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. 1983 May;80(10):3054–3058. doi: 10.1073/pnas.80.10.3054

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

K B Delclos, E Yeh, P M Blumberg
PMCID: PMC393972  PMID: 6304691

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 (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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bayley H., Knowles J. R. Photoaffinity labeling. Methods Enzymol. 1977;46:69–114. doi: 10.1016/s0076-6879(77)46012-9. [DOI] [PubMed] [Google Scholar]
  2. Blumberg P. M., Delclos K. B., Dunphy W. G., Jaken S. Specific binding of phorbol ester tumor promoters to mouse tissues and cultured cells. Carcinog Compr Surv. 1982;7:519–535. [PubMed] [Google Scholar]
  3. Blumberg P. M., Delclos K. B., Jaken S. Tissue and species specificity for phorbol ester receptors. Basic Life Sci. 1983;24:201–229. doi: 10.1007/978-1-4684-4400-1_11. [DOI] [PubMed] [Google Scholar]
  4. Blumberg P. M. In vitro studies on the mode of action of the phorbol esters, potent tumor promoters, part 2. Crit Rev Toxicol. 1981 Jun;8(3):199–234. doi: 10.3109/10408448109109658. [DOI] [PubMed] [Google Scholar]
  5. Blumberg P. M. In vitro studies on the mode of action of the phorbol esters, potent tumor promoters: part 1. Crit Rev Toxicol. 1980 Dec;8(2):153–197. doi: 10.3109/10408448009037493. [DOI] [PubMed] [Google Scholar]
  6. Bresch H., Kreibich G., Kubinyi H., Schairer H. U., Thielmann H. W., Hecker E. Uber die Wirkstoffe des Crotonols. IX. Partialsynthese von Wirkstoffen des Crotonols. Z Naturforsch B. 1968 Apr;23(4):538–546. [PubMed] [Google Scholar]
  7. Brunner J., Richards F. M. Analysis of membranes photolabeled with lipid analogues. Reaction of phospholipids containing a disulfide group and a nitrene or carbene precursor with lipids and with gramicidin A. J Biol Chem. 1980 Apr 25;255(8):3319–3329. [PubMed] [Google Scholar]
  8. Delclos K. B., Blumberg P. M. Identification of ascorbic acid as the heat-stable factor from brain which inactivates the phorbol ester receptor. Cancer Res. 1982 Apr;42(4):1227–1232. [PubMed] [Google Scholar]
  9. Diamond L., O'Brien T. G., Baird W. M. Tumor promoters and the mechanism of tumor promotion. Adv Cancer Res. 1980;32:1–74. doi: 10.1016/s0065-230x(08)60360-7. [DOI] [PubMed] [Google Scholar]
  10. Dowhan W., Wickner W. T., Kennedy E. P. Purification and properties of phosphatidylserine decarboxylase from Escherichia coli. J Biol Chem. 1974 May 25;249(10):3079–3084. [PubMed] [Google Scholar]
  11. Driedger P. E., Blumberg P. M. Specific binding of phorbol ester tumor promoters. Proc Natl Acad Sci U S A. 1980 Jan;77(1):567–571. doi: 10.1073/pnas.77.1.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dunphy W. G., Delclos K. B., Blumberg P. M. Characterization of specific binding of [3H]phorbol 12,13-dibutyrate and [3H]phorbol 12-myristate 13-acetate to mouse brain. Cancer Res. 1980 Oct;40(10):3635–3641. [PubMed] [Google Scholar]
  13. Etemadi A. H. Membrane asymmetry. A survey and critical appraisal of the methodology. II. Methods for assessing the unequal distribution of lipids. Biochim Biophys Acta. 1980 Dec 31;604(3):423–475. doi: 10.1016/0005-2736(80)90579-9. [DOI] [PubMed] [Google Scholar]
  14. Fisher P. B., Flamm M., Schachter D., Weinstein I. B. Tumor promoters induce membrane changes detected by fluorescence polarization. Biochem Biophys Res Commun. 1979 Feb 28;86(4):1063–1068. doi: 10.1016/0006-291x(79)90225-0. [DOI] [PubMed] [Google Scholar]
  15. Fontaine R. N., Harris R. A., Schroeder F. Aminophospholipid asymmetry in murine synaptosomal plasma membrane. J Neurochem. 1980 Feb;34(2):269–277. doi: 10.1111/j.1471-4159.1980.tb06592.x. [DOI] [PubMed] [Google Scholar]
  16. Grove R. I., Schimmel S. D. Generation of 1,2-diacylglycerol in plasma membranes of phorbol ester-treated myoblasts. Biochem Biophys Res Commun. 1981 Sep 16;102(1):158–164. doi: 10.1016/0006-291x(81)91502-3. [DOI] [PubMed] [Google Scholar]
  17. Gupta C. M., Radhakrishnan R., Gerber G. E., Olsen W. L., Quay S. C., Khorana H. G. Intermolecular crosslinking of fatty acyl chains in phospholipids: use of photoactivable carbene precursors. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2595–2599. doi: 10.1073/pnas.76.6.2595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hebdon G. M., LeVine H., 3rd, Sahyoun N. E., Schmitges C. J., Cuatrecasas P. Specific phospholipids are required to reconstitute adenylate cyclase solubilized from rat brain. Proc Natl Acad Sci U S A. 1981 Jan;78(1):120–123. doi: 10.1073/pnas.78.1.120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hecker E., Schmidt R. Phorbolesters--the irritants and cocarcinogens of Croton Tiglium L. Fortschr Chem Org Naturst. 1974;31(0):377–467. doi: 10.1007/978-3-7091-7094-6_7. [DOI] [PubMed] [Google Scholar]
  20. Horowitz A. D., Greenebaum E., Weinstein I. B. Identification of receptors for phorbol ester tumor promoters in intact mammalian cells and of an inhibitor of receptor binding in biologic fluids. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2315–2319. doi: 10.1073/pnas.78.4.2315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Horrocks L. A. The alk-1-enyl group content of mammalian myelin phosphoglycerides by quantitative two-dimensional thin-layer chromatography. J Lipid Res. 1968 Jul;9(4):469–472. [PubMed] [Google Scholar]
  22. Jaken S., Shupnik M. A., Blumberg P. M., Tashjian A. H., Jr Relationship between mezerein-mediated biological responses and phorbol ester receptor occupancy. Cancer Res. 1983 Jan;43(1):11–14. [PubMed] [Google Scholar]
  23. Jaken S., Tashjian A. H., Jr, Blumberg P. M. Characterization of phorbol ester receptors and their down-modulation in GH4C1 rat pituitary cells. Cancer Res. 1981 Jun;41(6):2175–2181. [PubMed] [Google Scholar]
  24. Kaibuchi K., Takai Y., Nishizuka Y. Cooperative roles of various membrane phospholipids in the activation of calcium-activated, phospholipid-dependent protein kinase. J Biol Chem. 1981 Jul 25;256(14):7146–7149. [PubMed] [Google Scholar]
  25. Kinzel V., Kreibich G., Hecker E., Süss R. Stimulation of choline incorporation in cell cultures by phorbol derivatives and its correlation with their irritant and tumor-promoting activity. Cancer Res. 1979 Jul;39(7 Pt 1):2743–2750. [PubMed] [Google Scholar]
  26. Kreibich G., Hecker E. Active principles of croton oil. X. Preparation of tritium labeled croton oil factor A1 and other tritium labeled phorbol derivatives. Z Krebsforsch. 1970;74(4):448–456. [PubMed] [Google Scholar]
  27. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  28. Lew K. K., Chritton S., Blumberg P. M. Biological responsiveness to the phorbol esters and specific binding of [3H]phorbol 12,13-dibutyrate in the nematode Caenorhabditis elegans, a manipulable genetic system. Teratog Carcinog Mutagen. 1982;2(1):19–30. doi: 10.1002/1520-6866(1990)2:1<19::aid-tcm1770020104>3.0.co;2-3. [DOI] [PubMed] [Google Scholar]
  29. Low M. G. Phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. Methods Enzymol. 1981;71(Pt 100):741–746. doi: 10.1016/0076-6879(81)71087-5. [DOI] [PubMed] [Google Scholar]
  30. Moonen P., Haagsman H. P., Van Deenen L. L., Wirtz K. W. Determination of the hydrophobic binding site of phosphatidylcholine exchange protein with photosensitive phosphatidylcholine. Eur J Biochem. 1979 Sep;99(3):439–445. doi: 10.1111/j.1432-1033.1979.tb13274.x. [DOI] [PubMed] [Google Scholar]
  31. Mufson R. A., Okin E., Weinstein I. B. Phorbol esters stimulate the rapid release of choline from prelabelled cells. Carcinogenesis. 1981;2(11):1095–1102. doi: 10.1093/carcin/2.11.1095. [DOI] [PubMed] [Google Scholar]
  32. NORTON W. T. Reaction of mercuric chloride with plasmalogen. Nature. 1959 Oct 10;184(Suppl 15):1144–1145. doi: 10.1038/1841144a0. [DOI] [PubMed] [Google Scholar]
  33. Nagle D. S., Jaken S., Castagna M., Blumberg P. M. Variation with embryonic development and regional localization of specific [3H]phorbol 12,13-dibutyrate binding to brain. Cancer Res. 1981 Jan;41(1):89–93. [PubMed] [Google Scholar]
  34. Ohuchi K., Levine L. Stimulation of prostaglandin synthesis by tumor-promoting phorbol-12, 13-diesters in canine kidney (MDCK) cells. Cycloheximide inhibits the stimulated prostaglandin synthesis, deacylation of lipids, and morphological changes. J Biol Chem. 1978 Jul 10;253(13):4783–4790. [PubMed] [Google Scholar]
  35. Op den Kamp J. A. Lipid asymmetry in membranes. Annu Rev Biochem. 1979;48:47–71. doi: 10.1146/annurev.bi.48.070179.000403. [DOI] [PubMed] [Google Scholar]
  36. Owens K. A two-dimensional thin-layer chromatographic procedure for the estimation of plasmalogens. Biochem J. 1966 Aug;100(2):354–361. doi: 10.1042/bj1000354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Quay S. C., Radhakrishnan R., Khorana H. G. Incorporation of photosensitive fatty acids into phospholipids of Escherichia coli and irradiation-dependent cross-linking of phospholipids to membrane proteins. J Biol Chem. 1981 May 10;256(9):4444–4449. [PubMed] [Google Scholar]
  38. Rohrschneider L. R., O'Brien D. H., Boutwell R. K. The stimulation of phospholipid metabolism in mouse skin following phorbol ester treatment. Biochim Biophys Acta. 1972 Sep 7;280(1):57–70. doi: 10.1016/0005-2760(72)90212-3. [DOI] [PubMed] [Google Scholar]
  39. Rothman J. E., Lenard J. Membrane asymmetry. Science. 1977 Feb 25;195(4280):743–753. doi: 10.1126/science.402030. [DOI] [PubMed] [Google Scholar]
  40. SCHMIDT G., OTTENSTEIN B., SPENCER W. A., KECK K., BLIETZ R., PAPAS J., PORTER D., LEVIN M. L., THANNHAUSER S. J. The partition of tissue phospholipides by phosphorus analysis. AMA J Dis Child. 1959 May;97(5 Pt 2):691–708. doi: 10.1001/archpedi.1959.02070010693007. [DOI] [PubMed] [Google Scholar]
  41. Sandermann H., Jr Regulation of membrane enzymes by lipids. Biochim Biophys Acta. 1978 Sep 29;515(3):209–237. doi: 10.1016/0304-4157(78)90015-1. [DOI] [PubMed] [Google Scholar]
  42. Singh O. M., Graham A. B., Wood G. C. The phospholipid-dependence of UDP glucuronosyltransferase. Purification, delipidation and reconstitution of microsomal enzyme from guinea-pig liver. Eur J Biochem. 1981 May 15;116(2):311–316. doi: 10.1111/j.1432-1033.1981.tb05335.x. [DOI] [PubMed] [Google Scholar]
  43. Slaga T. J., Fischer S. M., Nelson K., Gleason G. L. Studies on the mechanism of skin tumor promotion: evidence for several stages in promotion. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3659–3663. doi: 10.1073/pnas.77.6.3659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stoffel W., Metz P. Covalent cross-linking of photosensitive phospholipids to human serum high density apolipoproteins (apoHDL). Hoppe Seylers Z Physiol Chem. 1979 Feb;360(2):197–206. doi: 10.1515/bchm2.1979.360.1.197. [DOI] [PubMed] [Google Scholar]
  45. Sun G. Y., Horrocks L. A. The acyl and alk-1-enyl groups of the major phosphoglycerides from ox brain myelin and mouse brain microsomal, mitochondrial and myelin fractions. Lipids. 1970 Dec;5(12):1006–1012. doi: 10.1007/BF02533205. [DOI] [PubMed] [Google Scholar]
  46. Surewicz W. K., Leyko W. Interaction of propranolol with model phospholipid membranes. Monolayer, spin label and fluorescent spectroscopy studies. Biochim Biophys Acta. 1981 May 6;643(2):387–397. doi: 10.1016/0005-2736(81)90083-3. [DOI] [PubMed] [Google Scholar]

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