12] Purification and characterization of low-molecular-weight actin-depolymerizing proteins from brain and cultured cells

Abstract

Following the identification of a low-molecular-weight actin-depolymerizing factor (ADF) in embryonic chick and adult porcine brain) proteins with similar activity have been isolated from a number of sources and given a variety of names, such as depactin from starfish oocytes, destrin from porcine kidney, and actophorin from Acanthamoeba . This chapter reports the detailed methods used to purify and characterize this protein from embryonic chick brain. The concentration of purified chick brain ADF is determined from its absorbance at 280 nm using an extinction coefficient of E 0.1% = 0.645. However, studies on the regulation and function of these proteins in mammalian cells are of current interest, so modification of these methods have been included, which are useful in purifying and characterizing different isoforms of ADF from cultured mammalian cells. The purification of ADF from cultured baby hamster kidney cells and from cultured myocytes has been detailed. When working with mammalian cells, it is necessary to use a sensitive protein assay which was not subject to interference by other substances in the crude extracts.