RELEASE OF BETA-HEXOSAMINIDASE ISOENZYMES IN CULTURED HUMAN FIBROBLASTS

We have used enzyme immunoassay methods to study the intra- and extrac ellular isoenzyme pattern of beta-hexosaminidase (Hex) in human fibrob last cultures. The released activity into the medium during 24 h was a bout 10% of the intracellular activity. Intra- and extracellular ratio of Hex B to total Hex (Hex A plus Hex B) was about one-third. Estimat ion of the molecular weight of the released activity showed that it co rresponded to 150 kDa, which is equal to the high molecular mass precu rsor forms of the enzyme. NH4Cl is known to disturb the intracellular transport of lysosomal enzymes and increase the secretion of newly syn thesized precursor forms. Addition of NH4Cl even at 3 mM resulted in a n increased release of total Hex, which was already noted within 24 h. We speculate that the increased concentration of plasma NH4+ in patie nts with liver disease interferes with the intracellular distribution pathway of the lysosomal enzymes and this could contribute to the incr eased content of lysosomal enzymes present in plasma from these patien ts. Tunicamycin (0.3-3.0 mug/l) increased the percentage of Hex B in t he medium, whereas an increased release of Hex was noted only after 48 h. Tunicamycin is known to enhance the secretion of N-linked oligosac charide-free forms of lysosomal enzymes. Thus the oligosaccharide chai ns on alpha- and beta-subunits seem to be important for the normal for mation process of the Hex A isoenzyme.