The effect of chloroquine on the distribution of newly synthesized and old beta-hexosaminidase in fibroblasts.

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.