Characterisation and localisation of a 67 kDa calcium binding protein (p67) isolated from bovine hearts

Objective: A 67 kDa Ca²⁺ binding protein (p67) from bovine hearts has recently been purified. It bound Ca²⁺ in vitro and localised at the sarcolemma of both striated and smooth muscle cells. In this study, further biochemical and morphological experiments were carried out to examine whether the p67 is a member of the annexin family of proteins and how the protein is associated with the sarcolemma membrane. Methods: Phospholipid and biological membrane binding assays were used to determine if p67 has the properties of the annexin family of proteins. Extraction experiments with Triton X-114 and Na₂CO₃ were carried out to examine potential association of p67 with sarcolemma membrane vesicles isolated from bovine hearts. For characterisation of the protein, actin binding assays and immunoelectron microscopy were performed. Results: The p67 bound to acidic phospholipids and biological membranes in a Ca²⁺ dependent manner. The protein was exclusively recovered in the aqueous phase after Triton X-114 phase separation and it was released from sarcolemma membranes by Na₂CO₃ treatment. It bound to membrane cytoskeletons insoluble in Triton X-100 but failed to bind to F actin at physiological free Ca²⁺ concentrations. Immunoelectron microscopy showed that p67 seemed to be localised at the cytoplasmic side of the cardiac sarcolemma. Conclusion: The p67 that was purified from bovine hearts could be a member of the annexin family of proteins. It might be a peripheral membrane protein associated with the cytoplasmic side of sarcolemma membranes.