Digitonin
Dieses Steroidglykosid, ein natürlich vorkommendes Tensid, bietet ein breites Spektrum an Anwendungen.
Wie CHAPS und Octyl-ß-D-glucosid wurde auch das nicht-ionische Tensid Digitonin erfolgreich zur Solubilisierung von Rezeptoren eingesetzt.
Aufgrund der nicht-ionischen Natur seiner polaren Gruppe und seiner cholesterinähnlichen Struktur bleibt die native Konformation verschiedener Membranproteine während des Solubilisierungsprozesses intakt. So ist es gelungen, Opiat-, Adenosin-, Dopamin-, CCK-, CRF- und muscarinerge Rezeptoren auf äußerst schonende Weise zu isolieren.
Digitonin wird auch häufig für die Untersuchung anderer Membranproteine wie z.B. Photosystempartikel sowie für die Isolierung oder Fraktionierung von Mitochondrien eingesetzt.
Digitonin ist ein bekanntes Hämolysereagenz. Darüber hinaus wird es häufig zur Permeabilisierung bestimmter Zelltypen wie Blutplättchen, Hepatozyten, Spermatozoen und chromaffinen, Hefe- oder Tumorzellen eingesetzt.
Synonym: Digitin
CAS-Registriernummer: [11024-24-1]
Molekulare Formel: C56H92O29
Relative Molekülmasse (Mr): 1229.3
Klassifizierung: Nichtionisches Tensid
Literaturangabe:
- Kritische mizellare Konzentration (CMC): ca. 0,67 - 0,73 mM
- Aggregationszahl (Na): 60
- Hydrophil-Lipophil-Gleichgewicht (HLB): 0,4
BibliographIE
Solubilisierung und Charakterisierung von Rezeptoren
Grigoriadis, D.E., Zaczek, R., Pearsall, D.M. a. De Souza, E.B. (1989) Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: characterization of an active digitonin-solubized receptor complex. Endo¬crinology 125, 3068-77.
Hollis, C.M. a. Strange, P.G. (1989) Solubilization of the D1-dopamine receptor from the caudate nucleus of bovine brain. Biochem. Soc. Trans. 17, 774-5.
Leid, M., Schimerlik, M.I. a. Murray, T.F. (1989) Agonist radioligand interactions with the solu-bilized porcine atrial Ai-adenosine receptor. Mol. Pharmacol. 35, 450-7.
Poyner, D., Bansal, S., Bridsall, N.J.M., Curtis, C., Hulme, E.C. a. Pedder, E.K. (1989) Binding prop¬erties of reconstituted muscarinic receptors in the absence of G-proteins. Biochem. Soc. Trans. 17, 768-9.
Puget, A., Baillat, G., Mazarguil, H., Frances, B. a. Meunier, J. C. (1989) Progress in the purifi-cation of 'Op', the major type of opioid receptor in frog (Rana ridibunda) brain. Adv. Biosci. 75, 5-8.
Raidaru, G., Rinken, A., Jarv, J. a. Lohmus, M. (1989) Chromatographic analysis of Digitalis glycosides and their relation to solubilization of muscarinic receptor. Eesti NSV Tead. Akad. Toim., Keem. 38, 119-24.
Schonwetter, B.S., Luedtke, R.R., Kung, M.P., Billings, J., Kung,H.F. a. Molinoff,P.B. (1989) Characterization of membrane-bound and soluble D2 receptors in canine caudate using [125]IBZM. J. Pharmacol. Exp. Ther. 250, 110-6.
Szecowka, J., Hallden, G., Goldfine, I.D. a. Williams, J.A. (1989) Purification of the pancreatic cholecystokinin receptor. Regul. Pept 24, 215-24.
Zhang, X. a. Segawa, T. (1989) Investigation of rat striatal dopamine D-1 receptors solubilized by digitonin with a precipitation method. Eur. J. Pharmacol. 166, 401-10.
Arora, K.K. et al. (1992) Hexokinase receptors: Preferential enzyme binding in normal cells to nonmitochondrial sites and in transformed cells to mitochondrial sites. J. Bioenerg. Biomembr. 24, 47-53.
North, R.A. (2002) Molecular physiology of P2X receptors. Physiol. Rev. 82, 1013-67.
Paila, Y.D. et al. (2005) The cholesterol-complexing agent digitonin modulates ligand binding of the bovine hippocampal serotonin1A receptor. Mol. Membr. Biol. 22, 241-9.
Whitehurst, C.E. et al. (2006) Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry. J. Biomolec.. Screen. 11, 194-207.
Salom, D. et al. (2008) Heterologous expression and purification of the serotonin type 4 receptor from transgenic mouse retina. Biochemistry 47, 13296-307.
Solubilisierung und Charakterisierung von Enzymen
Eilenberg, H., Klinger, E., Przedecki, F. a. Shechter, I. (1989) Inactivation and activation of various membranal enzymes of the cholestreol biosynthetic pathway by digitonin. J. Lipid Res. 30, 1127-35.
Cesura, A.M., Imhof, R., Muggli-Maniglio, D., Picotti, G.B. a. Da Prada, M. (1990) Characteriza-tion of [3H]Ro 16-6491 binding to digitonin solubilized monoamine oxidase-B and purification of the enzyme from human platelets by affinity chromatography. Biochem. Pharmacol. 39, 216-20.
Lanubile, R. et al. (1997) Effect of Brefeldin A on the synthesis and transport of cell wall polysaccharides and proteins in pea root seedlings. J. Experiment. Bot. 48, 1925-33.
Ivanov, A. et al. (2000) Packing of the transmembrane helices of Na,K-ATPase: direct contact between ß-subunits and H8 segment of α-subunit revealed by oxidative cross-linking. Biochemistry 39, 9778-9785.
Solubilisierung und Charakterisierung von Photosystemen/Photosynthese-Pigmenten
Hermann, P., Hladik, J. a. Sofrova, D. (1988) Effect of detergents on thylakoid membranes of chloroplasts. Photosynthetica 22, 411-22.
Mansfield, R.W. a. Evans, M. C. W. (1988) EPR characteristics of the electron acceptors A0, A1, and (iron-sulfur)X in digitonin and Triton X-100 solubilized pea Photosystem I. Isr. J. Chem. 28, 97-102.
Apostolova, E. a. lvanov, A. (1989) Energy transfer in pea chloroplast membranes after treatment with non-ionic and ionic detergents. Photosynthetica 23, 372-5.
Nelson, N. a. Ben-Shem, A. (2002) Photosystem I reaction center: past and future. Photosynth. Res. 73, 193-206.
Satoh, K. (2003) The identification of the photosystem II reaction center: a personal story. Photosynth. Res. 76, 233-40.
Daum, B. et al. (2010) Arrangement of photosystem II and ATP synthase in chloroplast membranes of spinach and pea. Plant Cell 22, 1299-1312.
Järvi, S. et al. (2011) Optimized native gel systems for separation of thylakoid protein complexes: novel super- and mega-complexes. Biochem. J. 439, 207-14.
Solubilisierung und Charakterisierung von anderen Membranproteinen
Dhugga, K.S. and Ray, P.M. (1991) Isoelectric Focusing of plant plasma membrane proteins. Plant Physiol. 95, 1302-5.
Banerjee, P. et al. (1995) Differential solubilization of lipids along with membrane proteins by different classes of detergents. Chem. Phys. Lipids 77, 65-78.
Reisinger, V. a. Eichacker, L.A. (2006) Analysis of membrane protein complexes by Blue Native PAGE. Practical Proteomics 1-2/2006. Wiley-VCH Verlag, Weinheim.
Wittig, I. et al. (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol. Cell. Proteomics 6, 1215-25.
Permeabilisierung von Zellen
Kiaira, J.K., Njogu, R.M. (1988) Trypanosoma brucei brucei: the catabolism of glycolytic in-termediates by digitonin-permeabilized bloodstream trypomastigotes and some aspects of regulation of anaerobic glycolysis. Int. J. Biochem. 20, 1165-70.
Bittner, M.A., Habig, W.H. a. Holz, R.W. (1989) Isolated light chain of tetanus toxin inhibits exocy¬tosis: studies in digitonin-permeabilized cells. J. Neurochem. 53, 966-8.
Boschmann, M., Halangk, W. a. Bohnensack, R. (1989) Characterization of mitochondrial func-tions in digitonin-permeabilized rat liver cells. Biomed. Biochim. Acta 48, 645-52.
Haemmerle, T. a. Loeffler, M. (1989) Simultaneous analysis of mitochondrial activity and DNA content in Ehrlich ascites tumor cells by dual parameter flow cytometry. Histochemistry 93, 207-12.
Joshi, M.S., Gowda, L.R., Katwa, L.C. a. Bhat, S.G. (1989) Permeabilization of yeast cells (Kluy-veromyces fragilis) to lactose by digitonin. Enzyme Microb. Technol. 11, 439-43.
Lineberger, B., Dawicki, D.D., Agarwal, K.C., Kessimian, N., a. Steiner, M. (1989) Permeabiliza-tion of platelets: an investigation of biochemical, ultrastructural and functional aspects. Biochim. Biophys. Acta 1012, 36-45.
Noland, T.D. a. Olson, G.E. (1989) Calcium-induced modification of the acrosomal matrix in digitonin-permeabilized guinea pig spermatozoa. Biol. Reprod. 40, 1057-66.
Vercesi, A.E. et al. (1991) Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ. J. Biol. Chem. 266, 14431-4.
Hagstrom, J.E. et al. (1997) Nuclear import of DNA in digitonin-permeabilized cells. J. Cell Science 110, 2323-31.
Liu, J. et al. (1999) Use of digitonin-permeabilized cells in studies of steroid receptor subnuclear trafficking. Methods 19, 403-9.
Wilson, G.L. et al. (1999) Nuclear import of plasmid DNA in digitonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences. J. Biol. Chem. 274, 22025-32.
Yamada, H. et al. (2001) Ca2+-dependent exocytosis of L-glutamate by αTC6, clonal mouse pancreatic α-cells. Diabetes 50, 1012-20.
Miyamoto, K. et al. (2008) Reversible membrane permeabilization of mammalian cells treated with digitonin and its use for inducing nuclear reprogramming by Xenopus egg extracts. Cloning Stem Cells 10, 535-42.
Berlowska, J. et al. (2009) Pyruvate Decarboxylase activity assay in situ of different industrial yeast strains. Food Technol. Biotechnol. 47, 96-100.
Weitere Anwendungen
Bai, C., Slife, C.W., Aw, T.Y. a. Jones, D.P. (1989) Fractionation and analysis of mitochondria with polycarbonate membrane filters. Anal. Biochem. 179, 114-9.
Hovius, R., Lambrechts, H., Nicolay, K. a. De Kruijff, B. (1990) Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane. Biochim. Biophys. Acta 1021, 217-26.
Ottolenghi, C. et al. (1991) Separation of two populations of fish hepatocytes by digitonin infusion: some metabolic patterns and hormonal responsiveness. Can. J. Zoology 69, 427-35
Yamada, H. et al. (1994) Dystroglycan is a binding protein of laminin and merosin in peripheral nerve. FEBS Lett. 352, 49-53.