[5] F-actin affinity chromatography of detergent-solubilized plasma membranes: Purification and initial characterization of ponticulin from Dictyostelium discoideum

Publisher Summary This chapter describes the characteristics of ponticulin and describes the procedure of isolating it from detergent-solubilized Dictyostelium discoideum plasma membranes by elution with high salt from F-actin affinity columns followed by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Ponticulin purified by these methods has been used to obtain the amino acid composition and amino-terminal sequence data. The details for the preparation of 5 ml packed F-actin beads are presented. Ponticulin is a developmentally regulated protein that may be important for increased actin association with the plasma membrane during aggregation. The amount of ponticulin in the plasma membrane increases two- to threefold when D. discoideum amebas are forming aggregation streams, suggesting that this protein may play a role in the enhanced cell motility and/or cell-cell adhesion characteristic of this developmental stage. A relatively abundant protein in the plasma membrane, ponticulin constitutes about 1% of the total membrane protein, and appears to span the membrane. Ponticulin eluted from F-actin columns with high salt binds again to F-actin columns after the salt concentration is lowered to physiological levels by dialysis against 1% OG, CB. Furthermore, salt-eluted ponticulin subjected to SDS-polyacrylamide gel electrophoresis and electrotransfer to a nitrocellulose membrane directly and specifically binds 125 I-labeled F-actin.