A novel 53 kDa actin binding protein from porcine brain--further biochemical and immunological characterization

We have previously described some of the characteristics of an actin binding protein, 53 K protein, purified from porcine brains. The purification procedure was revised in order to investigate of this actin binding protein further. A Scatchard plot analysis showed that the association constant between actin and the 53 K protein has around the same value as those reported for the fascin-actin and for the filamin-actin interactions. The binding experiments also demonstrated the occurrence of competitive binding with other actin binding proteins such as filamin, alpha-actinin, caldesmon and tropomyosin for the actin filament. Antibody was produced against brain 53 K protein and further purified on an affinity column. Immunoblot analysis using the antibody showed that this protein is localized in both the soluble and membraneous fraction of the brain. Other tissues such as liver and lung also contain 53 K protein. The immunoblot analysis also revealed that the gelation product of rat brain extract described by Palmer et al. contains immunoreactive polypeptides having slightly lower molecular weights and more basic isoelectric points than porcine brain 53 K protein. Immunological localization of the 53 K protein within HeLa and BS-C-1 cells showed that this protein is distributed throughout the cell in small granules, and in some regions of the cell, these granules were aggregated into much larger granules.