PROTEOLYTIC PROCESSING OF THE BETA-SUBUNIT OF THE LYSOSOMAL-ENZYME, BETA-HEXOSAMINIDASE, IN NORMAL HUMAN-FIBROBLASTS

  • 25 February 1989
    • journal article
    • research article
    • Vol. 264  (6) , 3380-3384
Abstract
We have characterized the proteolytic processing of the .beta.-subunit of .beta.-hexosaminidase by identifying the amino termini of the various forms synthesized in cell-free translation and in cultured human fibroblasts. The procedures used had been developed for similar studies of the .alpha.-subunit (Little, L. E., Lau, M. M. H., Quon, D. V. K., Fowler, A. V., and Neufeld, E. F. (1988) J. Biol. Chem. 263, 4288-4292). Radioactive amino acids were incorporated biosynthetically into the different forms of the .beta.-subunit, which were isolated by immunoprecipitation, gel electrophoresis, and electroelution, and analyzed by automated Edman degradation. Translation by reticulocyte lysate in the presence of canine pancreas microsomes gave a product with alanine 43 at the amino terminus. The lysate could initiate translation at methionine 1 or methionine 13, depending on the SP6 mRNA provided. The product of signal peptidase action, the precursor form of the .beta.-subunit with amino-terminal alanine 43, was found in NH4+-induced secretions of cultured fibroblasts; intracellularly, this form was trimmed of two additional amino acids. The mature form was found to consist of three polypeptides joined by disulfide bonds; the amino termini were found to be valine 48, threonine 122, and lysine 315. Thus, in contrast to the .alpha.-subunit, the mature form of the .beta.-subunit of .beta.-hexosaminidase is derived from the precursor by internal proteolytic nicking rather than by removal of a large amino-terminal peptide segment.

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