leupeptins and Lactose-Intolerance

beta-Galactosidase was normalized by a serine-thiol protease inhibitor, leupeptin with concentration of 10 micrograms/ml in cultured skin fibroblasts from patients with beta-galactosidase-alpha-neuraminidase deficiency (beta-Gal-/Neu-). The induction of this enzyme was not observed in normal cells. Because the enzymic activity of cathepsin B1 increased significantly both in beta-Gal-/Neu- and normal cells by leupeptin loading, the restoration of beta-galactosidase in beta-Gal-/Neu- cells can not be explained by the theory that leupeptin inhibited intracellular degradation of beta-galactosidase molecules. The effects of leupeptin and sucrose on lysosomal hydrolase induction were compared.

Topics: beta-Galactosidase; Cathepsin B; Cathepsins; Cell Line; Enzyme Induction; Fibroblasts; Galactosidases; Humans; Hydrolases; Lactose Intolerance; Leupeptins; Lysosomes; Neuraminidase; Oligopeptides; Sucrose

In normal human fibroblasts, an enzymically active 85,000-dalton precursor form of beta-galactosidase is processed, via a number of intermediates, into a mature 64,000-dalton form. In addition there is an enzymically inactive 32,000-dalton component and its 54,000-dalton precursor. In fibroblasts from patients with a combined deficiency of beta-galactosidase and neuraminidase these last two components are absent and hardly any mature beta-galactosidase can be demonstrated. Nevertheless, in the mutant fibroblasts, precursor beta-galactosidase is synthesized and processed normally. The excessive intralysosomal degradation that is responsible for the deficiency of mature beta-galactosidase can be partially corrected by addition of the protease inhibitor leupeptin, which results in the accumulation of 85,000-dalton precursor beta-galactosidase and of a partially processed 66,000-dalton form. When mutant cells were grown in the presence of a "corrective factor" purified from the medium of NH4Cl-stimulated cell cultures, both beta-galactosidase and neuraminidase activities were restored to low control levels. The immunoprecipitation pattern was completely normal after addition of the corrective factor, and mature 64,000-dalton beta-galactosidase accumulated in the mutant fibroblasts. We propose that the combined beta-galactosidase/neuraminidase deficiency is caused by a defective 32,000-dalton glycoprotein which is normally required to protect beta-galactosidase and neuraminidase against excessive intralysosomal degradation and to give these enzymes their full hydrolytic activity.

Topics: beta-Galactosidase; Cells, Cultured; Enzyme Precursors; Fibroblasts; Humans; Lactose Intolerance; Leupeptins; Lysosomes; Molecular Weight; Neuraminidase

Topics: beta-Galactosidase; Cathepsin B; Cathepsins; Cells, Cultured; Endopeptidases; Enzyme Activation; Fibroblasts; Humans; Kinetics; Lactose Intolerance; Leupeptins; Neuraminidase; Protease Inhibitors; Proteins