Abstract
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 K m for the substrate at pH 8.0 was 0.3 m m. 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, Ca 2+, Mg 2+, and Ba 2+ had essentially no effect, whereas Mn 2+, Ni 2+, and Cu 2+ were inhibitory and Co 2+ stimulated activity at low concentrations but inhibited above 5 m m. An increase in the ionic strength of the reaction mixture to 0.3 m decreased the activity by 40%.
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