Improved methods for determination of guanylyl cyclase activity and synthesis of [α-32P]GTP

Birnbaumer, Lutz; Torres, Héctor N.; Flawiá, Mirtha M.; Fricke, Robert F.

doi:10.1016/s0003-2697(79)80125-6

Cited by 37 publications

(7 citation statements)

References 11 publications

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“…The reaction was stopped by transferring a 100-l reaction mixture into the phosphate assay reagent (pH Ϸ 0; 1 ml final volume) containing molybdate and malachite green. The phosphate content in commercial GTP products had to be reduced from Ϸ5% to Ͻ0.5% by anion exchange chromatography to reduce background absorption (17).…”

Section: Methodsmentioning

confidence: 99%

Structural basis for the activity and allosteric control of diguanylate cyclase

Chan¹,

Paul²,

Samoray³

et al. 2005

Acta Cryst Sect A

148

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Recent discoveries suggest that a novel second messenger, bis-(335)-cyclic di-GMP (c-diGMP), is extensively used by bacteria to control multicellular behavior. Condensation of two GTP to the dinucleotide is catalyzed by the widely distributed diguanylate cyclase (DGC or GGDEF) domain that occurs in various combinations with sensory and͞or regulatory modules. The crystal structure of the unorthodox response regulator PleD from Caulobacter crescentus, which consists of two CheY-like receiver domains and a DGC domain, has been solved in complex with the product cdiGMP. PleD forms a dimer with the CheY-like domains (the stem) mediating weak monomer-monomer interactions. The fold of the DGC domain is similar to adenylate cyclase, but the nucleotidebinding mode is substantially different. The guanine base is Hbonded to Asn-335 and Asp-344, whereas the ribosyl and ␣-phosphate moieties extend over the ␤2-␤3-hairpin that carries the GGEEF signature motif. In the crystal, c-diGMP molecules are crosslinking active sites of adjacent dimers. It is inferred that, in solution, the two DGC domains of a dimer align in a two-fold symmetric way to catalyze c-diGMP synthesis. Two mutually intercalated c-diGMP molecules are found tightly bound at the stem-DGC interface. This allosteric site explains the observed noncompetitive product inhibition. We propose that product inhibition is due to domain immobilization and sets an upper limit for the concentration of this second messenger in the cell.allosteric regulation ͉ signal transduction ͉ x-ray crystallography

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Section: Methodsmentioning

confidence: 99%

Structural basis for the activity and allosteric control of diguanylate cyclase

Chan¹,

Paul²,

Samoray³

et al. 2005

Acta Cryst Sect A

148

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“…1, enzyme b) activity was assayed immediately with pools of 8-10 pineal glands by a modification ~f the method of Kimura ee al. (24) as used by Birnbaumer et al (25). The pineals were homogenized in 5 pL/gland of 125 mM Tris-HC1 buffer (pH 7.5) containing 10 mM MnC12 and 2.5 mM NaN3.…”

Section: Methodsmentioning

confidence: 99%

Enzymes of guanine nucleotide metabolism and the diurnal cycle in cGMP content of the chick pineal gland

Wainwright¹,

Wainwright²

1983

Can. J. Biochem. Cell Biol.

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Levels of cGMP phosphodiesterase, guanylate cyclase, and GTPase activities were determined in homogenates of chick pineal glands. Only small variations in vivo were observed with glands removed at different times of the day from birds under a standard cycle of illumination. Glands cultured under the cycle of illumination from late in the photoperiod showed a progressive loss of about half the phosphodiesterase activity in 24 h, and an increase of roughly 75% in GTPase activity within 12 h. No simple correlations were found between variations in levels of enzyme activity and the diurnal cycles in pineal content of cGMP and level of serotonin N-acetyltransferase (NAT) activity. However, onset of rapid increases in 3',5'-cyclic GMP (cGMP) content and NAT activity was correlated with a transient decrease of about 30% in the phosphodiesterase activity, both in vivo and in culture. Further, known inhibitors of phosphodiesterase activity previously shown to elicit increase of cGMP content and marked elevation of NAT activity in cultured glands only inhibited phosphodiesterase activity of homogenates by 25-30%. It was therefore concluded that the transient decrease in level of phosphodiesterase may facilitate onset of increase in pineal cGMP content. However, it seems improbable that changes in pineal content of enzymes of guanine nucleotide metabolism are essential to regulation of diurnal cycles in cGMP content or level of NAT activity.

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“…Both the ATP and the reagents used to regenerate it during the assays were purified from contaminating "GTP-like" materials as described elsewhere (21,22). …”

Section: Adenylyl Cyclase Assaysmentioning

confidence: 99%

Glucagon-stimulable adenylyl cyclase in rat liver. Effects of chronic uremia and intermittent glucagon administration.

Dighe¹,

Rojas²,

Birnbaumer³

et al. 1984

J. Clin. Invest.

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Astract. The effects of chronic uremia and glucagon administration on glucagon-stimulable adenylyl cyclase in rat liver were assessed by determinations of adenylyl cyclase activities, specific iodoglucagon binding, and the activity of the stimulatory regulatory component of adenylyl cyclase. Glucagon-stimulated adenylyl cyclase was reduced in uremia to 75-80% of control levels (P < 0.05), in the presence or absence of saturating levels of guanosine triphosphate (GTP) and 5'-guanylylimidodiphosphate [GMP-P(NH)P]. Although these changes were accompanied by a concomitant 20% reduction in sodium fluoride-stimulated activity, basal, GTP-, GMP-P(NH)P-, and manganese-dependent adenylyl cyclase activities were unchanged. Using [125I-Tyr'0]monoiodoglucagon as a receptor probe, the number of high affinity glucagon-binding sites was reduced 28% (P < 0.01) in uremic as compared with control liver membranes. However, the affinity ofthese binding sites was unaltered. The S49 cyC-reconstituting activity with respect to both GMP-P(NH)P-and isoproterenol plus GTP-stimulable adenylyl cyclase was unaltered in membranes from uremic as compared with control rats. Intermittent glucagon (80-100 ,g) injections administered at 8-h intervals to normal rats reproduced all ofthe above described effects ofchronic experimental uremia on the adenylyl cyclase system. It is concluded that changes in the hormone-stimulable adAddress all reprint requests to Dr. Garber.Receivedfor publication 22 December 1982 and in revisedform 29 December 1983. enylyl cyclase complex in uremia and with glucagon treatment result primarily from a decrease in the number of hormone-specific receptor sites in hepatic plasma membranes. Since the changes in liver adenylyl cyclase are qualitatively and quantitatively the same in glucagontreated and uremic rats, it is suggested that these may be the result of the hyperglucagonemia of uremia. Further, the data reveal an unexpected dissociation between guanine nucleotide and sodium fluoride stimulation of adenylyl cyclase. Possible causes for this dissociation based on the known subunit composition of cyclase coupling proteins are discussed. IntroductionAbnormal carbohydrate metabolism is a frequent concomitant of chronic renal disease (1-3). As the result, in part, of the diminished renal clearance of glucagon, hyperglucagonemia is frequently observed in renal insufficiency (4, 5). These increased immunoreactive glucagon levels result from increases in a number of molecular weight species, each of which may have glucagon-like immunoreactivity if not biological activity (6-8). Glucagon-stimulable adenylyl cyclase activity in liver of uremic rats has been found to be increased (9, 10). Other more indirect assessments have inferred a decreased activity of this enzyme in chronic uremia (1 1). In light of these discordant data and to determine the impact of the uremic state on the hormone-stimulable adenylyl cyclase complex, we have used a monoiodinated glucagon as a probe of glucagon receptor density and affinity in membrane p...

show abstract

Improved methods for determination of guanylyl cyclase activity and synthesis of [α-32P]GTP

Cited by 37 publications

References 11 publications

Structural basis for the activity and allosteric control of diguanylate cyclase

Structural basis for the activity and allosteric control of diguanylate cyclase

Enzymes of guanine nucleotide metabolism and the diurnal cycle in cGMP content of the chick pineal gland

Glucagon-stimulable adenylyl cyclase in rat liver. Effects of chronic uremia and intermittent glucagon administration.

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