Sabata Martino

β N-acetylhexosaminidases A and S have similar sub-cellular distributions in HL-60 cells

Abstract In HL-60 cells the most abundant isoenzymes of β-N-acetyl-hexosaminidase are A (ap) and S (αα ). 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 β-N-acetylhexosaminidase less acidic than A, but there was no S. Analysis of the α-subunits of β-N-acetylhexosaminidases A and S using Western blotting and immuno-detection with antisera raised to synthetic peptides showed that mature α-subunits were present in both A and S isolated from the lysosomal fraction. This observation establishes that the αα-dimer of β-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 NH4C1 and, unlike fibroblasts, are unlikely to use mannose-6-phosphate mediated transport of β-N-acetylhexosaminidases to lysosomes. Comparison of the sequence of the β-N-acetylhexosaminidase a-subunit with a 43 amino acid sequence of cathepsin D, thought 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.