A Fluorometric Assay for Cyclic Guanosine 3′,5′-Monophosphate Incorporating a Sep-Pak Cartridge and Enzymatic Cycling

Seya, Kazuhiko; Furukawa, Ken‐Ichi; Motomura, Shigeru

doi:10.1006/abio.1999.4167

Cited by 7 publications

(8 citation statements)

References 13 publications

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“…Although it has been proposed that the cytosolic cardiac NO/cGMP pathway contributes to the induction of cardiac remodelling, the downstream mechanism of cGMP-dependent protein kinase activation by this pathway is unknown [10]. In the present study, we have demonstrated the existence of another cGMP production system in cardiac mitochondria using a fluorometric assay developed in our laboratory [18]. Furthermore, we have also confirmed that cardiac mitochondrial cGMP stimulates cytochrome c release from mitochondria independent of MPT.…”

Section: Discussionsupporting

confidence: 90%

“…The reaction was stopped by rapid addition of ice-cold 50 % trichloroacetic acid to the reaction tube. Following centrifugation, the supernatant was collected and the cGMP level produced was measured using an assay developed by our laboratory, as described previously [18]. The level of cGMP was expressed in terms of the amount of cGMP produced per mg of cellular protein per 10 min.…”

Section: Measurement Of Mitochondrial Cgmpmentioning

confidence: 99%

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Cardiac mitochondrial cGMP stimulates cytochrome c release

2006

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Although the existence of cardiac mitochondrial cGMP has been reported previously [Kimura and Murad (1974) J. Biol. Chem. 249, 6910-6916], the physiological and pathophysiological properties of cGMP in cardiac mitochondria have remained unknown. The aim of the present study was to clarify whether cardiac mitochondrial cGMP regulates the apoptosis of cardiomyocytes. In the presence of GTP, the NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine; 1 mmol/l) and SNP (sodium nitroprusside; 1 mmol/l) each markedly increased the cGMP level in a highly purified mitochondrial protein fraction prepared from left ventricular myocytes of male Wistar rats, and these increases were inhibited by 1 micromol/l ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), an inhibitor of NO-sensitive guanylate cyclase. In purified mitochondria, both SNAP (1 mmol/l) and the membrane-permeant cGMP analogue 8-Br-cGMP (8-bromo-cGMP; 1 mmol/l), but not cGMP (1 mmol/l), increased cytochrome c release from succinate-energized mitochondria without inducing mitochondrial swelling and depolarization of the mitochondrial membrane as factors of activation of MPT (mitochondrial permeability transition). The cytochrome c release mediated by SNAP was inhibited in the presence of 1 micromol/l ODQ. On the other hand, 1 mmol/l SNAP induced apoptosis in primary cultured adult rat cardiomyocytes in a time-dependent manner, and this induction was significantly inhibited in the presence of ODQ. Furthermore, apoptosis induced in primary cultured cardiomyocytes by hypoxia/re-oxygenation was also inhibited by ODQ. These results suggest that the acceleration of cGMP production in cardiac mitochondria stimulates cytochrome c release from mitochondria in an MPT-independent manner, resulting in apoptosis.

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Section: Discussionsupporting

confidence: 90%

Section: Measurement Of Mitochondrial Cgmpmentioning

confidence: 99%

Cardiac mitochondrial cGMP stimulates cytochrome c release

2006

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“…After a time, an original ATP molecule produces a large number of glucose-6-P molecules, which are then determined by their enzymatic reaction with glucose-6-P dehydrogenase and NAD. This procedure has been applied recently to some phosphorylate compounds, such as adenosine-3' À 5'-monophosphate (26)(27) and guanosine-3'-5'-monophosphate (28).…”

Section: Classical Enzymatic±fluorometric Methodsmentioning

confidence: 99%

Intrinsic fluorescence of enzymes and fluorescence of chemically modified enzymes for analytical purposes: a review

et al. 2001

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In recent years our research group has developed new alternatives for fluorescence enzymatic determinations. First, we observed that the intrinsic fluorescence of enzymes changes during enzymatic reactions, proportionally to the substrate concentration, avoiding the combination of the enzymatic reaction with a fluorophore-involving reaction. The main disadvantage of this method is that the excitation and emission wavelengths of the enzymes are in the UV region of the spectrum. An alternative to overcome this problem consisted of covalently bonding the enzyme to a fluorophore. In this paper, an overview is given of all of the applications and future developments on both types of alternatives that we have developed. Apart from the analytical characteristics of the methods, we have also reviewed all of the information about mathematical models we have elaborated to date.

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“…When pyruvic acid produced by the GTP-GDP cycle was reduced by lactate dehydrogenase, a reduced form of nicotinamide adenine dinucleotide (NADH) was equivalently oxidized to NAD+. After acidic degradation of NADH excess NAD+ was converted into 2-hydroxynicotinealdehydea fluorescent compound, which was excited at 370 nm and emitted fluorescence at 460 nm, by a strong alkali [104,105]. This case is not actually classical kind of molecular recognition but a long cascade of subsequent enzymatic reactions usable only in-vitro.…”

Section: Miscellaneous Methodsmentioning

confidence: 99%

“…A sensitive fluorometric assay for cyclic guanosine 3',5'-monophosphate (cGMP) based on multi-step conversion of cGMP to GTP was described [104]. Guanine nucleotides except cGMP were enzymatically phosphorylated to GTP.…”

Section: Miscellaneous Methodsmentioning

confidence: 99%

Molecular Recognition of Cyclic-Nucleotides and Current Sensors for Their Detection

Šebestı́k¹,

Hlaváček²,

Stibor

2005

CPPS

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This work briefly describes available sensors for cAMP and cGMP. Many sensors are based on derivatization of naturally occurring products such as immunoglobulins, protein kinases, etc. Only a few published works deal with chemosensors, which are built up by "total" chemical synthesis. This field stays opened for combinatorial chemist. The best sensors are protein kinases genetically modified with mutants of green fluorescent protein, which allow screening of entire cell cultures.

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A Fluorometric Assay for Cyclic Guanosine 3′,5′-Monophosphate Incorporating a Sep-Pak Cartridge and Enzymatic Cycling

Cited by 7 publications

References 13 publications

Cardiac mitochondrial cGMP stimulates cytochrome c release

Cardiac mitochondrial cGMP stimulates cytochrome c release

Intrinsic fluorescence of enzymes and fluorescence of chemically modified enzymes for analytical purposes: a review

Molecular Recognition of Cyclic-Nucleotides and Current Sensors for Their Detection

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