Characterization and immunolocalization of rat liver annexin VI

Abstract

Annexin VI has been purified to homogeneity from rat liver and monospecific antibodies have been produced. The antibodies have been used for immunoblot analysis of rat tissues. Annexin VI is present in most tissues, with particularly high concentrations in liver, spleen, muscle, and intestine. In liver, annexin VI constitutes approximately 0.25% of total cellular protein. Immunohistochemical studies have located annexin VI on plasma membranes of hepatocytes with enhanced concentration on bile canaliculi. Annexin VI binds in a Ca 2+-dependent manner to a sub-cellular fraction containing membranes. In the presence of physiological concentrations of ATP, the free Ca 2+ concentration required for half-maximal binding of annexin VI to membranes is significantly reduced. While annexin VI binds in vitro to membranes in the presence of Ca 2+, in rat liver about 31% of the annexin VI is associated with membranes in a Ca 2+-independent manner and its solubilization requires the presence of Triton X-100. However, studies using Triton X-114 showed no increase in the hydrophobicity of this fraction of the protein compared to the purified EGTA-soluble annexin VI.