Purification to homogeneity and properties of UDP-GlcNAc (GalNAc) pyrophosphorylase.
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.
Szumilo
T
T
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Zeng
Y
Y
Pastuszak
I
I
Drake
R
R
Szumilo
H
H
Elbein
A D
AD
D000345:Affinity Labels; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification; D013379:Substrate Specificity; D013552:Swine; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D000345:Affinity Labels; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase