. 2008 Feb 8;190(8):2987–2996. doi: 10.1128/JB.01970-07

TABLE 2.

Reactions catalyzed by MJ1101

Substrates	Products	Sp act (μmol min⁻¹ mg⁻¹)
Forward reaction^a
GlcN-1-P, UTP	UDP-GlcN, PPi	0.25 ± 0.07
GlcN-1-P, AcCoA, UTP	UDP-GlcNAc, PPi, CoA	7.74 ± 1.37
GlcN-1-P, AcCoA, dTTP	dTDP-GlcNAc, PPi, CoA	2.64 ± 0.05
GlcN-1-P, AcCoA, dUTP	dUDP-GlcNAc, PPi, CoA	2.10 ± 0.14
Glc-1-P, UTP	UDP-Glc, PPi	1.38 ± 0.16
Glc-1-P, dTTP	dTDP-Glc, PPi	0.23 ± 0.02
Reverse reaction^b
UDP-GlcNAc, PPi	GlcNAc-1-P, UTP	12.51 ± 0.18
UDP-Glc, PPi	Glc-1-P, UTP	8.40 ± 1.20

Thirty-microliter reaction mixtures containing saturating substrate concentrations (0.5 mM of nucleotide triphosphate and 0.5 mM Ac-CoA) with 1 mM DTT, 5 mM MgCl₂ , 50 mM Tris-HCl (pH 7.5), 2 units of inorganic pyrophosphatase, and 50 ng of His₁₀-MJ1101 were preincubated at 50°C for 2 min before the reaction was started by the addition of 0.5 mM sugar phosphate substrate. After 5 min of incubation at the same temperature, the reactions were stopped by the addition of 10 mM EDTA. Nucleotide products were detected by HPLC as described previously.

Assays were performed in a manner similar to forward reactions. The 30-μl reaction mixtures contained 0.5 mM UDP-GlcNAc, 1 mM pyrophosphate, 1 mM DTT, 5 mM MgCl₂, 50 mM Tris-HCl (pH 7.5), and 50 ng His₁₀-MJ1101. UTP product formation was determined using HPLC.