The M1 subunit of rat liver ribonucleotide reductase appears to be modified by ubiquitination.
Using a combination of immunoblotting, double immunoprecipitation, immunoglobulin-affinity chromatography, and isoelectrofocusing, we have been able to identify a group of proteins that display CDP-reductase activity and contain antigenic epitopes recognized by anti-ribonucleotide reductase M1 subunit and anti-ubiquitin antibodies. In the cytoplasm of rat liver cells, we could detect a total of five proteins with molecular masses of 92, 89, 56, 45, and 37 kilodaltons which reacted with the anti-M1 subunit serum. All of them, except the 89-kilodalton protein (the nascent unmodified M1), were also recognized by the anti-ubiquitin antibody. In normal liver cells, all of the apparently ubiquitinated species of the M1 protein were found in the cytoplasm, but not in the nuclear envelope associated pool of the enzyme. However, we did not detect ubiquitinated M1 protein fragments in the cytoplasm of Morris hepatoma 5123tc. The level of the apparently ubiquitinated fragments of the M1 subunit increased in parallel to the DNA-synthetic activity of normal liver cells, suggesting that ubiquitination plays a key role in the regulation of the activity of the enzyme during the cell cycle.
Sikorska
M
M
Institute for Biological Sciences, National Research Council, Ottawa, Canada.
Kwast-Welfeld
J
J
Youdale
T
T
Richards
R
R
Whitfield
J F
JF
Walker
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000201:enzymology; D004261:DNA Replication; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000201:enzymology; D011499:Protein Processing, Post-Translational; D051381:Rats; D011919:Rats, Inbred Strains; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism*
D014452:Ubiquitins; D012264:Ribonucleotide Reductases