstream from the site of subunit association. In fibroblasts from a patient with Sandhoff disease, that produced no beta-chains, the alpha-chains self-associated but their maturation was markedly decreased. We suggest that association with beta-chains is necessary not only for acquisition of catalytic activity but also for transport of alpha-chains to lysosomes.
mesh_terms: D002460:Cell Line; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000201:enzymology; D006596:Hexosaminidases / Q000235:genetics*; D006801:Humans; D007106:Immune Sera; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D014176:Protein Biosynthesis / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D012497:Sandhoff Disease / Q000201:enzymology; D001619:beta-N-Acetylhexosaminidases
chemical_list: D007106:Immune Sera; D046911:Macromolecular Substances; D003513:Cycloheximide; D006596:Hexosaminidases; D001619:beta-N-Acetylhexosaminidases