Photoreactivity of some light-sensitive estrogen derivatives. Use of an exchange assay to determine their photointeraction with the rat uterine estrogen binding protein

Katzenellenbogen, John A.; Johnson, Howard J.; Carlson, Kathryn E.; Myers, Harvey N.

doi:10.1021/bi00711a031

Cited by 86 publications

(43 citation statements)

References 27 publications

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“…5-N3IAA reduces the available site concentration without altering the affinity, suggesting that it binds in a specific manner to a high affinity class of auxinbinding sites. Specific and nonspecific labeling can be described as photolytic reactions inside and outside the active site, respectively ( 13). The second line of evidence indicating specific labeling is that NAA and IAA, both active auxins, are able to protect the site from labeling; by contrast, benzoic acid and tryptophan, compounds with low affinities for the NAA-binding site (19,22) This study is prerequisite to in vitro labeling of auxin-binding proteins.…”

Section: Discussionmentioning

confidence: 99%

Azido Auxins

Jones¹,

Melhado²,

Ho³

et al. 1984

Plant Physiol.

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The binding constants of three auxin analogs, 4-, 5-, and 6-azidoindole-3-acetic acid (4-, 5-, and 6-N3IAA), and of the photoproducts of 5-N3IAAto the naphthalene-l-acetic acid (NAA) binding sites of Zea mays L. WF9 x BR38 were determined to evaluate the potential of these analogs as photoaffinity labeling agents. We have found that 4-and 5-N3IAA bind to these sites with affinities similar to that of IAA, while 6-N3IAA and the photoproducts of 5-N3IAA bind less tightly. This 3School of Chemical Sciences. 4Abbreviations: NAA, naphthalene-l-acetic acid; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; NAA, naphthalene-1-acetic acid; N3IAA, azidoindole-3-acetic acid; 4-N3IAA, 4-azidoindole-3-acetic acid; 5-N3IAA, 5-azidoindole-3-acetic acid; 6-N3IAA, 6-azidoindole-3-acetic acid; RAC, ratio of association constants.that avoids the requirement that conditions for equilibrium binding be maintained while searching among the membrane proteins for specific auxin-binding sites. Once binding proteins are labeled, they can be isolated using standard techniques without the need to maintain equilibrium binding conditions. Therefore, the advantage of photoaffinity labeling is that binding proteins can still be isolated after they have lost the capacity for binding. Photoaffinity analogs bind reversibly to a site in the dark but can be caused to bind irreversibly by irradiation with light. Light induces the formation of reactive intermediates that attack nearby groups to form covalent bonds. The effectiveness of a photoaffinity agent, therefore, depends upon its affinity for the binding site in the dark and upon the half-lives of these photogenerated intermediates (4,24). The half-life of the ligandbinding site complex must be considerably longer than the halflives of the intermediates to achieve specific labeling.The only previous attempt to label the auxin site by affinity labeling was by Venis (26). He tested the usefulness of the diazonium salts of two auxin analogs, 2-chloro-4-aminophenoxyacetic acid and 2,5-dichloro-3-aminobenzoic acid, and reached tentative conclusions about the involvement of several amino acids in the auxin active site. The potential photoaffinity labeling agents 4-azido-2-chlorophenoxyacetic acid and 3-azido-5-chlorophenoxyacetic acid were found to have only moderate auxin activity (15), unlike the azidoindole-3-acetic acids here described. Spring and Hager (25) have recently demonstrated that sesquiterpene lactones specifically label important growth sites only when an auxin is also bound. The experiments provide an example of a nonequilibrium binding technique useful in auxin receptor research and are important because the results support a molecular model for auxin binding. The authors propose that auxin induces the receptor to undergo a conformational change, important for cell growth. Most significantly, the technique offers an independent assay for identifying auxin receptors and could be useful in conjunction with an independent approach such as we propose in this report.We ...

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Section: Discussionmentioning

confidence: 99%

Azido Auxins

Jones¹,

Melhado²,

Ho³

et al. 1984

Plant Physiol.

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“…The lamp is cooled by a circulating cold water jacket, and the light is filtered through a 1.5-cm-thick saturated copper sulfate solution. This filter absorbs all light of wavelength Ͻ315 nm (Katzenellenbogen et al, 1974). The samples were routinely irradiated for 1 min and continuously cooled to 4°C.…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Cholest-4-en-3-one, Oxime (TRO19622), a Novel Drug Candidate for Amyotrophic Lateral Sclerosis

Bordet

Buisson²,

Michaud³

et al. 2007

J Pharmacol Exp Ther

244

211

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive death of cortical and spinal motor neurons, for which there is no effective treatment. Using a cell-based assay for compounds capable of preventing motor neuron cell death in vitro, a collection of approximately 40,000 low-molecular-weight compounds was screened to identify potential small-molecule therapeutics. We report the identification of cholest-4-en-3-one, oxime (TRO19622) as a potential drug candidate for the treatment of ALS. In vitro, TRO19622 promoted motor neuron survival in the absence of trophic support in a dose-dependent manner. In vivo, TRO19622 rescued motor neurons from axotomy-induced cell death in neonatal rats and promoted nerve regeneration following sciatic nerve crush in mice. In SOD1G93A transgenic mice, a model of familial ALS, TRO19622 treatment improved motor performance, delayed the onset of the clinical disease, and extended survival. TRO19622 bound directly to two components of the mitochondrial permeability transition pore: the voltage-dependent anion channel and the translocator protein 18 kDa (or peripheral benzodiazepine receptor), suggesting a potential mechanism for its neuroprotective activity. TRO19622 may have therapeutic potential for ALS and other motor neuron and neurodegenerative diseases.Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder that selectively affects motor neurons in the spinal cord, brainstem, and cortex. ALS affects people of all races and ethnic backgrounds with an incidence approximately 2 per 100,000 individuals (McGuire and Nelson, 2006). The onset of ALS is most common in the 55 to 75 year age range, and incidence rises with advancing age; men have a higher risk of developing the disease than women (Nelson, 1995). Common clinical features of ALS include muscle weakness and fasciculations. These occur predominantly in limbs, although bulbar onset pathology can also lead to tongue atrophy and dysphagia. Failure of the respiratory muscles and cardiac complications are generally the fatal event, occurring within an average of 3 years of disease onset, with only a 5% chance of survival 5 years after diagnosis (del Aguila et al., 2003). Although 5 to 10% of ALS This work was supported by the Association Française contre les Myopathies.1 Current affiliation: Center for Motor Neuron Biology and Disease, Columbia University, New York.Article, publication date, and citation information can be found at

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“…[26][27][28][29][30][31][32][33][34][35][36]. Several authors have also indicated that photoaffinity labelling is not a stoichiometric reaction ( [13], and references therein), but this problem has been overcome by using larger amounts of protein to obtain information about the active site.…”

Section: Discussionmentioning

confidence: 99%

Study of the rat liver S-adenosylmethionine synthetase active site with 8-azido ATP

Deigner

Mato

Pajares

1995

Biochemical Journal

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The active site of rat liver S-adenosylmethionine synthetase was studied using 8-azido ATP, a photolabile analogue of ATP. Both forms of the enzyme, tetramer and dimer, could be labelled by using concentrations of the analogue similar to the KmATP values for each form, 350 microM and 1 mM respectively. Labelling of both S-adenosylmethionine synthetase forms with 8-azido [alpha-32P]ATP, followed by tryptic digestion and purification by HPLC, afforded one specifically labelled peptide in each case. Identification of the labelled peptide by amino acid analysis and peptide sequencing, and comparison with the enzyme sequence, indicated that the same peptide (267-286) was modified in both enzyme forms. The results are discussed on the basis of the high degree of similarity that this peptide shows in all the known S-adenosylmethionine synthetase sequences.

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Photoreactivity of some light-sensitive estrogen derivatives. Use of an exchange assay to determine their photointeraction with the rat uterine estrogen binding protein

Abstract: The photoreactivity of a number of photosensitive estrogen derivatives with the estrogen binding protein of rat uterus can be ascertained using a cytosol exchange assay. The

Cited by 86 publications

References 27 publications

Azido Auxins

Azido Auxins

Identification and Characterization of Cholest-4-en-3-one, Oxime (TRO19622), a Novel Drug Candidate for Amyotrophic Lateral Sclerosis

Study of the rat liver S-adenosylmethionine synthetase active site with 8-azido ATP

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