Secretion and uptake of beta-N-acetylglucosaminidase by fibroblasts. Effect of chloroquine and mannose 6-phosphate.
The effects of chloroquine and mannose 6-hosphate on the secretion and uptake of the lysosomal enzyme, beta-N-acetylglucosaminidase (EC 3.2.1.30), by human fibroblasts have been compared. There was a reciprocal relationship between intracellular depletion, and extracellular accumulation, of enzyme at chloroquine concentrations ranging from 5 micrometers to 100 micrometers. A loss of enzyme activity from the system (intra- plus extracellular activity) with increasing concentrations of chloroquine was due to inhibition of the beta-N-acetylglucosaminidase. At a concentration of 50 micrometers, chloroquine elicited a three fold increase in the extracellular accumulation of beta-N-acetylglucosaminidase in 24 h whereas the addition of 5 micrometers mannose 6-phosphate (a competitive inhibitor of receptor-mediated uptake) resulted in only a 13% increase. Uptake of beta-N-acetylglucosaminidase by enzyme-deficient fibroblasts was completely inhibited by 5 micrometers mannose 6-phosphate. In the presence of chloroquine there was also no uptake of enzyme, however ther was a marked decrease in the residual activity of the cells. The results suggest that the effect of chloroquine on fibroblasts is to stimulate secretion rather than to inhibit uptake as previously reported. The isoenzyme pattern of the beta-N-acetylglucosaminidase from normal culture medium was compared with that accumulating in the medium following exposure of the cells to 50 micrometers chloroquine. In the presence of chloroquine, there was an increase in the A isoenzyme, however the activity was eluted in a broad peak which probably represents several closely related forms of the enzyme. There was an almost total loss of the A isoenzyme of beta-N-acetylglucosaminidase from fibroblasts cultured in the presence of chloroquine. A small peak of activity eluting at a similar position to the secreted, As, isoenzyme was present in extracts of chloroquine-treated fibroblasts, suggesting that the As isoenzyme is formed and/or stored at a site distinct from the intracellular isoenzyme.
D000118:Acetylglucosaminidase / Q000378:metabolism*; D002738:Chloroquine / Q000494:pharmacology*; D002852:Chromatography, Ion Exchange; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006596:Hexosaminidases / Q000378:metabolism*; D006600:Hexosephosphates / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008360:Mannosephosphates / Q000494:pharmacology*
D006600:Hexosephosphates; D007527:Isoenzymes; D008360:Mannosephosphates; D002738:Chloroquine; D006596:Hexosaminidases; D000118:Acetylglucosaminidase