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Biochimica et Biophysica Acta, 586 (1979) 442--452 © Elsevier/North-Holland Biomedical Press

BBA 29018

SECRETION AND UPTAKE OF ~-N-ACETYLGLUCOSAMINIDASE BY F I B R O B L A S T S E F F E C T O F C H L O R O Q U I N E AND MANNOSE 6-PHOSPHATE

PAUL WILLCOX and STEPHANIE RATTRAY

Department of Biochemistry, The University of Auckland, Auckland (New Zealand) (Received January 30th, 1979)

Key words: Secretion; ~J-N-Acetylglucosaminidase; Chloroquine effect; Pinocytosis; Mannose phosphate effect; (Fibroblast )

Summary The effects of chloroquine and mannose 6-phosphate on the secretion and uptake of the lysosomal enzyme, ~-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 ~M to 100 t~M. A loss of enzyme activity from the system (intra- plus extracellular activity) with increasing concentrations of chloroquine was due to inhibition of the fi-N-acetylglucosaminidase. At a concentration of 50 pM, chloroquine elicited a three fold increase in the extracellular accumulation of ~-N-acetylglucosaminidase in 24 h whereas the addition of 5 mM mannose 6-phosphate (a competitive inhibitor of receptormediated uptake) resulted in only a 13% increase. Uptake of fl-N-acetylglucosaminidase by enzyme-deficient fibroblasts was completely inhibited b y 5 mM mannose 6-phosphate. In the presence of chloroquine there was also no uptake of enzyme, however there 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 /3-N-acetylglucosaminidase from normal culture medium was compared with that accumulating in the medium following exposure of the cells to 50 pM 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 ~-N-acetylglucosaminidase from fibroblasts cultured in the presence of chloroquine. A small

443 peak of activity eluting at a similar position to the secreted, As, isoenzyme was present in extracts of chloroquine-treated fibroblasts, suggesting that the A s isoenzyme is formed and/or stored at a site distinct from the intracellular isoenzyme.

Introduction We have recently shown that the lysosomal enzyme, ~-N-acetylglucosaminidase (EC 3.2.1.30), is secreted by normal human fibroblasts in a linear manner with respect to time up to 96 h [1]. Furthermore, we have shown that the major form of the A isoenzyme which appears in the medium is different from the intracellular isoenzyme and resembles the isoenzyme found in other extracellular fluids such as plasma and tears [1]. This extraceUular isoenzyme has been designated A s [ 2 ]. In order to investigate the origin, and the mechanism of secretion, of ~-Nacetylglucosaminidase As by fibroblasts we have been studying the effect of a number of compounds known to, or suspected to, affect secretion in related systems. In 1975, Wiesmann et al. [3] reported that the addition of low concentrations of chloroquine to the culture medium of fibroblasts resulted in a marked increase in the levels of the lysosomal enzymes, arylsulphatase A and ~-glucuronidase, found in the medium. The extracellular accumulation of enzyme was interpreted as an inhibition of the uptake of normally secreted enzyme rather than to a direct effect on secretion. In view of preliminary studies in this laboratory on the effect of inhibitors of enzyme uptake on the accumulation of lysosomal enzymes in the culture medium, this explanation seemed unlikely. We therefore reinvestigated and extended the original observation and studied the effect of chloroquine and mannose 6-phosphate (a competitive inhibitor of receptor-mediated uptake [4]) on the secretion and uptake of the lysosomal enzyme ~-N-acetylglucosaminidase by human fibroblasts. Materials and Methods

Materials. 4-Methylumbelliferyl-2-acetamido-2

Secretion and uptake of beta-N-acetylglucosaminidase by fibroblasts. Effect of chloroquine and mannose 6-phosphate.

442 Biochimica et Biophysica Acta, 586 (1979) 442--452 © Elsevier/North-Holland Biomedical Press BBA 29018 SECRETION AND UPTAKE OF ~-N-ACETYLGLUCOS...
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