Isolation from the microsomal fraction of rat liver of a subfraction highly enriched in uncoated endocytic vesicles with high H+‐ATPase activity and a 50 kDa phosphoprotein

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Abstract
A subcellular fraction, highly enriched in uncoated vesicles (UCV) with high H+‐ATPase (EC 3.6.1.34) activity, was isolated from the crude microsomal fraction of rat liver homogenates by discontinuous sucrose gradient centrifugation. The UCV fraction, recovered at the interface of sucrose density 1.08 and 1.10 , was shown morphologically to be a mixture of small, smooth‐surfaced univesicular and a few multivesicular structures. A permeable anion (e.g. chloride) was required for internal acidification, indicating an electro‐neutral proton pump. Specific inhibitors of anion transport (pyridoxal 5'‐phosphate and 4‐acetamide‐4'‐isothiocyanostilbene‐2, 2'‐disulfonic acid) totally inhibit proton translocation. The proton pump activity was insensitive to oligomycin, but was completely inhibited by about 5 μM of the tridentate bathophenanthroline chelate of Fe(II). The activity was also inhibited 100% by low concentrations of the protonophore carbonyl cyanide p‐trifluoromethoxyphenylhydrazone, the proton conduction inhibitor N,N'‐dicyclohexyl‐carbodiimide and the ionophore monensin. The UCV fraction contained 2 proteins of M r 50000 (major) and 54000 (minor) which were phosphorylated by an endogenous cyclic nucleotide‐ and Ca2+‐independent protein kinase.