Coupling an alpha 2-adrenergic receptor peptide to G-protein: a new photolabeling agent.
A photoreactive derivative of a tetradecapeptide G-protein activator (peptide Q) derived from the alpha 2-adrenergic receptor was designed and used to label purified G-protein (Go/Gi). N-bromoacetyl-N'-(3-diazopyruvoyl)-m-phenylene-diamine (Br-DAP) was conjugated to the C-terminal cysteine of peptide Q. The DAP-modified peptide Q (DAP-Q) specifically incorporated into the a subunit of Go. The incorporation of DAP-Q into alpha o was blocked by unmodified Q peptide (IC50 = 15 +/- 6 microM; n = 4). Photolysis of sixfold higher concentrations of DAP-Q with ovalbumin or bovine albumin failed to produce cross-linked products. Br-DAP should prove useful in detecting mutual contact sites between peptides and their binding proteins.
Taylor
J M
JM
Department of Pharmacology, University of Michigan, Ann Arbor 48109.
Jacob-Mosier
G G
GG
Lawton
R G
RG
Neubig
R R
RR
D000345:Affinity Labels; D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*; D010777:Photochemistry; D011773:Pyruvates / Q000737:chemistry*; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry*; D013439:Sulfhydryl Reagents / Q000737:chemistry*
D000345:Affinity Labels; D003432:Cross-Linking Reagents; C090696:N-bromoacetyl-N'-(3-diazopyruvoyl)-1,3-phenylenediamine; D010455:Peptides; D011773:Pyruvates; D018341:Receptors, Adrenergic, alpha-2; D013439:Sulfhydryl Reagents; C090695:peptide Q