Release of beta-hexosaminidase isoenzymes in cultured human fibroblasts.
We have used enzyme immunoassay methods to study the intra- and extracellular isoenzyme pattern of beta-hexosaminidase (Hex) in human fibroblast cultures. The released activity into the medium during 24 h was about 10% of the intracellular activity. Intra- and extracellular ratio of Hex B to total Hex (Hex A plus Hex B) was about one-third. Estimation of the molecular weight of the released activity showed that it corresponded to 150 kDa, which is equal to the high molecular mass precursor forms of the enzyme. NH4Cl is known to disturb the intracellular transport of lysosomal enzymes and increase the secretion of newly synthesized precursor forms. Addition of NH4Cl even at 3 mM resulted in an increased release of total Hex, which was already noted within 24 h. We speculate that the increased concentration of plasma NH4+ in patients with liver disease interferes with the intracellular distribution pathway of the lysosomal enzymes and this could contribute to the increased content of lysosomal enzymes present in plasma from these patients. Tunicamycin (0.3-3.0 micrograms/l) increased the percentage of Hex B in the medium, whereas an increased release of Hex was noted only after 48 h. Tunicamycin is known to enhance the secretion of N-linked oligosaccharide-free forms of lysosomal enzymes. Thus the oligosaccharide chains on alpha- and beta-subunits seem to be important for the normal formation process of the Hex A isoenzyme.
Hultberg
B
B
Department of Clinical Chemistry, University Hospital, Lund, Sweden.
Isaksson
A
A
Nordström
M
M
Kjellström
T
T
D000643:Ammonium Chloride / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000201:enzymology; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D006801:Humans; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism*; D008247:Lysosomes / Q000201:enzymology; D008970:Molecular Weight; D014415:Tunicamycin / Q000494:pharmacology; D001619:beta-N-Acetylhexosaminidases / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D000643:Ammonium Chloride; D014415:Tunicamycin; D003513:Cycloheximide; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases