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

Abstract

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 (=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 100IC 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.The phorbol esters are potent tumor promoters, which have also been shown to induce dramatic cellular and biochemical changes in vivo and in vitro (1-4). This laboratory has demonstrated specific, saturable binding of the phorbol esters to tissue particulate preparations and intact cells from a variety of organisms (5-10). The structure-activity requirements for binding are consistent with the receptors mediating the biological responses to the phorbol esters. The highest level of binding activity, 28 pmol/mg of protein, is found in brain (7). This level, which is considerably higher than that typical for hormone or neurotransmitter receptors, led us to suggest that the phorbol esters may be binding to a modulatory site on an enzyme or transport protein (7).We previously have demonstrated that phorbol ester binding in brain was sensitive to heat, protease, and phospholipase A2 treatments (7). In addition, we have shown that low concentrations of ascorbate inactivated binding with a time course and dose-response curve paralleling that for induction of lipid peroxidation (11). These results suggested either that the phorbol ester receptor is dependent on its specific lipid environment or else that lipid plays a role in the binding site itself.In an effort to obtain fiurther evidence on the nature of the phorbol ester binding site, we prepared a radioactive phorbol ester photoaffinity derivative, phorbol 12-p-azidobenzoate 13-benzoate (PaBzBz). This derivative contains an aryl azide, which is one of the widely used classes of photoaffinity labels (12, 13). We describe here specific, covalent bind...

Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols.

“…Further, proteolysis of protein kinase C yields a 51-kDa fragment whose kinase activity is independent of both Ca2+ and lipids (7,34). Finally, the model is consistent with experiments employing a photoactivable phorbol diester (35), in which the major labeled products were phospholipids. This is consistent with phorbol esters occupying a site analogous to that depicted for acyl2Gro in Fig.…”

Competitive inhibition by diacylglycerol of specific phorbol ester binding.

“…The tissue distribution (10,11), evolutionary conservation (10), and phospholipid requirements (12) for the phorbol ester receptor markedly resembled those of the Ca2+-phospholipid dependent. protein kinase (protein kinase C) described by Nishizuka and co-workers (13) and by Kuo and co-workers (14,15).…”

Characterization of a specific phorbol ester aporeceptor in mouse brain cytosol.

“…Both the dose-response and time course of this inhibition of binding correlated with stimulation of lipid peroxidation, and a number of inhibitors of lipid peroxidation protected against the ascorbic acid-mediated inactivation. Further evidence for interaction between the phorbol ester receptor and phospholipids was provided by studies with the phorbol ester photoaffinity probe [20-3H]phorbol 12-p-azidobenzoate 13-benzoate (PaBzBz) (19). UV irradiation caused PaBzBz to bind irreversibly to the membranes.…”