Effect of ammonium chloride on subcellular distribution of lysosomal enzymes in human fibroblasts.
Three subcellular fractions enriched in lysosomal enzyme activities have been isolated recently from human cultured fibroblasts with Percoll gradients: the dense lysosomes (DL), light lysosomes (LL), and light membranous vesicles (LM). They were shown to have different morphological, cytochemical, biochemical, and immunological properties. We now report on the dramatic but different effects of a primary amine, NH4Cl, on these subfractions. The lysosomes, as detected with a specific ultrastructural cytochemical stain for the lysosomal enzyme, arylsulfatase A, were swollen significantly in all these fractions, increasing their volumes by 64% (DL), 53% (LL), and 95% (LM), respectively. When arylsulfatase A enzyme activity was monitored, about half of the DL content was diverted to the LL. However, when newly synthesized arylsulfatase A enzyme protein was monitored with metabolic labeling and immunoprecipitation, about 80% of the enzyme protein was depleted from both the DL and LL. In contrast, neither the enzyme activity nor the newly synthesized enzyme protein of arylsulfatase A was greatly altered in the LM fraction by the treatment. Since primary amines caused newly synthesized lysosomal enzymes to diverge from the lysosomal route to a secretory pathway, it was deduced that (i) the LM fraction corresponded to a prelysosomal compartment whose lysosomal enzyme content was not affected by the amine and was thus proximal to the point of diversion between the secretory and lysosomal pathways; (ii) the LL and DL fractions were distal to the point of diversion since both fractions were depleted of their newly synthesized lysosomal enzyme; and (iii) the sorting of newly synthesized lysosomal enzyme may be different from that of the preexisting pool of the same enzyme since the LL fraction was depleted of its newly synthesized enzyme protein while accumulating excessive enzyme activity.
D000643:Ammonium Chloride / Q000494:pharmacology*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002553:Cerebroside-Sulfatase / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D008247:Lysosomes / Q000145:classification / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D000643:Ammonium Chloride; D002553:Cerebroside-Sulfatase; D001619:beta-N-Acetylhexosaminidases