Resolution of phosphoglucomatase and the 62-kDA acceptor for the glucosylphosphotransferase.
The radioactive, photoactivatable labeling probe [beta-32P]5-azidouridine 5'-diphosphoglucose has recently been shown to label a 62-kDa protein in crude homogenates and in partially purified enzyme preparations without photoactivation. Here, we report that a portion of this radioactivity is due to labeling of phosphoglucomutase by contaminating levels of [32P]alpha Glc-1-P initially present at less than 1% of the total 32P. This conclusion is based in part on the ability of excess unlabeled alpha Glc-1-P and Glc-6-P, but not UDP-Glc, to block the labeling. In addition, the labeled protein in liver homogenates had a tryptic peptide pattern similar to that of authentic phosphoglucomutase. These findings, however, raised a second question. Assays for the UDP-Glc: glycoprotein glucosyl phosphotransferase (Glc phosphotransferase) have utilized [beta-32P]UDP-Glc and have resulted in the labeling of a small number of acceptors, including one of approximately 62 kDa. Despite the fact that these assays had routinely been performed in the presence of 1 mM alpha Glc-1-P, the coincidence in molecular weights led to these further studies. We conclude that the acceptor of approximately 62 kDa is distinct from phosphoglucomutase. This conclusion is based on differences in the time courses of incorporation, the specificity of blocking agents, the presence of covalently linked glucose, the products of acid hydrolysis and of beta-elimination, and isoelectric points.
Marchase
R B
RB
Department of Cell Biology and Anatomy, University of Alabama, Birmingham 35294.
Richardson
K L
KL
Srisomsap
C
C
Drake
R R
RR
Haley
B E
BE
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010733:Phosphoglucomutase / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases / Q000378:metabolism*; D011506:Proteins; D011865:Radioisotope Dilution Technique; D051381:Rats; D017855:Transferases (Other Substituted Phosphate Groups); D014357:Trypsin; D014532:Uridine Diphosphate Glucose / Q000031:analogs & derivatives / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D011506:Proteins; D014539:Uridine Diphosphate Sugars; C060227:5-azidouridine 5'-diphosphoglucose; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C037061:UDPglucose-glycoprotein glucose-1-phosphotransferase; D014357:Trypsin; D010733:Phosphoglucomutase; D014532:Uridine Diphosphate Glucose