Isotope-dilution studies of the effects of 5-fluorodeoxyuridine and hydroxyurea on the incorporation of deoxycytidine and thymidine by cultured thymus cells

1976 ◽  
Vol 54 (3) ◽  
pp. 238-248 ◽  
Author(s):  
F. W. Scott ◽  
D. R. Forsdyke
Keyword(s):  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Isotope Dilution ◽  
Inhibitory Effect ◽  
Intact Cells ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Radioactive Labelling ◽  
Salvage Pathways ◽  
Rate Limiting

From experimentally induced changes in the slope (Vmax) and intercept (pool) of isotope-dilution plots inferences may be drawn on the position and regulation of rate-limiting steps affecting the incorporation of pyrimidine deoxynucleosides by intact cells. 5-Fluorodeoxyuridine (FdUrd; 1 μM) reduced the Vmax of radioactive labelling with deoxy[5-3H]cytidine; this was reversed by thymidine (19 μM) suggesting that FdUrd makes the concentration of deoxythymidine triphosphate (dTTP) rate-limiting for DNA synthesis. With deoxy[U-14C]cytidine the reversal of FdUrd inhibition by thymidine was only partial; this was in keeping with (i) deoxy[U-14C]cytidine labelling both cytosine and thymine in DNA, and (ii) a continuing inhibition of thymidylate synthetase by FdUrd in the presence of thymidine (19 μM).The deoxycytidine competitor pool was increased by cytidine (10–50 μM) and decreased by (i) thymidine (19 μM), (ii) hydroxyurea (50 μM) and (iii) deoxycytidine (12 μM, in the presence of FdUrd). It is suggested that these pool-decreasing agents, or their derivatives (e.g., dTTP), inhibit ribonucleotide reductase and hence prevent the entry of pyrimidine ribonucleotide derivatives into the deoxycytidine competitor pool; because of this pool decrease, radioactive labelling with deoxy[5-3H]cytidine was enhanced by thymidine (19 μM) and hydroxyurea (50 μM). However, at the latter hydroxyurea concentration, labelling with [Me-3H]thymidine was inhibited, due to a decrease in the Vmax of the rate-limiting step for thymidine incorporation (probably thymidine kinase). This sensitivity of labelling with [Me-3H]thymidine to inhibition by hydroxyurea (50 μM) was reduced by adding FdUrd to prevent the accumulation of dTTP. At high hydroxyurea concentrations (0.1–1.0 mM), labelling with deoxy[5-3H]cytidine was also inhibited, due to a decrease in Vmax of the rate-limiting step, which was probably at the level of DNA polymerase.The results suggest that hydroxyurea inhibits DNA synthesis by making the concentration of purine deoxynucleotides rate-limiting. Pyrimidine deoxynucleotides are formed in sufficient quantities from deoxycytidine by way of salvage pathways. Indeed, dTTP accumulates and inhibits thymidine kinase, thus amplifying the inhibitory effect of hydroxyurea on labelling with [Me-3 H]thymidine.

scholarly journals Isotope-dilution analysis of rate-limiting steps and pools affecting the incorporation of thymidine and deoxycytidine into cultured thymus cells

1974 ◽  
Vol 138 (2) ◽  
pp. 253-262 ◽  
Author(s):  
Dolores A. Sjostrom ◽  
D. R. Forsdyke
Keyword(s):  
Thymidine Kinase ◽  
Isotope Dilution ◽  
Thymidylate Synthetase ◽  
Isotope Dilution Analysis ◽  
Experimental Conditions ◽  
Intact Cells ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Thymus Cells ◽  
Rate Limiting

1. Rat thymus cells were incubated in homologous serum (10%) and medium 199. The effects of various quantities of thymidine or deoxycytidine (0–30μm) on the radioactive labelling of cells with the corresponding radioactive deoxynucleoside were examined. From plots of the reciprocal of the radioactivity incorporated against the total deoxynucleoside concentration (`isotope-dilution plots'), values were obtained for (a) the Vmax. of the rate-limiting step governing incorporation of the deoxynucleoside, and (b) the concentration of the pool of compounds competing with the radioactive deoxynucleoside at that rate-limiting step. From changes in these values under different experimental conditions inferences were drawn on the position and control of the rate-limiting step within intact cells. 2. Isotope-dilution plots for deoxycytidine were linear, whereas plots for thymidine were bimodal, indicating an abrupt increase in both the Vmax. and pool concentration at a critical thymidine concentration (approx. 5μm). The bimodality was removed by amethopterin. The Vmax. determined with deoxy[U-14C]cytidine was approximately equal to the sum of the Vmax. determined with deoxy[5-3H]cytidine and the Vmax. determined with [Me-3H]thymidine at thymidine concentrations above 5μm. 3. The thymidine competitor pool at thymidine concentrations above 5μm was approximately equal to the sum of the deoxycytidine competitor pool and the thymidine competitor pool at thymidine concentrations below 5μm. The pools were independent of cell concentration and dependent on serum concentration. 4. These results were explained on the following basis. Deoxycytidine in serum (16μm) is the major source of both cytosine and, by way of thymidylate synthetase, thymine, in the DNA of thymus cells. Serum deoxycytidine normally maintains a sufficient intracellular concentration of dTTP to inhibit partially the activity of thymidine kinase. When the dTTP concentration is lowered, either by decreasing the concentration of deoxycytidine or by inhibiting thymidylate synthetase, the activity of thymidine kinase increases. The activity of thymidine kinase may also be increased by concentrations of thymidine greater than 5μm, which overcome the inhibition of the enzyme by dTTP. At concentrations of thymidine below 5μm, thymidine kinase limits the rate of labelling with [Me-3H]thymidine and the radioactivity is diluted by a pool of unlabelled thymidine in serum (4μm). At thymidine concentrations greater than 5μm, the activity of DNA polymerase limits the rate of labelling and the radioactivity is diluted both by serum thymidine and, indirectly, by serum deoxycytidine.

scholarly journals Analysis of Efficiency and Fidelity of HIV-1 (+)-Strand DNA Synthesis Reveals a Novel Rate-limiting Step during Retroviral Reverse Transcription

2000 ◽  
Vol 276 (9) ◽  
pp. 6711-6719 ◽  
Author(s):  
Matthias Götte ◽  
Masanori Kameoka ◽  
Nathan McLellan ◽  
Luciano Cellai ◽  
Mark A. Wainberg
Keyword(s):  
Dna Synthesis ◽  
Reverse Transcription ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Hiv 1

scholarly journals Effect of metal ions on the kinetics of tyrosine oxidation catalysed by tyrosinase

1985 ◽  
Vol 228 (3) ◽  
pp. 647-651 ◽  
Author(s):  
A Palumbo ◽  
G Misuraca ◽  
M D'Ischia ◽  
G Prota
Keyword(s):  
Inhibitory Effect ◽  
Buffer System ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Marked Inhibitory Effect ◽  
Tyrosine Oxidation ◽  
Hydroxylation Reaction ◽  
Lag Period ◽  
Rate Limiting ◽  
Kinetics Of

The conversion of tyrosine into dopa [3-(3,4-dihydroxyphenyl)alanine] is the rate limiting step in the biosynthesis of melanins catalysed by tyrosinase. This hydroxylation reaction is characterized by a lag period, the extent of which depends on various parameters, notably the presence of a suitable hydrogen donor such as dopa or tetrahydropterin. We have now found that catalytic amounts of Fe2+ ions have the same effect as dopa in stimulating the tyrosine hydroxylase activity of the enzyme. Kinetic experiments showed that the shortening of the induction time depends on the concentration of the added metal and the nature of the buffer system used and is not suppressed by superoxide dismutase, catalase, formate or mannitol. Notably, Fe3+ ions showed only a small delaying effect on tyrosinase activity. Among the other metals which were tested, Zn2+, Co2+, Cd2+ and Ni2+ had no detectable influence, whereas Cu2+ and Mn2+ exhibited a marked inhibitory effect on the kinetics of tyrosine oxidation. These findings are discussed in the light of the commonly accepted mechanism of action of tyrosinase.

scholarly journals Inhibition of ferrochelatase during differentiation of murine erythroleukaemia cells

1987 ◽  
Vol 243 (2) ◽  
pp. 419-424 ◽  
Author(s):  
A Fadigan ◽  
H A Dailey
Keyword(s):  
Biosynthetic Pathway ◽  
Protoporphyrin Ix ◽  
Inhibitory Effect ◽  
Dimethyl Sulphoxide ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Bivalent Cations ◽  
Haem Biosynthesis ◽  
Low Levels ◽  
Rate Limiting

During dimethyl sulphoxide-induced differentiation of DS-19 murine erythroleukaemia (MEL) cells, the activity of the terminal enzyme of the haem-biosynthetic pathway, ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), is thought to be the rate-limiting step for haem production. Differentiation of induced MEL cells in the presence of exogeneously supplied protoporphyrin IX showed that total haem production was affected by added porphyrin only after 48 h. These data suggest that iron insertion, the terminal step, is rate-limiting during the first 48 h of differentiation. Addition of low levels of diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine to differentiating cultures resulted in decreased haem production and decreased ferrochelatase activity. N-Methylprotoporphyrin at nanomolar concentrations also strongly inhibited ferrochelatase activity, but had no inhibitory effect on cellular haem production. The bivalent cations Co2+, Cd2+ and Mn2+ were tested for their effect on haem production and ferrochelatase activity. All three metals were found to inhibit both haem formation and ferrochelatase activity, with Mn2+ being the strongest effector. These data, together with those previously published, suggest that the terminal step in haem biosynthesis is rate-limiting during the early stages of differentiation in MEL cells.

scholarly journals Schnyder corneal dystrophy-associated UBIAD1 is defective in MK-4 synthesis and resists autophagy-mediated degradation

2020 ◽  
Vol 61 (5) ◽  
pp. 746-757
Author(s):  
Dong-Jae Jun ◽  
Marc M. Schumacher ◽  
Seonghwan Hwang ◽  
Lisa N. Kinch ◽  
Nick V. Grishin ◽  
...  
Keyword(s):  
Autosomal Dominant ◽  
Corneal Dystrophy ◽  
Inhibitory Effect ◽  
Synthetic Activity ◽  
Autosomal Dominant Disorder ◽  
Intact Cells ◽  
Rate Limiting Step ◽  
Coa Reductase ◽  
Schnyder Corneal Dystrophy ◽  
Rate Limiting

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.

Ornithine Decarboxylase Activity in Cultured Endothelial Cells Stimulated by Serum, Thrombin and Serotonin

1978 ◽  
Vol 39 (02) ◽  
pp. 496-503 ◽  
Author(s):  
P A D’Amore ◽  
H B Hechtman ◽  
D Shepro
Keyword(s):  
Endothelial Cells ◽  
Ornithine Decarboxylase ◽  
Decarboxylase Activity ◽  
Aortic Endothelial Cells ◽  
Ornithine Decarboxylase Activity ◽  
Rate Limiting Step ◽  
Bovine Aortic Endothelial Cells ◽  
Limiting Step ◽  
Rate Limiting ◽  
Serum Serotonin

SummaryOrnithine decarboxylase (ODC) activity, the rate-limiting step in the synthesis of polyamines, can be demonstrated in cultured, bovine, aortic endothelial cells (EC). Serum, serotonin and thrombin produce a rise in ODC activity. The serotonin-induced ODC activity is significantly blocked by imipramine (10-5 M) or Lilly 11 0140 (10-6M). Preincubation of EC with these blockers together almost completely depresses the 5-HT-stimulated ODC activity. These observations suggest a manner by which platelets may maintain EC structural and metabolic soundness.

Dynamics of hepatic and peripheral insulin effects suggest common rate-limiting step in vivo

Diabetes ◽  
1993 ◽  
Vol 42 (2) ◽  
pp. 296-306 ◽  
Author(s):  
D. C. Bradley ◽  
R. A. Poulin ◽  
R. N. Bergman
Keyword(s):  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting
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