Isolation of rat liver endocytic vesicles using the proton pump as a marker.
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.
Sabolić
I
I
Max-Planck-Institute für Biophysik, Frankfurt am Main, F.R.G.
Haase
W
W
Burckhardt
G
G
D000165:Acridine Orange; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D002712:Chlorides / Q000494:pharmacology; D003911:Dextrans / Q000493:pharmacokinetics; D004024:Dicyclohexylcarbodiimide; D005033:Ethylmaleimide / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins / Q000493:pharmacokinetics; D007454:Iodides / Q000494:pharmacology; D007978:Levamisole / Q000494:pharmacology; D008099:Liver / Q000648:ultrastructure*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008564:Membrane Potentials; D009840:Oligomycins / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D011522:Protons; D051381:Rats; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism
D002712:Chlorides; D003911:Dextrans; D005452:Fluoresceins; D007454:Iodides; D009840:Oligomycins; D011522:Protons; C015219:fluorescein isothiocyanate dextran; D007978:Levamisole; D008345:Manganese; D004024:Dicyclohexylcarbodiimide; D010042:Ouabain; D000165:Acridine Orange; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium; D005033:Ethylmaleimide