Beta-N-acetylhexosaminidases A and S have similar sub-cellular distributions in HL-60 cells.
In HL-60 cells the most abundant isoenzymes of beta-N-acetyl-hexosaminidase are A (alpha beta) and S (alpha alpha). Sub-cellular fractionation of HL-60 cells by differential centrifugation showed that both A and S forms were present in the lysosomal and post-lysosomal (soluble) fractions in approximately equal abundance. Ion-exchange chromatography showed that a fraction enriched with plasma membranes had the A form, and a form of beta-N-acetylhexosaminidase less acidic than A, but there was no S. Analysis of the alpha-subunits of beta-N-acetylhexosaminidases A and S using Western blotting and immuno-detection with antisera raised to synthetic peptides showed that mature alpha-subunits were present in both A and S isolated from the lysosomal fraction. This observation establishes that the alpha alpha-dimer of beta-N-acetyl-hexosaminidase (S) can be transported to lysosomes in HL-60 cells whereas there is good evidence that this does not take place in fibroblasts. HL-60 cells were not stimulated to secrete lysosomal enzymes by incubating them with NH4Cl and, unlike fibroblasts, are unlikely to use mannose-6-phosphate mediated transport of beta-N-acetylhexosaminidases to lysosomes. Comparison of the sequence of the beta-N-acetylhexosaminidase alpha-subunit with a 43 amino acid sequence of cathepsin D, though to function in the mannose-6-phosphate independent targeting of this enzyme to lysosomes, showed alignment in a region towards the C-terminus in which 21% of the residues were identical with the interposition of a one amino acid gap.
Martino
S
S
Division of Life Sciences, King's College London, Campden Hill, UK.
Emiliani
C
C
Orlacchio
A
A
Hosseini
R
R
Stirling
J L
JL
D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015473:Leukemia, Promyelocytic, Acute / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology; D008361:Mannosidases / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D043323:alpha-Mannosidase; D001619:beta-N-Acetylhexosaminidases / Q000032:analysis*
D007527:Isoenzymes; D000643:Ammonium Chloride; D008361:Mannosidases; D043323:alpha-Mannosidase; D001619:beta-N-Acetylhexosaminidases