Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester.

Publisher Website
Google Scholar
Abstract
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 Kd of 0.81 .+-. 0.09 .times. 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 PaBzBa), irradiation caused 45% of the PaBzBa binding to become irreversible. Most of the binding (.apprxeq. 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 PaBzBa specifically labeled lipid, labeling was blocked by pretreatment of membranes at 100.degree. C for 5 min or by papain digestion. The identified lipids evidently 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.

This publication has 47 references indexed in Scilit: