Measurement of tissue purine, pyrimidine, and other nucleotides by radial compression high-performance liquid chromatography

Reiss, Paul D.; Zuurendonk, Peter F.; Veech, Richard L.

doi:10.1016/0003-2697(84)90148-9

Cited by 76 publications

(35 citation statements)

References 28 publications

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“…The concentration we found for UTP is similar to that found by others [27][28][29] The "4C specific radioactivities of the UDP-sugars became relatively high, taking into account that the label is substantial diluted in the large pools. This is indicative of a high turnover and/or an input of label from an UTP pool with a higher specific radioactivity than the measured average UTP value.…”

Section: Discussionsupporting

confidence: 90%

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Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Rijcken

Hooghwinkel

Ferwerda

1990

Biochemical Journal

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With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 1'C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.

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Section: Discussionsupporting

confidence: 90%

“…The values reported for CDP-choline, determined enzymically [26], and cytidine phosphates obtained with the h.p.l.c. technique [27,28] [26,29]. For the concentrations of the UDP-sugars we obtained higher values as most other investigators.…”

Section: Discussionsupporting

confidence: 59%

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Rijcken

Hooghwinkel

Ferwerda

1990

Biochemical Journal

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“…According to our kit results, HeLa cells without any treatment have the NADH and NAD levels of 158 nM and 206 nM /10 6 cells respectively. The value of NAD of 206 nM/10 6 cells is comparable with the value reported for rat liver cells of 1μM per gram of wet weight (approximately 10 6 cells) [34]. The levels of NADH and NADPH generally decrease with the increase in FCCP concentration.…”

Section: Nad + /Nadh and Nadp+/nadph Quantificationsupporting

confidence: 83%

Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence

Rehman¹,

Anwer²,

Gosnell³

et al. 2017

Biomed. Opt. Express

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Carbonyl cyanide-p-trifluoro methoxyphenylhydrazone (FCCP) is a well-known mitochondrial uncoupling agent. We examined FCCP-induced fluorescence quenching of reduced nicotinamide adenine dinucleotide / nicotinamide adenine dinucleotide phosphate (NAD(P)H) in solution and in cultured HeLa cells in a wide range of FCCP concentrations from 50 to 1000µM. A non-invasive label-free method of hyperspectral imaging of cell autofluorescence combined with unsupervised unmixing was used to separately isolate the emissions of free and bound NAD(P)H from cell autofluorescence. Hyperspectral image analysis of FCCP-treated HeLa cells confirms that this agent selectively quenches fluorescence of free and bound NAD(P)H in a broad range of concentrations. This is confirmed by the measurements of average NAD/NADH and NADP/NADPH content in cells. FCCP quenching of free NAD(P)H in cells and in solution is found to be similar, but quenching of bound NAD(P)H in cells is attenuated compared to solution quenching possibly due to a contribution from the metabolic and/or antioxidant response in cells. Chemical quenching of NAD(P)H fluorescence by FCCP validates the results of unsupervised unmixing of cell autofluorescence. Hidalgo, "Ultraviolet excitation fluorescence spectroscopy: a noninvasive method for the measurement of redox changes in ischemic myocutaneous flaps," Plast. Reconstr. Surg. 96(3), 673-680 (1995). 31. J. Eng, R. M. Lynch, and R. S. Balaban, "Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes," Biophys. J. 55(4), 621-630 (1989). 32. A. S. Galkin, V. G. Grivennikova, and A. D. Vinogradov, "→H+/2e-stoichiometry in NADH-quinone reductase reactions catalyzed by bovine heart submitochondrial particles," FEBS Lett. 451(2), 157-161 (1999). 33. J. P. Brennan, R. G. Berry, M. Baghai, M. R. Duchen, and M. J. Shattock, "FCCP is cardioprotective at concentrations that cause mitochondrial oxidation without detectable depolarisation," Cardiovasc.

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“…The reaction occurs in homogenates and crude supernatants at pH values close to 7. The apparent K m value for the substrate is close to its intracellular concentration in rat liver [21][22][23], and is of the same order of magnitude as that of many enzymes involved in nucleotide metabolism [24][25][26][27][28]. The AdK, MK and ADA concentrations used in these experiments coincide with the concentrations found in rat liver tissue [11,12].…”

Section: Physiological Rolesupporting

confidence: 60%

Evidence of a new phosphoryl transfer system in nucleotide metabolism

et al. 2008

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Purine nucleotides are precursors of nucleic acids and participate in many metabolic pathways as substrates, coenzymes and energy sources. Much attention has been focused on the roles and intracellular levels of AMP, ADP and ATP in various tissues under normal and pathological conditions [1][2][3]. Their relationships to genetic diseases, blood disorders, drugs, tumours and other pathologies have been studied extensively [4][5][6].Although ATP formation occurs via several wellknown mechanisms, ADP is thought to be formed only from ATP, either by the adenylate kinase reaction or by ATPase-mediated hydrolysis. The possibility that ADP can be formed by de novo synthesis from low-energy precursors has never been investigated fully. Even less studied is the possibility that ADP might be synthesized from low-energy precursors under special conditions, such as ischaemia and hypoxia, in which massive depletion of ATP and elevation of AMP are known to occur [7][8][9][10].In this article, it is shown that, under physiological conditions, ADP may be formed in mammalian tissues by the disproportionation of AMP, consistent with an AMP-AMP phosphotransferase reaction. This reaction is carried out by enzymes of purine metabolism which, under specific cellular conditions, associate in a biological network and cooperate in a reaction not reported previously. Crude rat liver extract showed AMP-AMP phosphotransferase activity which, on purification, was ascribed to a novel interaction between adenylate kinase, also known as myokinase (EC 2.7.4.3), and adenosine kinase (EC 2.7.1.20). The activity was duplicated using the same enzymes purified from recombinant sources. The reaction requires physical contact between myokinase and adenosine kinase, and the net reaction is aided by the presence of adenosine deaminase (EC 3.5.4.4), which fills the gap in the energy balance of the phosphoryl transfer and shifts the equilibrium towards ADP and inosine synthesis. The proposed mechanism involves the association of adenosine kinase and myokinase through non-covalent, transient interactions that induce slight conformational changes in the active site of myokinase, bringing two already bound molecules of AMP together for phosphoryl transfer to form ADP. The proposed mechanism suggests a physiological role for the enzymes and for the AMP-AMP phosphotransferase reaction under conditions of extreme energy drain (such as hypoxia or temporary anoxia, as in cancer tissues) when the enzymes cannot display their conventional activity because of substrate deficiency.

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Measurement of tissue purine, pyrimidine, and other nucleotides by radial compression high-performance liquid chromatography

Cited by 76 publications

References 28 publications

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Pyrimidine metabolism and sugar nucleotide synthesis in rat liver

Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence

Evidence of a new phosphoryl transfer system in nucleotide metabolism

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