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.

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.

scholarly journals Effects of taurolithocholate, a Ca2+-mobilizing agent, on cell Ca2+ in rat hepatocytes, human platelets and neuroblastoma NG108-15 cell line

1991 ◽  
Vol 273 (1) ◽  
pp. 153-160 ◽  
Author(s):  
J F Coquil ◽  
B Berthon ◽  
N Chomiki ◽  
L Combettes ◽  
P Jourdon ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Bile Acid ◽  
Cell Line ◽  
Rat Liver ◽  
Neuronal Cell ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Liver Cells ◽  
Human Platelets ◽  
Rat Liver Cells

The monohydroxy bile acid taurolithocholate permeabilizes the endoplasmic reticulum to Ca2+ in rat liver cells. To assess whether this action on the endoplasmic reticulum was restricted to this tissue, the effects of bile acid were investigated in two cell types quite unrelated to rat hepatocyte, namely human platelets and neuronal NG108-15 cell line. The results showed that taurolithocholate (3-100 microM) had no effect on free cytosolic [Ca2+] in human platelets and NG108-15 cells. whereas it increased it from 180 to 520 nM in rat hepatocytes. In contrast, in cells permeabilized by saponin, taurolithocholate initiated a profound release of the stored Ca2+ from the internal Ca2+ pools in the three cell types. The bile acid released 90% of the Ca2+ pools, with rate constants of about 5 min-1 and half-maximal effects at 15-30 microM. The results also showed that, in contrast with liver cells, which displayed an influx of [14C]taurolithocholate of 2 nmol/min per mg, human platelets and the neuronal cell line appeared to be resistant to [14C]taurolithocholate uptake. The influx measured in these latter cells was about 100-fold lower than in rat liver cells. Taken together, these data suggest that human platelets and NG108-15 cells do not possess the transport system for concentrating monohydroxy bile acids into cells. However, they show that human platelets and neuronal NG108-15 possess, in common with liver cells, the intracellular system responsible for taurolithocholate-mediated Ca2+ release from internal stores.

'Liver-type' 11β-hydroxysteroid dehydrogenase cDNA encodes reductase but not dehydrogenase activity in intact mammalian COS-7 cells

1994 ◽  
Vol 13 (2) ◽  
pp. 167-174 ◽  
Author(s):  
S C Low ◽  
K E Chapman ◽  
C R W Edwards ◽  
J R Seckl
Keyword(s):  
Dehydrogenase Activity ◽  
Rat Liver ◽  
Mammalian Cells ◽  
Reductase Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Intact Cells ◽  
Mmtv Ltr

ABSTRACT 11β-Hydroxysteroid dehydrogenase (11β-HSD) catalyses the metabolism of corticosterone to inert 11-dehydrocorticosterone, thus preventing glucocorticoid access to otherwise non-selective renal mineralocorticoid receptors (MRs), producing aldosterone selectivity in vivo. At least two isoforms of 11β-HSD exist. One isoform (11β-HSD1) has been purified from rat liver and an encoding cDNA cloned from a rat liver library. Transfection of rat 11β-HSD1 cDNA into amphibian cells with a mineralocorticoid phenotype encodes 11 β-reductase activity (activation of inert 11-dehydrocorticosterone) suggesting that 11β-HSD1 does not have the necessary properties to protect renal MRs from exposure to glucocorticoids. This function is likely to reside in a second 11β-HSD isoform. 11β-HSD1 is co-localized with glucocorticoid receptors (GRs) and may modulate glucocorticoid access to this receptor type. To examine the predominant direction of 11β-HSD1 activity in intact mammalian cells, and the possible role of 11β-HSD in regulating glucocorticoid access to GRs, we transfected rat 11β-HSD1 cDNA into a mammalian kidney-derived cell system (COS-7) which has little endogenous 11β-HSD activity or mRNA expression. Homogenates of COS-7 cells transfected with increasing amounts of 11β-HSD cDNA exhibited a dose-related increase in 11 β-dehydrogenase activity. In contrast, intact cells did not convert corticosterone to 11-dehydrocorticosterone over 24 h, but showed a clear dose-related 11β-reductase activity, apparent within 4 h of addition of 11-dehydrocorticosterone to the medium. To demonstrate that this reflected a change in functional intracellular glucocorticoids, COS-7 cells were co-transfected with an expression vector encoding GR and a glucocorticoid-inducible MMTV-LTR luciferase reporter construct, with or without 11β-HSD. Corticosterone induced MMTV-LTR luciferase expression in the presence or absence of 11β-HSD. 11-Dehydrocorticosterone was without activity in the absence of 11β-HSD, but induced MMTV-LTR luciferase activity in the presence of 11β-HSD. These results indicate that rat 11β-HSD1 can behave exclusively as a reductase in intact mammalian cells. Thus in some tissues in vivo, 11β-HSD1 may regulate ligand access to GRs by reactivating inert glucocorticoids.

scholarly journals Polyribosomes in isolated liver cells. Preparative procedures, effects of incubation and correlation with protein synthesis

1980 ◽  
Vol 186 (1) ◽  
pp. 35-45 ◽  
Author(s):  
A J Dickson ◽  
C I Pogson
Keyword(s):  
Protein Synthesis ◽  
Rat Liver ◽  
Isolated Hepatocytes ◽  
Liver Cells ◽  
Liver Protein ◽  
Isolated Cells ◽  
Isolated Liver Cells ◽  
Rat Liver Cells ◽  
Isolated Liver

Methods have been derived which permit the isolation of undergraded polyribosomes from isolated rat liver cells. Under the conditions used the polyribosome profile of hepatocytes immediately after isolation was essentially identical with that from intact liver. However, during incubation of cells in complex physiological media there was a progressive dissociation of polyribosomes. The addition of a variety of factors that produce reaggregation of polyribosomes in rat liver in vivo did not prevent dissociation during cell incubations. Although large polyribosomes were lost most rapidly, the albumin-synthesizing capacity of isolated cells was not selectively lost when compared with total protein synthesis. The significance of these results for the use of isolated hepatocytes in the study of liver protein synthesis is discussed.

Antiketogenic effect of glucose per se in vivo in man and in vitro in isolated rat liver cells

Metabolism ◽  
1986 ◽  
Vol 35 (7) ◽  
pp. 608-613 ◽  
Author(s):  
J.P. Riou ◽  
M. Beylot ◽  
M. Laville ◽  
L. De Parscau ◽  
J. Delinger ◽  
...  
Keyword(s):  
Rat Liver ◽  
Liver Cells ◽  
Isolated Rat Liver ◽  
Rat Liver Cells ◽  
Per Se ◽  
Effect Of Glucose ◽  
Isolated Rat

scholarly journals MTrasT24, a metallothionein-ras fusion gene, modulates expression in cultured rat liver cells of two genes associated with in vivo liver cancer.

1988 ◽  
Vol 85 (2) ◽  
pp. 344-348 ◽  
Author(s):  
Y. C. Li ◽  
T. Seyama ◽  
A. K. Godwin ◽  
T. S. Winokur ◽  
R. M. Lebovitz ◽  
...  
Keyword(s):  
Liver Cancer ◽  
Rat Liver ◽  
Fusion Gene ◽  
Liver Cells ◽  
Rat Liver Cells

Vasopressin promotes the metabolism of near-physiological concentration of glutamine in isolated rat liver cells

1984 ◽  
Vol 4 (2) ◽  
pp. 171-176 ◽  
Author(s):  
Guy Martin ◽  
Gabriel Baverel
Keyword(s):  
Rat Liver ◽  
Liver Cells ◽  
Hepatic Gluconeogenesis ◽  
Physiological Concentration ◽  
Isolated Rat Liver ◽  
Rat Liver Cells ◽  
Isolated Rat

In isolated rat liver cells, vasopressin, like glucagon, promotes the metabolism of glutamine used at nearphysiological concentration (1 mM). These findings indicate that, in vivo, both hormones might participate in the control of hepatic gluconeogenesis and ureogenesis from glutamine.

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