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The effect of monensin on beta-hexosaminidase transport in normal and I-cell fibroblasts.

ABSTRACT: The carboxylic ionophore, monensin, blocks the migration of glycoprotein-containing vesicles from the Golgi region to the plasma membrane in fibroblasts resulting in an accumulation of secretory products in the Golgi cisternae. Treatment of cultured I-cell fibroblasts with monensin (0.5 muM) decreased the abnormal excretion of beta-hexosaminidase to 40% of untreated cultures within 15 min. A corresponding intracellular accumulation of the enzyme to greater than 200% of untreated cultured by 24 h was also observed. A small intracellular accumulation and slightly enhanced excretion of beta-hexosaminidase occurred in treated normal fibroblasts cultures. The intra- and extra-cellular distribution of newly synthesized beta-hexosaminidase in both monensin-treated normal and I-cell fibroblasts were electrophoretically indistinguishable from the four bands characteristic of I-cell intracellular beta-hexosaminidase. The excreted enzyme from both cultures was found to be a low- or no-uptake form. This form of beta-hexosaminidase may have been excreted from a secondary route preceding the site of the monensin effect. The similar findings in monensin-treated normal and I-cell cultures suggest that the subcellular site of the biochemical defect in I-cell disease is at a location after the site of the monensin effect i.e. late in the Golgi region or at a post-Golgi-region location.

SUBMITTER: Vladutiu GD

PROVIDER: S-EPMC1162404 | biostudies-other | 1980 Dec

REPOSITORIES: biostudies-other

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Project description:Most of the newly synthesized beta-N-acetyl-d-glucosaminidase (EC 3.2.1.30; beta-hexosaminidase) in normal fibroblast cultures is excreted during 24h incubation with serum-free medium. In this study, this new enzyme only comprises about one-half of the excreted pool as determined by a near total inhibition of [(14)C]leucine incorporation into the excreted enzyme in the presence of cycloheximide, with only a 46% reduction in enzyme activity. These data indicate that nearly equal fractions of new and old enzyme are normally excreted by fibroblasts. Incubation of normal fibroblast cultures with chloroquine (25 mum) for 24h doubled the amount of extracellular beta-hexosaminidase activity from 15% to 37% of total culture activity while reducing the incorporation of [(14)C]leucine into intra- and extracellular enzyme by 66 and 29% of control, respectively. Therefore, it appears that chloroquine inhibited enzyme synthesis while enhancing the excretion of old as well as newly synthesized enzyme. Chloroquine and cycloheximide together reduced the [(14)C]leucine incorporation into intracellular enzyme by more than either agent alone, indicating a combined effect on enzyme synthesis and/or degradation. beta-Hexosaminidase-deficient fibroblasts that contained endocytosed enzyme spontaneously excreted 10% of their enzyme during 24h incubation with serum-free medium and 18% in the presence of mannose 6-phosphate (2 mm). These results indicated that about one-half of the excreted enzyme still possessed its phosphomannosyl recognition residues and actually re-entered the cells. Chloroquine stimulated the excretion of an addition 15% of the total endocytosed enzyme at 48 and 72h after endocytosis. These data suggest that new, old and endocytosed beta-hexosaminidase are all excreted by fibroblasts, that this excretion is enhanced by chloroquine, and that a fraction of the excreted enzyme retains its phosphomannosylated residues needed for re-uptake and transport inside the cells.

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The effect of monensin on beta-hexosaminidase transport in normal and I-cell fibroblasts.

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