N-Acetyl-ß-ᴅ-hexosaminidases of the Brine Shrimp Artemia: Partial Purification and Characterization
2014
N-Acetyl-β-ᴅ-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. Kₘ between 0.16 and 0.72 mmol/1) for the three substrates (p-nitrophenyl-N-acetyl-β-ᴅ-glucosamine or p-nitrophenyl-N-acetyl-β-ᴅ-galactosamine and N ,N′-diacetyl-chitobiose) and are therefore N-acetyl-β-ᴅ-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-β-ᴅ-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-β-ᴅ-glucosamine, enzyme I in a competitive manner, both enzymes II₁ and II₂ in a non-competitive way. 2-Acetamido-2-deoxy-ᴅ-galactonolactone is a strong inhibitor for enzyme I (Kᵢ = 13 μtmol/l) with much lower affinities towards enzymes II₁ and II₂ (Kᵢ = 0.63 and 1.03 mmol/l). All three enzymes are inhibited in a dose-dependent way and completely reversible by α-methyl-mannoside.
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