[30] Assay of guanylyl cyclase catalytic activity

Domino, Steven E.; Tubb, D. Janette; Garbers, D L

doi:10.1016/0076-6879(91)95179-n

Cited by 65 publications

(55 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Forty-eight hours after transfection, guanylyl cyclase activity (40.4 ± 6.8 pmol of cGMP per min per mg of protein), assayed in the presence of Triton X-100 and Mn2+ to elicit ligand-independent enzyme activity, was detectable in membrane but not cytosolic fractions (22). COS We next performed whole-mount in situ hybridization to examine the pattern of expression of GC-D in the olfactory epithelium (23).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A receptor guanylyl cyclase expressed specifically in olfactory sensory neurons.

Fülle

Vassar

Foster

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

252

188

View full text Add to dashboard Cite

show abstract

Section: Resultsmentioning

confidence: 99%

“…Transient transfections of COS cells with GC-D were performed by the DEAE-dextran method (21), and guanylyl cyclase activity (given as mean ± SEM of three independent experiments) was measured as described (22).…”

Section: Methodsmentioning

confidence: 99%

A receptor guanylyl cyclase expressed specifically in olfactory sensory neurons.

Fülle

Vassar

Foster

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

252

188

View full text Add to dashboard Cite

show abstract

“…cGMP production was also measured by radioimmunoassay (RIA) as previously described (12,17). All samples were assayed in duplicate, and each experiment was repeated at least three times.…”

Section: Sgc Expression and Purificationmentioning

confidence: 99%

Characterization of a Novel Type of Endogenous Activator of Soluble Guanylyl Cyclase

Balashova

Chang

Lamothe

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

Nitric oxide (NO) remains the only firmly established endogenous modulator of soluble guanylyl cyclase (sGC) activity, but physiological, structural, and biochemical evidence now suggests that in vivo regulation of sGC involves direct interaction with other factors. We searched for such endogenous modulators in human umbilical vein endothelial cells and COS-7 cells. The cytosolic fraction of both cell types stimulated the activity of semipurified sGC severalfold in the absence or presence of a saturating concentration of NO. The cytosolic factor was sensitive to proteinase K and destroyed by boiling, suggesting that it contains a protein component. Size exclusion chromatography revealed peaks of activity between 40 and 70 kDa. The sGC-activating effect was further purified by ion exchange chromatography. In the presence of the benzylindazole YC-1 or NO, the partially purified factor synergistically activated sGC, suggesting that this factor had a mode of activation different from that of YC-1 or NO. Four candidate activators were identified from the final purification step by matrix-assisted laser desorption ionization mass spectrometry analysis. Using an sGC affinity matrix, one of them, the molecular chaperone Hsp70, was shown to directly interact with sGC. This interaction was further confirmed by co-immunoprecipitation in lung tissues and by co-localization in smooth muscle cells. sGC and Hsp70 co-localized at the plasma membrane, supporting the idea that sGC can be translocated to the membrane. Hsp70 co-purifies with the sGC-activating effect, and immunodepletion of Hsp70 from COS-7 cytosol coincided with a marked attenuation of the sGC-activating effect, yet the effect was not rescued by the addition of pure Hsp70. Thus, Hsp70 is a novel sGC-interacting protein that is responsible for the sGC-activating effect, probably in association with other factors or after covalent modification.

show abstract

“…The reactions were terminated at the appropriate time points by transferring 100-l aliquots to 0.5 ml of 110 mM zinc acetate on ice. cGMP accumulation was quantitated as described by Domino et al (1991).…”

Section: Guanylyl Cyclase Assaysmentioning

confidence: 99%

A Constitutively “Phosphorylated” Guanylyl Cyclase-linked Atrial Natriuretic Peptide Receptor Mutant Is Resistant to Desensitization

Potter

Hunter

1999

MBoC

View full text Add to dashboard Cite

Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5Ј-(␤,␥-imino)-triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process.

show abstract

[30] Assay of guanylyl cyclase catalytic activity

Cited by 65 publications

References 7 publications

A receptor guanylyl cyclase expressed specifically in olfactory sensory neurons.

A receptor guanylyl cyclase expressed specifically in olfactory sensory neurons.

Characterization of a Novel Type of Endogenous Activator of Soluble Guanylyl Cyclase

A Constitutively “Phosphorylated” Guanylyl Cyclase-linked Atrial Natriuretic Peptide Receptor Mutant Is Resistant to Desensitization

Contact Info

Product

Resources

About