Assay and properties of N-acetylglucosamine-6-phosphate deacetylase from rat liver.
A single-vial assay has been developed for N-acetylglucosamine-6-phosphate deacetylase, in which [3H]acetate released from 3H-acetyl-labeled substrate is measured in a biphasic liquid scintillation counting system after acidification of the reaction mixture. The deacetylase was partially purified from rat liver, and some of its properties were determined. Chromatography on a calibrated Sepharose CL-6B column indicated a molecular weight of 345,000. The Km for the substrate at pH 8.0 was 0.3 mM. Glucosamine 6-phosphate and glucose 6-phosphate inhibited the enzyme, whereas N-acetylgalactosamine, N-acetylglucosamine, N-acetylglucosamine 1-phosphate, and glucosamine 1-phosphate were without effect. The effects of several divalent cations were also examined. Under the conditions tested, Ca2+, Mg2+, and Ba2+ had essentially no effect, whereas Mn2+, Ni2+, and Cu2+ were inhibitory and Co2+ stimulated activity at low concentrations but inhibited above 5 mM. An increase in the ionic strength of the reaction mixture to 0.3 M decreased the activity by 40%.
Campbell
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P
Institute of Dental Research, University of Alabama, Birmingham 35294.
Laurent
T C
TC
Rodén
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L
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D002845:Chromatography / Q000379:methods; D003300:Copper / Q000494:pharmacology; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009532:Nickel / Q000494:pharmacology; D051381:Rats
D008345:Manganese; D003300:Copper; D009532:Nickel; D000581:Amidohydrolases; C018717:N-acetylglucosamine-6-phosphate deacetylase