The M1 subunit of rat liver ribonucleotide reductase appears to be modified by ubiquitination

1992 ◽  
Vol 70 (3-4) ◽  
pp. 215-223 ◽  
Author(s):  
Marianna Sikorska ◽  
Joanna Kwast-Welfeld ◽  
Tony Youdale ◽  
Robert Richards ◽  
James F. Whitfield ◽  
...  
Keyword(s):  
Rat Liver ◽  
Ribonucleotide Reductase ◽  
Reductase Activity ◽  
Normal Liver ◽  
Liver Cells ◽  
Synthetic Activity ◽  
M1 Protein ◽  
Rat Liver Cells ◽  
Molecular Masses ◽  
Morris Hepatoma

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.Key words: ribonucleotide reductase turnover, proteolysis, ubiquitin, DNA replication.

Evidence that mammalian ribonucleotide reductase is a nuclear membrane associated glycoprotein

1990 ◽  
Vol 68 (5) ◽  
pp. 880-888 ◽  
Author(s):  
Marianna Sikorska ◽  
Linda M. Brewer ◽  
Tony Youdale ◽  
Robert Richards ◽  
James F. Whitfield ◽  
...  
Keyword(s):  
Rat Liver ◽  
Ribonucleotide Reductase ◽  
Nuclear Membrane ◽  
Mammalian Cells ◽  
Reductase Activity ◽  
Cell Types ◽  
Liver Cells ◽  
Affinity Column ◽  
Rat Liver Cells

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.Key words: replication, DNA, synthesis, glycosylation, liver.

Changes in the cytoplasmic and nuclear activities of the ribonucleotide reductase holoenzyme and its subunits in regenerating liver cells in normal and thyroparathyroidectomized rats

1984 ◽  
Vol 62 (9) ◽  
pp. 914-919 ◽  
Author(s):  
T. Youdale ◽  
L. Frappier ◽  
J. F. Whitfield ◽  
R. H. Rixon
Keyword(s):  
Dna Synthesis ◽  
Ribonucleotide Reductase ◽  
Maximum Level ◽  
Liver Cells ◽  
Synthetic Activity ◽  
Regenerating Liver ◽  
Nuclear Level ◽  
Rat Liver Cells ◽  
Specific Effector ◽  
High Level

The level of the cytoplasmic ribonucleotide reductase nonheme-iron-containing L2 subunit in regenerating rat liver cells began rising about 2 h before the onset of DNA synthesis, rose sharply to a maximum level about 4 h before the DNA-synthetic activity reached its peak, and then stayed at this high level even after the cells had finished replicating their DNA. The cytoplasmic level of the CDP-specific, effector-binding L1 subunit and the holoenzyme activity began rising together about 2 h after the L2 subunit began increasing and at the same time as the DNA-synthetic activity, but subsequently rose much more slowly than the L2 subunit and continued rising even after the cells had finished making DNA. The nuclear level of the L2 subunit did not rise in the regenerating liver cells, but the nuclear level of the L1 subunit and the holoenzyme activity began rising together about the same time as the DNA-synthetic activity, peaked briefly 4–6 h before the peak DNA-synthetic activity, and dropped sharply back to the basal levels by the time the DNA-synthetic activity reached its peak, but then rose again slowly as the cells finished making DNA. Thyroparathyroidectomy 72 h before partial hepatectomy prevented the cytoplasmic and nuclear subunits and holoenzyme activity from rising and prevented most of the remaining liver cells from initiating DNA synthesis.

Effects of Hypophysectomy on the Fine Structure of Rat Liver Cells

1967 ◽  
Vol 25 ◽  
pp. 156-157
Author(s):  
Robert R. Cardell
Keyword(s):  
Electron Microscopy ◽  
Fine Structure ◽  
Rat Liver ◽  
Liver Cell ◽  
Anterior Pituitary ◽  
Liver Cells ◽  
Biochemical Studies ◽  
Liver Functions ◽  
Rat Liver Cells ◽  
And Control

Hypophysectomy of the rat renders this animal deficient in the hormones of the anterior pituitary gland, thus causing many primary and secondary hormonal effects on basic liver functions. Biochemical studies of these alterations in the rat liver cell are quite extensive; however, relatively few morphological observations on such cells have been recorded. Because the available biochemical information was derived mostly from disrupted and fractionated liver cells, it seemed desirable to examine the problem with the techniques of electron microscopy in order to see what changes are apparent in the intact liver cell after hypophysectomy. Accordingly, liver cells from rats which had been hypophysectomized 5-120 days before sacrifice were studied. Sham-operated rats served as controls and both hypophysectomized and control rats were fasted 15 hours before sacrifice.

Heterogeneity of carboxylesterases in rat liver cells

1992 ◽  
Vol 44 (4) ◽  
pp. 827-829 ◽  
Author(s):  
Rolf Gaustad ◽  
Trond Berg ◽  
Frode Fonnum
Keyword(s):  
Rat Liver ◽  
Liver Cells ◽  
Rat Liver Cells
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