Fast protein liquid chromatography (FPLC) of leukaemic cell N-acetyl beta-D hexosaminidases

Leukaemic myeloid and lymphoid cell N-acetyl beta-D glucosaminidase (hexosaminidase) enzymes were fractionated by Fast Protein Liquid Chromatography (FPLC) using high-resolution ion exchange (Mono-S and Mono-Q), gel filtration (Superose-6) and chromatofocusing (Mono-P) columns. Although only one molecular weight species was detected in haemopoietic cells, with an apparent mass of 129 kD, "isoenzyme" variants defined by differences in molecular charge were considerably more diverse than previously thought. Separation of the major Hex forms (A and B) by chromatofocusing indicated that intermediate (I) components were present in most acute leukaemias irrespective of lineage supporting the concept that the I form is a non-specific marker of haemopoietic immaturity. Substrate and inhibitor studies further revealed that leukaemic cell hexosaminidases hydrolyse galactopyranosides at significantly lower rates than glucopyranosides and that the hydrolysis of N-acetyl beta-D glucosamine is inhibited by both glucosamine and galactosamine products.