[42] Peptide-based affinity labeling of adenosine cyclic monophosphate-dependent protein kinase

Wt, Miller

doi:10.1016/0076-6879(91)00166-t

Cited by 11 publications

(10 citation statements)

References 11 publications

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“…C-H bonds that are predicted to be particularly reactive include tertiary centers such as the C␥-H bond of leucine, the C␤-H bond of valine, and C-H bonds adjacent to heteroatoms. Residues that have been reported to be sites of covalent attachment for a chemically diverse group of benzophenone containing ligands include methionine (15)(16)(17), glycine (16,17), glutamine (18), asparagine (18), and serine (19).…”

Section: Discussionmentioning

confidence: 99%

Identification of Methionine as the Site of Covalent Attachment of a p-Benzoyl-Phenylalanine-containing Analogue of Substance P on the Substance P (NK-1) Receptor

Kage

Leeman

Krause

et al. 1996

Journal of Biological Chemistry

137

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Previously we have been able to restrict the site of covalent attachment of a photolabile and radiolabeled derivative of substance P (SP), p-benzoylphenylalanine8-SP (Bpa8-SP), to residues 178-183 located on the second extracellular loop (E2) of the SP (NK-1) receptor (Boyd, N. D., Kage, R., Dumas, J. J., Krause, J. E., and Leeman, S. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 433-437). To ascertain the specific amino acid in this sequence that serves as the site of covalent attachment for 125I-Bolton-Hunter reagent (BH)-Bpa8-SP, we have employed here a novel solid-phase approach to cyanogen bromide cleavage of the photolabeled receptor followed by mass spectrometric analysis of a purified labeled fragment. SP receptors on transfected Chinese hamster ovary cells were photolabeled with isotopically diluted 125I-BH-Bpa8-SP. A membrane preparation of the photolabeled receptors was adsorbed onto C-18-derivatized silica gel and cleaved with cyanogen bromide. A single radiolabeled fragment containing 63% of the photoincorporated radioactivity was generated and purified by high performance liquid chromatography. Mass spectrometric analysis identified a single molecular ion with a molecular mass of 1751.4 +/- 2, establishing that upon irradiation the bound photoligand forms a covalent link with the methyl group of a methionine residue at the peptide binding site. In view of our previous findings, this methionine is Met-181 on the primary sequence of the SP receptor.

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Section: Discussionmentioning

confidence: 99%

Identification of Methionine as the Site of Covalent Attachment of a p-Benzoyl-Phenylalanine-containing Analogue of Substance P on the Substance P (NK-1) Receptor

Kage

Leeman

Krause

et al. 1996

Journal of Biological Chemistry

137

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“…5) is noteworthy in this context, because this labeled protein has a similar molecular mass to that predicted for the 54-kD syntide-2 kinase coupled to syntide-2. Peptide-based photoaffinity probes have previously been used to successfully photoaffinity label protein kinases for which they are a specific substrate (Miller, 1991). These data led us to the hypothesis that the 54-kD kinase activity was responsible for the phosphorylation of syntide-2 in vitro and, hence, was a possible target for the effect of syntide-2 on the GA response in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…To generate photoaffinity forms of syntide-2 and malantide, the fluorescently labeled peptides were stirred for 4 h in the dark with benzophenone-isothiocyanate (Molecular Probes, Eugene, OR) at a 10:1 molar excess in PBS, pH 8.0 (Miller, 1991;Banks and Paquette, 1995). Labeled peptide was purified by preparative TLC as described above.…”

Section: Fluorescent and Photoaffinity Labeling Of Peptidesmentioning

confidence: 99%

Calcium-Dependent Protein Phosphorylation May Mediate the Gibberellic Acid Response in Barley Aleurone1

Ritchie

Gilroy

1998

Plant Physiology

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Peptide substrates of well-defined protein kinases were microinjected into aleurone protoplasts of barley (Hordeum vulgare L. cv Himalaya) to inhibit, and therefore identify, protein kinase-regulated events in the transduction of the gibberellin (GA) and abscisic acid signals. Syntide-2, a substrate designed for Ca2+- and calmodulin (CaM)-dependent kinases, selectively inhibited the GA response, leaving constitutive and abscisic acid-regulated events unaffected. Microinjection of syntide did not affect the GA-induced increase in cytosolic [Ca2+], suggesting that it inhibited GA action downstream of the Ca2+ signal. When photoaffinity-labeled syntide-2 was electroporated into protoplasts and cross-linked to interacting proteins in situ, it selectively labeled proteins of approximately 30 and 55 kD. A 54-kD, soluble syntide-2 phosphorylating protein kinase was detected in aleurone cells. This kinase was activated by Ca2+ and was CaM independent, but was inhibited by the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (250 mum), suggesting that it was a CaM-domain protein kinase-like activity. These results suggest that syntide-2 inhibits the GA response of the aleurone via an interaction with this kinase, implicating the 54-kD kinase as a Ca2+-dependent regulator of the GA response in these cells.

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“…To identify which peak from the separation of inhibitor 1 corresponded to which diastereomer, the HPLC peaks were analyzed by leucine aminopeptidase digestion (10). Leucine aminopeptidase was preactivated by incubation at 37°C for 1 hour in lOOmM Tris pH 8.5,20mM MnC12.…”

Section: Task 3 Determining the Sequences Of Neu Substratesmentioning

confidence: 99%

Peptide-Bassed Inhibitors of Neu Tyrosine Kinase

Miller¹

1999

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[42] Peptide-based affinity labeling of adenosine cyclic monophosphate-dependent protein kinase

Cited by 11 publications

References 11 publications

Identification of Methionine as the Site of Covalent Attachment of a p-Benzoyl-Phenylalanine-containing Analogue of Substance P on the Substance P (NK-1) Receptor

Identification of Methionine as the Site of Covalent Attachment of a p-Benzoyl-Phenylalanine-containing Analogue of Substance P on the Substance P (NK-1) Receptor

Calcium-Dependent Protein Phosphorylation May Mediate the Gibberellic Acid Response in Barley Aleurone1

Peptide-Bassed Inhibitors of Neu Tyrosine Kinase

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