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.


CONCLUSIONS
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.
Wang
L
L
Department of Anatomy, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Iida
H
H
Shibata
Y
Y
D000818:Animals; D001681:Biological Assay; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D009206:Myocardium / Q000737:chemistry* / Q000166:cytology; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012508:Sarcolemma / Q000737:chemistry / Q000648:ultrastructure
D002135:Calcium-Binding Proteins; D010743:Phospholipids