Cytidylate cyclase: development of assay and determination of kinetic properties of a cytidine 3′,5′-cyclic monophosphate-synthesizing enzyme

Newton, Russell P.; Salvage, Barbara J.; Hakeem, N. A.

doi:10.1042/bj2650581

Cited by 17 publications

(12 citation statements)

References 22 publications

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“…Moreover, in experiments with [␣-32 P]UTP as substrate, [ 32 P]cUMP was identified as a bona fide product of sGC (Gille et al, 2004), but direct structural confirmation of cUMP production by sGC with mass spectrometry techniques is still missing. The structural identity of the putative mammalian cytidylyl cyclase is elusive as well (Newton et al, 1990).…”

Section: Discussionmentioning

confidence: 99%

Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2′,3′-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5′-Triphosphates

Suryanarayana¹,

Göttle²,

Hübner³

et al. 2009

J Pharmacol Exp Ther

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Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In a recent study (Mol Pharmacol 70: 878 -886, 2006), we reported that 2Ј,3Ј-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5Ј-triphosphates (TNP-NTPs) are potent inhibitors (K i values in the 10 nM range) of the purified catalytic subunits VC1 and IIC2 of membranous AC (mAC). The crystal structure of VC1:IIC2 in complex with TNP-ATP revealed that the nucleotide binds to the catalytic site with the TNP-group projecting into a hydrophobic pocket. The aims of this study were to analyze the interaction of TNP-nucleotides with VC1:IIC2 by fluorescence spectroscopy and to analyze inhibition of mAC isoforms, soluble AC (sAC), soluble guanylyl cyclase (sGC), and G-proteins by TNP-nucleotides. Interaction of VC1:IIC2 with TNP-NDPs and TNP-NTPs resulted in large fluorescence increases that were differentially reduced by a water-soluble forskolin analog. TNP-ATP turned out to be the most potent inhibitor for ACV (K i , 3.7 nM) and sGC (K i , 7.3 nM). TNP-UTP was identified as the most potent inhibitor for ACI (K i , 7.1 nM) and ACII (K i , 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by G s -and G iproteins only with low potencies. Molecular modeling revealed that TNP-GTP and TNP-ATP interact very similarly, but not identically, with VC1:IIC2. Collectively, our data show that TNPnucleotides are useful fluorescent probes to monitor conformational changes in VC1:IIC2 and that TNP-NTPs are a promising starting point to develop isoform-selective AC and sGC inhibitors. TNP-ATP is the most potent sGC inhibitor known so far.ACs catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In mammals, nine membranous AC isoforms (ACI-IX) have been identified (Defer et al., 2000;Sunahara and Taussig, 2002). mACs differ from each other in regulation and tissue expression, and studies with transgenic and gene-knock-out mice have revealed different functions for the various AC isoforms. Most strikingly, ACV is an important AC isoform in the heart (Göttle et al., 2009), and the knock-out of ACV protects mice against heart failure and induces longevity (Yan et al., 2007). Thus, ACV inhibitors may be valuable drugs for the treatment of cardiovascular diseases and ageing (Iwatsubo et al., 2004;Göttle et al., 2009). In addition to mACs, mammals express a structurally distinct sAC that is predominantly found in testis and is important for sperm maturation (Chen et al., 2000). Thus, sAC inhibitors may be promising candidates for male contraceptives (Schlicker et al., 2008). Finally, sGC, catalyzing the conversion of GTP into O-(2,4,6-trinitrophenyl); VC1 and IIC2, the N-and C-terminal catalytic domains from canine type V mAC and rat type II mAC, respectively, expressed as soluble proteins; GTP␥S, guanosine 5Ј-[␥-thio]triphosphate.

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Section: Discussionmentioning

confidence: 99%

Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2′,3′-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5′-Triphosphates

Suryanarayana¹,

Göttle²,

Hübner³

et al. 2009

J Pharmacol Exp Ther

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“…These four previously unsuspected cytidine cyclic phosphates provide a feasible explanation of the controversy which existed relating to cytidylyl cyclase and cyclic CMP; their identification has enabled the development of a novel bilayer column capable of separating cyclic CMP from the other products of the cytidylyl cyclase reaction," and application of this column in enzyme assays has led to the demonstration that cytidylyl cyclase is a specific enzyme, distinct from adenylyl and guanylyl cycla~e. '~~ 25 Knowledge of the additional cytidine compounds has also permitted development of a trilayer-column system capable of separating cyclic CMP from each of the other endogenous cytidine compounds and from the other cyclic nucleotides; incorporation of this system into a radioimmunoassay p r o t~c o l '~~~~*~~ has produced a specific, sensitive assay method used to demonstrate the elevation of cyclic CMP concentrations in the urine of leukaemic patientsz6.". 17, and to monitor turnover of cyclic CMP in a brain tumour cell line.…”

Section: Identification Of Synthetic Derivativesmentioning

confidence: 99%

Contributions of fast‐atom bombardment mass spectrometry to studies of cyclic nucleotide biochemistry

Newton

1993

Rapid Comm Mass Spectrometry

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Cyclic nucleotides are biochemical second messengers which mediate the actions of many hormones and neurotransmitters. Fast-atom bombardment (FAB) mass spectrometry and mass-analysed ion kinetic energy (MIKE) spectra have provided a means of unambiguous identification of these compounds, and in addition to identifying endogenous putative cyclic nucleotides and their synthetic analogues FABIMIKE analysis has now been applied to good effect to both qualitative and quantitative studies of cyclic nucleotide-related enzymes. Here the application of this mass spectrometric technique to cyclic nucleotides is described and its impact upon this field reviewed.At the onset of the work reviewed here, two cyclic nucleotides, adenosine 3',5'-cyclic monophosphate and guanosine 3 ' 3 '-cyclic monophosphate, had been demonstrated to be key regulators of metabolism in mammalian cells. Initially our application of mass spectrometric techniques was confined to attempting to establish whether these two compounds were also present in higher plant tissues, and if a third cyclic nucleotide, cytidine 3',5'-cyclic monophosphate, was also endogenous to mammalian and plant tissues. The use of mass spectrometric analysis has proved not only productive and reproducible but also enjoyable, with the result that our utilization of the techniques in studying cyclic nucleotides has continued over the past decade, being developed and extended to include both qualitative and quantitative analysis of cyclic nucleotides, their derivatives, and the enzymes responsible for their metabolism.CYCLIC NUCLEOTLDES Structure, function and metabolism Cyclic nucleotides are of immense biochemical significance because of their role as secondary messengers, the intracellular mediators of the action of the extracellular primary messenger, a hormone or neurotransmitter.' The structure of a cyclic nucleotide comprises one of six heterocyclic bases, a ribose sugar and a cyclic phosphate moiety (Fig. 1) with the phosphate moiety cyclized at ribose positions 3' and 5' (1); a second series of isomers, the 2',3'-cyclic nucleotides (2), also exist as products of nucleic acid breakdown, but as such do not have any significant regulatory function analogous to that of the 3',5'-isomers.* Three sets of enzyme systems are involved in the metabolism and mechanism of action of the 3',5'-cyclic nucleotides (referred to hereafter simply as cyclic nucleotides) . Nucleotidylyl cyclases are responsible for the conversion of a nucleotide triphosphate to a cyclic nucleotide (Reaction (1)); the number of different intracellular enzymes and other proteins, thereby altering their activity by introducing a change in protein conformation. A third set of enzymes, the cyclic nucleotide phosphodiesterases, hydrolyse the cyclic nucleotide into the corresponding \

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“…7 In brief, the enzyme preparation was incubated at 37 °C for 35 min. in a total volume of 500 µL 50 mM Tris-HCl (pH 7.…”

Section: Radiometric Assaymentioning

confidence: 99%

“…1). 6 The identification of these side products by FAB/MIKES analysis provided an explanation of the earlier controversy, since these four compounds show cross-reactivity with antiserum raised against cyclic CMP and co-chromatographed with cyclic CMP in some systems previously used, and has consequently enabled the development of unambiguous methods for the radiometric estimation of cytidylyl cyclase 7 and for the radioimmunoassay of cyclic CMP. 8 The generation of these compounds as side products has not yet been explained.…”

mentioning

confidence: 98%

Estimation of Cytidylyl Cyclase Activity and Monitoring of Side-product Formation by Fast-atom Bombardment Mass Spectrometry

Newton

Groot

Geyschem

et al. 1997

Rapid Commun. Mass Spectrom.

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The enzyme cytidylyl cyclase catalyses the conversion of cytidine 5'-triphosphate into cytidine 3',5'-cyclic monophosphate, a third naturally occurring cyclic nucleotide currently under investigation to assign a biochemical function. Quantitation of the activity of this enzyme has been carried out by the positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The data obtained are in good agreement with those obtained from the conventional radiometric and radioimmunoassays of the same enzyme preparations. The advantage of the mass spectrometer-based assay is the facility for multiple component monitoring. Thus, the production of the cytidine diphosphates and monophosphates, and the production of four cytidine 3',5'-cyclic monophosphate analogues as side-products, were simultaneously estimated. The identities of two of the side-products, 2'-O-glutamyl- and 2'-O-aspartyl-cytidine-3',5'-cyclic monophosphate, and of the cytidine 3',5'-cyclic monophosphate product, were confirmed by mass-analysed ion kinetic energy spectra from the collision-induced dissociation of the protonated molecules.

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Cytidylate cyclase: development of assay and determination of kinetic properties of a cytidine 3′,5′-cyclic monophosphate-synthesizing enzyme

Cited by 17 publications

References 22 publications

Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2′,3′-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5′-Triphosphates

Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2′,3′-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5′-Triphosphates

Contributions of fast‐atom bombardment mass spectrometry to studies of cyclic nucleotide biochemistry

Estimation of Cytidylyl Cyclase Activity and Monitoring of Side-product Formation by Fast-atom Bombardment Mass Spectrometry

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