N-ACETYL-BETA-D-HEXOSAMINIDASES OF THE BRINE SHRIMP ARTEMIA - PARTIAL-PURIFICATION AND CHARACTERIZATION

N-Acetyl-beta-D-hexosaminidases (EC 3.2.1.52) from Artemia nauplii were isolated and characterized. Three different enzymes I, II1 and II2 were separated according to their behaviour on anion exchange chromatography and gel filtration columns. Their apparent molecular masses were 83,000 +/- 7000, 110,000 +/- 10,000 and 56,000 +/- 5000 Da with corresponding S-values of 8.6, 11.9 and 7.9. All three enzymes also differ in their apparent pH-optima (5.1, 4.5 and 6.1) and they all bind to concanavalin A. The three enzymes have about the same affinities (app. K(m) between 0.16 and 0.72 mmol/l) for the three substrates (p-nitrophenyl-N-acetyl-beta-D-glucosamine or p-nitrophenyl-N-acetyl-beta-D-galactosamine and N,N'-diacetyl-chitobiose) and are therefore N-acetyl-beta-D-hexosaminidases. In contrast, the three enzymes behave quite differently, both in terms of their inhibitor constants and the type of inhibition. The substrates inhibit both enzymes II1 and II2 but not enzyme I. On the other hand, N-acetyl-beta-D-galactosamine inhibits enzyme I in a non-competitive way but not enzymes II1 and II2. All three enzymes are inhibited by the end product N-acetyl-beta-D-glucosamine, enzyme I in a competitive manner, both enzymes II1 and II2 in a non-competitive way. 2-Acetamido-2-deoxy-D-galactonolactone is a strong inhibitor for enzyme I (K(i) = 13-mu-mol/l) with much lower affinities towards enzymes II1 and II2 (K(i) = 0.63 and 1.03 mmol/l). All three enzymes are inhibited in a dose-dependent way and completely reversible by alpha-methyl-mannoside.