Use of Resonance Energy Transfer To Determine the Proximity of the Guanine Nucleotide Binding Site of Transducin Relative to a Conformationally-Sensitive Site on the .gamma. Subunit of the Cyclic GMP Phosphodiesterase

Biochemistry ◽  
1995 ◽  
Vol 34 (27) ◽  
pp. 8693-8700 ◽  
Author(s):  
Jon W. Erickson ◽  
Rohit Mittal ◽  
Richard A. Cerione
Keyword(s):  
Energy Transfer ◽  
Binding Site ◽  
Cyclic Gmp ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Gamma Subunit ◽  
Sensitive Site ◽  
Guanine Nucleotide Binding

scholarly journals A cGMP-Dependent Protein Kinase Assay for High Throughput Screening Based on Time-Resolved Fluorescence Resonance Energy Transfer

2001 ◽  
Vol 6 (4) ◽  
pp. 255-264 ◽  
Author(s):  
Benjamin Bader ◽  
Elke Butt ◽  
Alois Palmetshofer ◽  
Ulrich Walter ◽  
Thomas Jarchau ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
Cyclic Gmp ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Protein Kinase ◽  
Time Resolved ◽  
Dependent Protein

Activation of cyclic GMP-dependent protein kinase (cGK) is an important event in the regulation of blood pressure and platelet function. Upstream signals are the generation of nitric oxide (NO) by NO syntheses and the subsequent rise in cyclic GMP levels mediated by NO-dependent guanylyl cyclases (GCs). The identification of new cGK activators by high throughput sreening (HTS) may lead to the development of a novel class of therapeutics for the treatment of cardiovascular diseases. Therefore, a homogeneous, nonradioactive assay for cGK activity was developed using a biotinylated peptide derived from vasodilator-stimulated phosphoprotein (VASP), a well-characterized natural cGK substrate. The phosphorylated peptide could be detected by a VASP-specific monoclonal phosphoserine antibody and a fluorescent detection system consisting of a europium-labeled secondary antibody and allophycocyanin (APC)-labeled streptavidin. Fluorescence resonance energy transfer (FRET) from europium to APC was detected in a time-resolved fashion (TR-FRET). Activation and inhibition constants for known substances determined by this new fluorescence-based assay correlated well with published results obtained by conventional radioactive cGK activity assays. The assay proved to be sensitive, robust, highly specific for cGK, and suitable for HTS in 96- and 384-well formats. This assay is applicable to purified enzymes as well as to complex samples such as human platelet extracts.

Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer

Biochemistry ◽  
1979 ◽  
Vol 18 (21) ◽  
pp. 4505-4516 ◽  
Author(s):  
Anjana Rao ◽  
Paul Martin ◽  
Reinhart A. F. Reithmeier ◽  
Lewis C. Cantley
Keyword(s):  
Energy Transfer ◽  
Binding Site ◽  
Anion Exchange ◽  
Human Erythrocyte ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Exchange System

scholarly journals Influence of gamma subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes.

1992 ◽  
Vol 3 (1) ◽  
pp. 49-61 ◽  
Author(s):  
K H Muntz ◽  
P C Sternweis ◽  
A G Gilman ◽  
S M Mumby
Keyword(s):  
Membrane Fraction ◽  
Plasma Membranes ◽  
Nucleotide Binding ◽  
Site Directed Mutagenesis ◽  
Regulatory Proteins ◽  
Guanine Nucleotide ◽  
Gamma Subunit ◽  
Cos Cells ◽  
Gamma Subunits ◽  
Guanine Nucleotide Binding

Two approaches were taken to address the possible role of gamma-subunit prenylation in dictating the cellular distribution of guanine nucleotide-binding regulatory proteins. Prenylation of gamma subunits was prevented by site-directed mutagenesis or by inhibiting the synthesis of mevalonate, the precursor of cellular isoprenoids. When beta or gamma subunits were transiently expressed in COS-M6 simian kidney cells (COS) cells, the proteins were found in the membrane fraction by immunoblotting. Immunofluorescence experiments indicated that the proteins were distributed to intracellular structures in addition to plasma membranes. Replacement of Cys68 of gamma with Ser prevented prenylation of the mutant protein and association of the protein with the membrane fraction of COS cells. Immunoblotting results demonstrated that some of the beta subunits were found in the cytoplasm when coexpressed with the nonprenylated mutant gamma subunit. When Neuro 2A cells were treated with compactin to inhibit protein prenylation, a fraction of endogenous beta and gamma was distributed in the cytoplasm. It is concluded that prenylation facilitates association of gamma subunits with membranes, that the cellular location of gamma influences the distribution of beta, and that prenylation is not an absolute requirement for interaction of beta and gamma.

Spin-labeling proton NMR study on aromatic amino acid residues in the guanine nucleotide binding site of human c-Ha-ras(1-171) protein

Biochemistry ◽  
1989 ◽  
Vol 28 (24) ◽  
pp. 9550-9556 ◽  
Author(s):  
Akiko Hata-Tanaka ◽  
Gota Kawai ◽  
Kazuhiko Yamasaki ◽  
Yutaka Ito ◽  
Hiroko Kajiura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Aromatic Amino Acid ◽  
Proton Nmr ◽  
Nucleotide Binding ◽  
Spin Labeling ◽  
Guanine Nucleotide ◽  
Amino Acid Residues ◽  
Nmr Study ◽  
Guanine Nucleotide Binding

Aggregation and fusion of lipid vesicles induced by diphtheria toxin at low pH: Possible involvement of the P site and the NAD+ binding site

1985 ◽  
Vol 5 (3) ◽  
pp. 243-250 ◽  
Author(s):  
Veronique Cabiaux ◽  
Michel Vandenbranden ◽  
Paul Falmagne ◽  
Jean-Marie Ruysschaert
Keyword(s):  
Energy Transfer ◽  
Binding Site ◽  
Fluorescent Probes ◽  
Diphtheria Toxin ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Lipid Vesicles ◽  
Low Ph ◽  
Model Membranes

Model membranes have been used to study the interaction between diphtheria toxin and lipids. We report here on the ability of this toxin to induc% at low pH, fusion and aggregation of asolectin lipid vesicles. Resonance energy transfer experiments using lipid fluorescent probes make it possible to discriminate between these two processes.

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