Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester.
As part of our effort to characterize receptors for the phorbol ester tumor promoters, a phorbol ester photoaffinity probe, [20-3H]phorbol 12-p-azidobenzoate 13-benzoate (PaBzBz), was synthesized. In the dark, PaBzBz bound reversibly to brain particulate fractions with a dissociation constant (Kd) of 0.81 +/- 0.09 x 10(-9) M. Specific binding of PaBzBz, at a concentration equal to its Kd, represented 85% of the total bound. At saturation, 24 +/- 5 pmol of PaBzBz were bound per mg of brain protein, a level similar to that observed with [20-3H]phorbol 12,13-dibutyrate. Under the conditions used (concentrations greater than the Kd for PaBzBz), irradiation caused 45% of the PaBzBz binding to become irreversible. Most of the binding (approximately equal to 60%), including most of the specific irreversible binding, was to phospholipid rather than to protein. Based on susceptibility to enzymatic digestion and on chromatographic mobility, the specifically labeled phospholipids were identified as phosphatidylserine, phosphatidylethanolamine, and phosphatidylethanolamine plasmalogen. Although the PaBzBz specifically labeled lipid, labeling was blocked by pretreatment of membranes at 100 degrees C for 5 min or by papain digestion. Therefore, it seems likely that the identified lipids are specifically associated with a protein receptor and are preferentially labeled either because of the location or reactivity of the nitrene generated on the photoaffinity probe.
D000345:Affinity Labels; D000818:Animals; D001921:Brain / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism*; D051379:Mice; D010703:Phorbol Esters / Q000378:metabolism*; D010704:Phorbols / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010777:Photochemistry; D011493:Protein Kinase C; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011955:Receptors, Drug
D000345:Affinity Labels; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D008563:Membrane Lipids; D010703:Phorbol Esters; D010704:Phorbols; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D011956:Receptors, Cell Surface; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D011493:Protein Kinase C