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Isolation of rat liver endocytic vesicles using the proton pump as a marker

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Sabolic,  Ivan
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;
Department of Physiology, Faculty of Medicine, University of Zagreb, Zagreb Yugoslavia;

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Haase,  Winfried
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Burckhardt,  Gerhard
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Sabolic, I., Haase, W., & Burckhardt, G. (1988). Isolation of rat liver endocytic vesicles using the proton pump as a marker. Biochimica et Biophysica Acta-Biomembranes, 944(2), 191-201. doi:10.1016/0005-2736(88)90432-4.


Cite as: http://hdl.handle.net/21.11116/0000-0007-DE1C-1
Abstract
ATP-driven acidification visualized by the delta pH indicator acridine orange was used as marker for isolation of endocytic vesicles from rat liver. By differential and Percoll density gradient centrifugation, a vesicle fraction was obtained with an approx. 80-fold enriched H+-pump activity. The preparation contained vesicles that had taken up fluorescein isothiocyanate-labeled dextran or horseradish peroxidase injected into rats in vivo, proving the presence of endosomes. The H+-pump in these vesicles showed: (a) strict preference for ATP; (b) stimulation by Mg2+ and Mn2+, but not by monovalent cations; (c) stimulation by Cl-, I- and Br-; (d) electrogenicity; (e) insensitivity to vanadate, slight inhibition by oligomycin and strong inhibition by N-ethylmaleimide (NEM) and N,N'-dicyclohexylcarbodimide (DCCD). The vesicles exhibited an ouabain-, oligomycin- and levamisole-resistant ATPase activity, which was slightly stimulated by Cl-, unaffected by vanadate and inhibited by NEM and DCCD. Thus, a simple and efficient high-speed centrifugation method is available for isolation of endocytic vesicles from mammalian liver.