Evidence that mammalian ribonucleotide reductase is a nuclear membrane associated glycoprotein.
Epitope-specific antibodies to the M1 and M2 subunits of mammalian ribonucleotide reductase were prepared using peptides predicted to have a high antigenic index. Western blotting demonstrated that the anti-M1 antibody was specific for the 89-kilodalton M1 subunit (and its degradation fragments) and the anti-M2 antibody specifically recognized the 45-kilodalton M2 subunit. Both antibodies inhibited the CDP-reductase activity of the holoenzyme. Using these antibodies, both the M1 and M2 subunits were shown to be localized in the cytoplasm and in the nuclear regions of a number of cell types, including B77 avian sarcoma virus transformed NRK cells, T51B rat liver cells, 5123tc hepatoma cells, and rat liver cells in vivo. In addition, the M1 subunit was found to be localized as a halo around isolated rat liver nuclei. Biochemical analysis of the cytoplasmic fraction of liver cells and a Triton X-100 wash of nuclei from these cells confirmed the location of the enzyme activity in these cellular compartments. The M1 subunit appears to be glycosylated, as indicated by its retention on a Affi-Gel-concanavalin A affinity column. Therefore, in mammalian cells ribonucleotide reductase appears to be not only in the cytoplasm, but is also associated with the nuclear membrane or nuclear lamina. The activity of the enzyme in the membrane fraction changes dynamically during the cell cycle.
Division of Biological Sciences, National Research Council, Ottawa, Ont., Canada.
M
M
Sikorska
L M
LM
Brewer
T
T
Youdale
R
R
Richards
J F
JF
Whitfield
R A
RA
Houghten
P R
PR
Walker
D000818:Animals; D002460:Cell Line; D002461:Cell Line, Transformed; D003593:Cytoplasm / Q000201:enzymology; D004261:DNA Replication; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D008099:Liver / Q000201:enzymology / Q000648:ultrastructure; D008114:Liver Neoplasms, Experimental; D008115:Liver Regeneration / Q000502:physiology; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000378:metabolism; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D012264:Ribonucleotide Reductases / Q000032:analysis* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D008562:Membrane Glycoproteins; D012264:Ribonucleotide Reductases