Biochimica et Biophysica Acta 899 (1987) 143-150 1,t3 Elsevier BBA 73537 Low pH fusion of mouse liver nuclei with liposomes bearing covalently bound lysozyme Tudor Arvinte, Philippe Wahl and Claude Nicolau Centre de Biophysique Mol~culaire du C.N.R.S., IA, Avenue de la Recherche Scientifique, F-45071 Orleans Cedex 2 (France) (Received 11 September 1986) (Revised manuscript received 5 January 1987) Key words: Membrane fusion; Liposome; Lysozyme; Hepatocyte nuclei; Resonance energy transfer; Fluorescence photobleaching recovery; (Mouse) Lysozyme covalently bound to liposomes induces the fusion of iiposomes with isolated mouse liver nuclei. The fusion behavior is very similar to the case of erythrocyte ghosts (Arvinte, T., Hildenhrand, K., Wahl, P. and Nicolau, C. (1986) Proc. Natl. Acad. Sci. USA 83, 962-966). Kinetic studies showed that membrane lipid mixing was completed within 15 min, as indicated from the resonance energy transfer (RET) measurements. For the resonance energy transfer kinetic measurements the iiposomes contained L-a-di- paimitoylphosphatidylethanolamine 0DPPE), labeled at the free amino group with the energy donor 7.nitrobenz-2-oxa-l,3-diazol-,l-yi (NBD) or with the energy acceptor tetramethylrhodamine. The lipid mixing at equilibrium was studied by the fluorescence recovery after photobleaching technique (FRAP). Liposomes (with/without lysozyme) containing Rh-labeled DPPE in their membranes were incubated with nuclei at 37°C, pH 5.2, for 30 min. After washing of nuclei by three centrifugations, 60-70% of the initial amount of labeled DPPE was associated with the nuclei in the ease of liposomes bearing lysozyme and only 7-10% in the case of iiposomes without lysozyme. For the nuclei incubated with liposomes having iysozyme, about 70% of the total Rh-labeled iipids present in the nuclei diffused in the nuclear membrane(s) (lateral diffusion constant of D = (1.4 + 0.5). 10 -9 cm2//s). By encapsulating fluorescein isothiocyanate-labeled dextran of 150 kDa molecular mass into the liposomes and using a microfiuorimetric method, it was shown that after the fusion a part of the liposome contents is found in the nuclei interior. In this lysozyme-induced fusion process between liposomes and nuclei or erythrocyte ghosts, the binding of lysozyme to the glycoconjugates contained in the biomemhranes at acidic pH seems to be the determining step which explains the high fnsogenic property of the iiposomes bearing iysozyme. Abbreviations: DPPE, L-a-dipalmitoylphosphatidylethanola- mine; FRAP, fluorescence recovery after photobleaching; FITC-D150, fluorescein isothiocyanate-labeled dextran of 150 kDa molecular mass; GlcNAc, N-acetyl-D-glucosamine; G- protein, membrane glycoprotein from vesicular stomatitis virus; NBD, 7-nitrobenz-2-oxa-l,3-diazol-4-yl; R.h-labeled, tetra- methylrhodamine labeled; PE, phosphatidylethanolamine; PC, phosphatidylcholine; Tris-HCl, tris(hydroxymethyl)amino- methane hydrochloride; TKM buffer, 50 mM Tris-HCl/25 mM KC1/3.3 mM CAC12/5 mM MgCI 2. Correspondence (present address): C. Nicolau, Department of Medical Biochemistry and Genetics, Texas A&M University, College of Medicine, College Station, TX 77843-1112, U.S.A. Introduction Membrane fusion is a fundamental cell-biologi- cal process, but its mechanism is not yet well understood. Many recent studies have focused on the fusogenic properties of biological molecules due to the widely accepted viewpoint that mem- brane functions are strongly dependent on their composition. It is possible to study the role of biological molecules in the fusion process by in- vestigating liposome-liposome or liposome-bio- 0005-2736/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)