Studies of mammalian ribonucleotide reductase activity in intact permeabilized cells: A genetic approach

Wright, Jim A.; Hards, Robert G.; Dick, John E.

doi:10.1016/0065-2571(81)90011-x

Cited by 24 publications

(8 citation statements)

References 44 publications

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“…When CDP or ADP reductase activity is examined in permeabilized cells or cell-free extracts, a control experiment is performed consisting of the standard assay plus heatinactivated enzyme [3,10]. On completion of the assay, nucleotides are converted to nu cleosides by the action of snake venom en zymes, and labelled compounds are sepa rated on Dowex-1-borate columns as de scribed in Materials and Methods.…”

Section: Resultsmentioning

confidence: 99%

“…Ribonucleotide Reductase CHO cells were made permeable to nucleotides using the Tween 80 method as previously reported [3,10]. Exponentially growing cells were plated at a den sity of 5 X 106/150 mm plastic tissue culture plate with a-MEM + 10% FCS or 8% HS + 2% FCS and incubated at 37 °C.…”

Section: Cell Permeabilization and Assay Ofmentioning

confidence: 99%

“…Chinese hamster ovary (CHO) cells were cultured at 37 °C on the surface of glass bottles (Brockway Glass Co.) or on plastic tissue culture plates (Lux Scientific Ltd.) in a-minimal essential medium (a-MEM; Flow Laboratories, Ltd.) supplemented with antibiotics and 10% fetal calf serum (FCS; Gibco Ltd.) as previously described [3,10]. In some experiments 8% horse serum (HS; Gibco Ltd.) plus 2% FCS was substituted for FCS.…”

Section: Cells and Growth Conditionsmentioning

confidence: 99%

“…For ex ample, a common system for analyzing the products of a crucial rate-limiting step in DNA synthesis, ribonucleotide reduction [3,4], involves the conversion of labelled nu cleotides to nucleosides by snake venom activities followed by separation of deoxyribonucleosides from ribonucleosides on Dowex-1-borate columns [3,5]. Various laborato ries, including our own, have observed that…”

Section: Introductionmentioning

confidence: 99%

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An Iron-Dependent 5'-Nucleotide Phosphoribohydrolase Activity in the Venom of Crotalus atrox and Its Effect upon the Determination of Mammalian Ribonucleotide Reductase

Rg¹,

Ja²

1983

Enzyme

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There is an iron-dependent activity in the venom of Crotalus atrox which appears to hydrolyze the N-glycosidic sugar-base bond of pyrimidine and purine nucleotides. Maximal activation of this activity occurred at 1.0 mmol/1 and 0.8 mmol/1 FeCl(3) for cytidine diphosphate (CDP) and ADP substrates, respectively. The release of free base affects the background values of control experiments carried out during nucleotide-labelled assays of ribonucleotide reductase which require the conversion of nucleotides to nucleosides by snake venom treatment. When the reductase is examined in intact cells, a situation closely resembling normal physiological conditions for the enzyme, FeCl(3) was found to be an inhibitor of ADP reductions and varied from a mild stimulator to a significant inhibitor of CDP reductions depending upon FeCl(3) concentration. Possible explanations for previously observed variability in ribonucleotide reductase activity in the presence of iron are suggested.

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Section: Resultsmentioning

confidence: 99%

Section: Cell Permeabilization and Assay Ofmentioning

confidence: 99%

Section: Cells and Growth Conditionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

An Iron-Dependent 5'-Nucleotide Phosphoribohydrolase Activity in the Venom of Crotalus atrox and Its Effect upon the Determination of Mammalian Ribonucleotide Reductase

Rg¹,

Ja²

1983

Enzyme

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“…This method requires column monitoring, and the product is obtained in a large volume. The fourth method, which is the most common method in current use, employs an Affigel 601 column [30] or a high resolution HPLC C 18 column [32] to separate deoxycytidine and cytidine after dephosphorylation. All these methods are tedious and expensive.…”

Section: Introductionmentioning

confidence: 99%

A Simple and Sensitive Ribonucleotide Reductase Assay

Jong

Zhou

et al. 1998

J Biomed Sci

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Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [¹⁴C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg²⁺ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler, faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.

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Functional and mechanistic studies on the toxicity of deoxyguanosine for the in vitro proliferation and differentiation of human peripheral blood B lymphocytes

et al. 1986

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Deoxyguanosine (dGuo) has been implicated as the toxic metabolite causing a severe impairment of cellular immunity in children with a genetic deficiency of purine nucleoside phosphorylase (PNP). In peripheral blood T cells of normal donors both the pathway which leads to phosphorylation of dGuo (ultimately resulting in deoxyguanosine triphosphate, dGTP) and the salvage pathway which starts with degradation of dGuo by PNP (resulting in the formation of guanosine triphosphate, GTP) contribute to the inhibition of proliferation. In normal peripheral blood B cells, addition of dGuo leads to an inhibition of proliferation and differentiation. The concentrations of dGuo needed to cause a 50% inhibition are equivalent for peripheral blood T cells and B cells. Inhibition of B cell differentiation can be observed at the level of intracytoplasmic as well as secreted Ig and concerns all Ig isotypes. The early phase of B cell activation which takes place during a 24-h preculture with formalinized Cowan I Staphylococci is not affected by dGuo; it is not until proliferation and differentiation of B cells, brought about by culturing in the presence of crude concanavalin A supernatant, occurs that inhibitory effects of dGuo become evident. Addition of dGuo to B cell cultures results in an intracellular accumulation of GTP and dGTP. Addition of 8-aminoguanosine, a PNP inhibitor, next to dGuo, completely prevents the dGuo-mediated inhibition. Under these circumstances the dGuo-mediated increase in intracellular GTP is abrogated while dGTP accumulation still occurs. This indicates that the inhibitory effect of dGuo on the proliferation and differentiation of peripheral blood B lymphocytes of normal donors is independent of dGTP accumulation.

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Studies of mammalian ribonucleotide reductase activity in intact permeabilized cells: A genetic approach

Cited by 24 publications

References 44 publications

An Iron-Dependent 5'-Nucleotide Phosphoribohydrolase Activity in the Venom of Crotalus atrox and Its Effect upon the Determination of Mammalian Ribonucleotide Reductase

An Iron-Dependent 5'-Nucleotide Phosphoribohydrolase Activity in the Venom of Crotalus atrox and Its Effect upon the Determination of Mammalian Ribonucleotide Reductase

A Simple and Sensitive Ribonucleotide Reductase Assay

Functional and mechanistic studies on the toxicity of deoxyguanosine for the in vitro proliferation and differentiation of human peripheral blood B lymphocytes

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