Purification to Homogeneity and Properties of UDP-GlcNAc (GalNAc) Pyrophosphorylase

Abstract

The pyrophosphorylase that condenses UTP and GlcNAc-1-P was purified 9500-fold to near homogeneity from the soluble fraction of pig liver extracts. At the final stage of purification, the enzyme was quite stable and could be kept for at least 4 months in the freezer with only slight loss of activity. On native gels, the purified enzyme showed a single protein band, and this band was estimated to have a molecular mass of approximately125 kDa on Sephacryl S-300. SDS-polyacrylamide gel electrophoresis analysis of the enzyme gave three protein bands of 64, 57, and 49 kDa, but these polypeptides are all closely related based on the following. 1) All three polypeptides show strong cross-reactivity with antibody prepared against the 64-kDa band. 2) All three proteins become labeled with either the UDP-GlcNAc photoaffinity probe azido-125I-salicylate-allylamine-UDP-GlcNAc or a similar UDP-GalNAc photoaffinity probe, and either labeling was inhibited in a specific and concentration-dependent manner by unlabeled UDP-GlcNAc or UDP-GalNAc. Thus, the enzyme is probably a homodimer composed of two 64-kDa subunits. The purified enzyme had an unusual specificity in that, at higher substrate concentrations, it utilized UDP-GalNAc as a substrate as well as UDP-GlcNAc in the reverse direction and GalNAc-1-P as well as GlcNAc-1-P in the forward direction. However, the Km for the GalNAc substrates was considerably higher than that for GlcNAc derivatives. This activity 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.

Kinetic and Physical Characterization of the Inducible UDP-N-acetylglucosamine Pyrophosphorylase from Giardia intestinalis

“…However, the bifunctionality of the prokaryotic GlmU is not observed in eukaryotes, which have evolved with two distinct enzymes: a pyrophosphorylase and an acetyltransferase. In contrast to the trimeric form found in the prokaryotic GlmUs (43,46), the eukaryotic UAPs usually exist as a monomer (13,47) or dimer (14,37). Yet the native GiUAP was reported to have a dimeric structure consisting of two ϳ33 kDa subunits (19), the rGiUAP was found to exist as a monomer of ϳ50 kDa, which corresponds to the GiUAP gene (17,18) and is more comparable with the subunit size of other eukaryotic UAPs.…”

“…The activities of these enzymes have been shown to increase from 8-to 4000-fold during encystation of this parasite (5). To date UAP has been purified and characterized from bacteria (11), yeast (12), Neurospora crassa (13), calf liver (11), pig liver (14), and human (12,15,16). However, only the UAP from G. intestinalis has been reported to be developmentally controlled, with 20-fold increase in activity during encystment (5), which makes it distinct from its counterparts in other systems.…”

Hormonal regulation ofmummyis needed for apical extracellular matrix formation and epithelial morphogenesis inDrosophila

“…Mmy (CG9535) is the predicted Drosophila orthologue of yeast and human UDP-GlcNAc-Pyp, and is thus expected to be responsible for UDP-GlcNAc synthesis in Drosophila. UDP-GlcNAc-Pyp from different species have also been shown to have UDP-GalNAc-Pyp activity (Szumilo et al, 1996;Peneff et al, 2001). In addition, UDP-GlcNAc is converted to UDP-GalNAc by the UDP-N-acetylglucosamine 4-epimerase (Winans and Bertozzi, 2002).…”

Identification of the ATP Binding Domain of Recombinant Human 40-kDa 2′,5′-Oligoadenylate Synthetase by Photoaffinity Labeling with 8-Azido-[α-32P]ATP

“…Azido photoprobes of purine and pyrimidine nucleotides covalently cross-link following UV irradiation such that photolabeled peptide fragments and/or specific amino acids have been identified (13)(14)(15)(16)(17)(18)(19)(20). Upon UV irradiation, a reactive nucleophilic nitrene is produced, which forms a covalent bond with an electrophilic group in juxtaposition for covalent bonding (13,16).…”