Luminescent immobilized enzyme test systems for inorganic pyrophosphate: Assays using firefly luciferase and nicotinamide-mononucleotide adenylyl transferase or adenosine-5′-triphosphate sulfurylase

Barshop, Bruce A.; Adamson, David T.; Vellom, Daniel C.; Rosen, Fred; Epstein, Benita L.; Seegmiller, J. Edwin

doi:10.1016/0003-2697(91)90387-9

Cited by 45 publications

(27 citation statements)

References 17 publications

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“…It is possible that the PP i concentration may be elevated in certain circumstances, resulting in the transient reversal of PP i -generating reactions; however, in a general sense, biochemical or cellular mechanisms must exist that limit the extent of the reverse reactions to assure that biosynthetic reactions will go forward. The subcellular distribution of PP i and pyrophosphatases has not been extensively described; yet a wide range of PP i concentrations, ranging from 4 M to 400 M, has been reported in cell extracts (3,8,18,24,41,44). This range is explained, at least in part, by events that occur during or after preparation of the cell extracts.…”

Section: Discussionmentioning

confidence: 99%

Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations

Smith¹,

Meyer²,

Asthana

et al. 2005

Antimicrob Agents Chemother

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Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with 3-azido-3-deoxythymidine (AZT) selects for mutant forms of viral reverse transcriptase (RT) with increased ability to remove chain-terminating nucleotides from blocked DNA chains. We tested various cell extracts for the presence of endogenous acceptor substrates for this reaction. ؉ or CD8 ؉ T cells, while PP i was present at 7 to 15 M. Under these conditions, the ATP-dependent reaction predominated, and excision by the AZT-resistant mutant RT was more efficient than wild type RT. Activated CD4 ؉ or CD8 ؉ T cells contained 1.4 to 2.7 mM ATP and 55 to 79 M PP i . These cellular PP i concentrations are lower than previously reported; nonetheless, the PP i -dependent reaction predominated in extracts from activated T cells, and excision by mutant and wild-type RT occurred with similar efficiency. While PP i -dependent excision may contribute to AZT resistance in vivo, it is likely that selection of AZT-resistant mutants occurs primarily in an environment where the ATP-dependent reaction predominates.Human immunodeficiency virus type 1 (HIV-1) replication is inhibited by the incorporation of chain-terminating nucleotide analogs at the 3Ј end of the nascent DNA chain during reverse transcription. Viral replication in the presence of these drugs results in selection of resistance mutations in the viral sequences encoding reverse transcriptase (RT), the replicative enzyme for HIV. Mutations at six codons in RT (M41L, D67N, K70R, L210W, T215Y or -F, and K219Q) are commonly selected by the nucleoside inhibitor 3Ј-azido-3Ј-deoxythymidine (AZT) and have been shown to confer AZT resistance through an increased ability to remove the chain terminator after it has been incorporated into DNA (1, 29, 46). These mutations are also correlated with significant resistance to other chain-terminating nucleosides (33,34,47).Excision has been shown to occur by RT-catalyzed transfer of the chain-terminating residue to a variety of acceptor substrates in vitro by a reaction that is related to pyrophosphorolysis (1,5,16,29,31,42,43). The intracellular acceptor for this reaction is unknown, but likely candidates include nucleoside triphosphates and nucleoside diphosphates as well as inorganic pyrophosphate (PP i ).In this report, we show that cell extracts contain a mixture of acceptor substrates for the excision reaction. ATP or PP i predominates depending on the cell type and its activation status. For these experiments, it was necessary to avoid errors in the measurement of PP i levels in the extracts, which can be affected by a number of factors: the breakdown of unstable intracellular compounds to form PP i during the extraction procedure, contaminating platelets, and cellular enzymes that alter PP i levels during the extraction process. Results obtained with an optimized extraction procedure disagree with widely quoted values of 130 to 150 M for intracellular PP i concentration in unstimulated lymphocytes (3); our results are more in agreement with the dat...

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Section: Discussionmentioning

confidence: 99%

Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations

Smith¹,

Meyer²,

Asthana

et al. 2005

Antimicrob Agents Chemother

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“…The reaction was started by adding sGC and was stopped by adding 50 mM EDTA. ATP was then determined by the firefly luciferase method (modified from Barshop et al, 1991). The microtiter plates were placed in a luminometer (Lumistar Fa.…”

Section: Methodsmentioning

confidence: 99%

Biochemistry and Pharmacology of Novel Anthranilic Acid Derivatives Activating Heme-Oxidized Soluble Guanylyl Cyclase

Schindler

Strobel²,

Schönafinger³

et al. 2006

Molecular Pharmacology

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The heme-enzyme soluble guanylyl cyclase (sGC) is an ubiquitous NO receptor, which mediates NO downstream signaling by the generation of cGMP. We studied the mechanism of action of the anthranilic acid derivatives 5-chloro-2-(5-chloro-thiophene-2-sulfonylamino-N-(4-(morpholine-4-sulfonyl)-phenyl)-benzamide sodium salt (HMR1766) (proposed international nonproprietary name, ataciguat sodium) and 2-(4-chloro-phenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)-phenyl)-benzamide (S3448) as a new class of sGC agonists. Both compounds activated different sGC preparations (purified from bovine lung, or crude from human corpus cavernosum) in a concentrationdependent and quickly reversible fashion (EC 50 ϭ 0.5-10 M), with mixed-type activation kinetics. Activation of sGC by these compounds was additive to activation by NO donors, but instead of being inhibited, it was potentiated by the heme-iron oxidants 1H-[1,2,4]-oxdiazolo[3,4-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028), suggesting that the new compounds target the ferric heme sGC isoform. Protoporphyrin IX acted as a competitive activator, and zinc-protoporphyrin IX inhibited activation of hemeoxidized sGC by HMR1766 and S3448, whereas heme depletion of sGC by Tween 20 treatment reduced activation. Both compounds increased cGMP levels in cultured rat aortic smooth muscle cells; induced vasorelaxation of isolated endothelium-denuded rat aorta, porcine coronary arteries, and human corpus cavernosum (EC 50 1 to 10 M); and elicited phosphorylation of the cGMP kinase substrate vasodilator-stimulated phosphoprotein at Ser239. HMR1766 intravenous bolus injection decreased arterial blood pressure in anesthetized pigs. All of these pharmacological responses to the new compounds were enhanced by ODQ and NS2028. Our findings suggest that HMR1766 and S3448 preferentially activate the NO-insensitive heme-oxidized form of sGC, which exists to a variable extent in vascular tissues, and is a pharmacological target for these new vasodilator drugs.The heterodimeric heme-protein soluble guanylyl cyclases (E.C. 4.6.1.2. pyrophosphate lyase, cyclizing; sGC) function as a receptor for the ubiquitous signaling molecule NO. Binding of NO to the ferrous heme activates the enzyme for rapid catalysis of cGMP formation from GTP (Koesling and Friebe, 1999). The second-messenger cGMP triggers several biological processes, such as a decrease in vascular tone and platelet activity, through an interaction with cGMP-specific protein kinases, phosphodiesterases, and ion channels (Munzel et al., 2003). A unifying concept of the molecular requisites for sGC activation has been put forward (Ignarro et al., 1984; Ballou A.M. was supported by grants from Hoechst-Marion-Roussel/Aventis and the German Research Council (SFB 553, project C10).Part of this work was published previously as a thesis (by A.T.) titled Kontrolle der glattmuskulä ren GC-Aktivitä t via NO-Sensitivitä t, Hä m-RedoxStatus und Proteinexpression, 2001 (ISBN 3-930...

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“…AZT-resistant RT has been reported to exhibit the same (34), a higher (11,22), or lower (21) pyrophosphorolysis activity than wild-type RT. During DNA synthesis, the incorporation of regular dNTPs is favored about 100-fold over pyrophosphorolysis (28), and the presence of potent pyrophosphatases maintaining an intracellular concentration of PP i around 150 M impedes pyrophosphorolysis (35). However, when DNA polymerization is stopped by the incorporation of a chain terminator, PP i present in the medium is able to restore a functional 3Ј-OH and rescue the blocked DNA.…”

Section: Atp-and Pp I -Mediated Repair Of the D4tmp-terminated Primermentioning

confidence: 99%

The Valine-to-Threonine 75 Substitution in Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Its Relation with Stavudine Resistance

Selmi

Boretto

Navarro

et al. 2001

Journal of Biological Chemistry

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The amino acid change V75T in human immunodeficiency virus type 1 reverse transcriptase confers a low level of 2,3-didehydro-2,3-dideoxythymidine (stavudine, d4T) resistance in vivo and in vitro. Valine 75 is located at the basis of the fingers subdomain of reverse transcriptase between the template contact point and the nucleotide-binding pocket. V75T reverse transcriptase discriminates 3.6-fold d4T 5-triphosphate relative to dTTP, as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. In addition, V75T increases the DNA polymerization rate up to 5-fold by facilitating translocation along nucleic acid single-stranded templates. V75T also increases the reverse transcriptase-mediated repair of the d4TMP-terminated DNA by pyrophosphate but not by ATP. The V75T/Y146F double substitution partially suppressed both increases in rate of polymerization and pyrophosphorolysis, indicating that the hydroxyl group of Thr-75 interacts with that of Tyr-146. V75T recombinant virus was 3-4-fold d4T-resistant and 3-fold resistant to phosphonoformic acid relative to wild type, confirming that the pyrophosphate traffic is affected in V75T reverse transcriptase. Thus, in addition to nucleotide selectivity V75T defines a type of amino acid change conferring resistance to nucleoside analogues that links translocation rate to the traffic of pyrophosphate at the reverse transcriptase active site. Antiretroviral therapy directed against the human immunodeficiency virus (HIV)1 is currently increasing the life expectancy of infected individuals. To replicate in infected cells, HIV relies on reverse transcriptase (RT), an essential RNA-and DNA-dependent DNA polymerase encoded by the viral pol gene (1). RT is a major target for drugs acting as inhibitors of retroviral replication. Because dNTPs are natural substrates for RT, more than 20 nucleoside analogues are currently used in vitro, in clinical trials, and in the clinic to inhibit retroviral replication (2, 3). To act as specific RT inhibitors, nucleoside analogues must be converted into 5Ј-triphosphate nucleosides upon phosphorylation by intracellular nucleoside/nucleotide kinases. Most nucleotide analogues do not possess a 3Ј-OH group required for phosphodiester bond formation during DNA synthesis. Therefore, they act as DNA chain terminators once incorporated into the nascent viral DNA.The efficacy of antiretroviral chemotherapy is limited by at least three factors. First, nucleoside analogues have to cross the cellular membrane to be activated to the 5Ј-triphosphate form by cellular kinases. This series of phosphorylation reactions is generally not efficient enough to produce adequate levels of the active nucleoside 5Ј-triphosphate able to compete efficiently with natural dNTPs. Second, RT incorporates nucleotide analogues with, at best, equal efficiency to that of their natural counterparts, i.e. dTTP for both AZTTP and d4TTP. The consequence of these two points is that retroviral replication is not completely suppressed. Third, mutant viruses esca...

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Luminescent immobilized enzyme test systems for inorganic pyrophosphate: Assays using firefly luciferase and nicotinamide-mononucleotide adenylyl transferase or adenosine-5′-triphosphate sulfurylase

Cited by 45 publications

References 17 publications

Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations

Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations

Biochemistry and Pharmacology of Novel Anthranilic Acid Derivatives Activating Heme-Oxidized Soluble Guanylyl Cyclase

The Valine-to-Threonine 75 Substitution in Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Its Relation with Stavudine Resistance

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