A Colorimetric Method for Estimating the Pyridine Nucleotide Content of Small Amounts of Animal Tissue

Slater, T. F.; Sawyer, Barbara

doi:10.1038/193454a0

Cited by 107 publications

(33 citation statements)

References 9 publications

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“…A blue formazan precipitate is formed with a characteristic absorption maximum of 540 nm (van Noorden & Tas, 1980). For details of this cycling reaction see Slater and Sawyer (1962) and Slater et al (1964). The intensity of staining is proportional to the enzyme activity and varies in each individual cell.…”

Section: Processingmentioning

confidence: 99%

Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia

Jonas

Benedetto²,

Flatman³

et al. 1992

Br J Cancer

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The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected]. Images Figure 1

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Section: Processingmentioning

confidence: 99%

Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia

Jonas

Benedetto²,

Flatman³

et al. 1992

Br J Cancer

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“…Samples were withdrawn at 30, 60 and 120 min and frozen at 270 uC. Pyridine nucleotides were estimated by the method of Slater & Sawyer (1962), with the following modifications. Cellular pellets corresponding to 15 ml of culture were resuspended in 200 ml 0.2 M HCl or in 400 ml 0.2 M NaOH, and centrifuged at 16 000 g for 10 min.…”

Section: Determination Of Nadpmentioning

confidence: 99%

The soxRS response of Escherichia coli can be induced in the absence of oxidative stress and oxygen by modulation of NADPH content

2011

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The soxRS regulon protects Escherichia coli cells against superoxide and nitric oxide. Oxidation of the SoxR sensor, a [2Fe-2S]-containing transcriptional regulator, triggers the response, but the nature of the cellular signal sensed by SoxR is still a matter of debate. In vivo, the sensor is maintained in a reduced, inactive state by the activities of SoxR reductases, which employ NADPH as an electron donor. The hypothesis that NADPH levels affect deployment of the soxRS response was tested by transforming E. coli cells with genes encoding enzymes and proteins that lead to either build-up or depletion of the cellular NADPH pool. Introduction of NADP + -reducing enzymes, such as wheat non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase or E. coli malic enzyme, led to NADPH accumulation, inhibition of the soxRS regulon and enhanced sensitivity to the superoxide propagator methyl viologen (MV). Conversely, expression of pea ferredoxin (Fd), a redox shuttle that can oxidize NADPH via ferredoxin-NADP(H) reductase, resulted in execution of the soxRS response in the absence of oxidative stress, and in higher tolerance to MV. Processes that caused NADPH decline, including oxidative stress and Fd activity, correlated with an increase in total (NADP + +NADPH) stocks. SoxS expression can be induced by Fd expression or by MV in anaerobiosis, under conditions in which NADPH is oxidized but no superoxide can be formed. The results indicate that activation of the soxRS regulon in E. coli cells exposed to superoxide-propagating compounds can be triggered by depletion of the NADPH stock rather than accumulation of superoxide itself. They also suggest that bacteria need to finely regulate homeostasis of the NADP(H) pool to enable proper deployment of this defensive response.

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“…Concentrations of CTP were calculated from the weight of dissolved material, in conjunction with the purity stated by the manufacturers. NAD kina,se assays [I] were performed a t 30" in 0.1 M triethaloamine-Cl buffer, pH 7.4; NADP was assayed by the method of Slater and Sawyer [8] and APADP with isocitric dehydrogenase [9]. The enzyme as prepared was free from activities which interfere with its assay [I].…”

Section: Chemicalsmentioning

confidence: 99%

“…The cycling assay was used for NADPH [8], and reduction with yeast glucose 6-phosphate dehydrogenase or pig heart isocitrate Double reciprocal plots were constructed with APAD and ATP * Mg= as substrates: the h e t i c constants derived from these are shown in Table 3. Since the K m values were dependent on second substrate concentrations (Fig.…”

Section: Pyridine Nucleotide Specificitymentioning

confidence: 99%

The Substrate Specificity of Pigeon Liver NAD Kinase

Apps

1969

European Journal of Biochemistry

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1. The specificity of pigeon liver NAD kinase for both substrates and for divalent cations has been investigated. 2. The enzyme is activated by Mg++, Co++, Mn++, and Zn++ only: the ATPmetal complexes have different Km, values but the maximum velocities of the reactions are the same. 3. All nucleoside triphosphates investigated will act as substrates, although the Km values and maximum velocities differ. 4. The only NAD analogue found to act as a substrate was 3-acetyl pyridine-adenine dinucleotide (APAD). 5. The enzyme is inhibited by free Mg and free ATP. 6. The independence of substrate binding sites confirms that a random-addition, rapid equilibrium mechanism applies to NAD kinase.I n a previous paper [I] some kinetic work on NAD kinase (ATP : NAD 2'-phosphotransferase) was reported, and it was concluded that the enzyme acts by a mechanism in which the substrates bind rapidly, and in random order, to independent binding sites, the rate-limiting step of the reaction being interconversion of central ternary complexes. The case for such a mechanism would be strengthened if it could be shown that replacement of one substrate by an analogue with a different K , or Vmax had no effect on the binding of the other substrate: with this in mind, all three participants in the forward component of the reaction ATP * M= + +NAD= + ADP -M-+ +NADP4-+ H+ viz. the metal ion, the phosphate donor (nucleoside triphosphate) and the phosphate acceptor (pyridine nucleotide coenzyme) have been independently varied, and the kinetic parameters tabulated.

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A Colorimetric Method for Estimating the Pyridine Nucleotide Content of Small Amounts of Animal Tissue

Cited by 107 publications

References 9 publications

Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia

Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia

The soxRS response of Escherichia coli can be induced in the absence of oxidative stress and oxygen by modulation of NADPH content

The Substrate Specificity of Pigeon Liver NAD Kinase

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