PURIFICATION TO HOMOGENEITY AND PROPERTIES OF UDP-GLCNAC (GALNAC) PYROPHOSPHORYLASE

The pyrophosphorylase that condenses UTP and GlcNAc-1-P was purified 9 500-fold to near homogeneity from the soluble fraction of pig liver ex tracts. At the final stage of purification, the enzyme was quite stabl e and could be kept for at least 4 months in the freezer with only sli ght loss of activity. On native gels, the purified enzyme showed a sin gle protein band, and this band was estimated to have a molecular mass of similar to 125 kDa on Sephacryl S-300. SDS-polyacrylamide gel elec trophoresis analysis of the enzyme gave three protein bands of 64, 57, and 49 kDa, but these polypeptides are all closely related based on t he following, 1) All three polypeptides show strong cross-reactivity w ith antibody prepared against the 64-kDa band, 2) All three proteins b ecome labeled with either the UDP-GlcNAc photoaffinity probe azido-I-1 25-salicylate-allylamine-UDP-GlcNAc or a similar UDP-GalNAc photoaffin ity probe, and either labeling was inhibited in a specific and concent ration-dependent manner by unlabeled UDP-GlcNAc or UI)PGalNAc. Thus, t he enzyme is probably a homodimer composed of two 64-kDa subunits, The purified enzyme had an unusual specificity in that, at higher substra te concentrations, it utilized UDP-GalNAc as a substrate as well as UD P-GlcNAc in the reverse direction and GalNAc-1-P as well as GlcNAc-1-P in the forward direction, However, the K-m for the GalNAc substrates was considerably higher than that for GlcNAc derivatives. This activit y for synthesizing UDP-GalNAc was not due to epimerase activity since no UDP-GalNAc could be detected when the enzyme was incubated with UDP -GlcNAc for various periods of time. The pyrophosphorylase required a divalent cation, with Mn2+ being best at 0.5-1 mM, and the pH optimum was between 8.5 and 8.9.