[Show abstract][Hide abstract] ABSTRACT: Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-dependent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamburg and D. Bray. 1987. J. Cell Biol. 105: 2817-2825). To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF species with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombinant ADF. We have purified both ADF isoforms from myocytes and have shown by peptide mapping and partial sequence analysis that the new isoform is structurally related to ADF. Immunoprecipitation of both isoforms from extracts of cells prelabeled with [32P]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADF. pADF can be converted into a form which comigrates with ADF on 1-D and 2-D gels by treatment with alkaline phosphatase. pADF has been quantified in a number of cells and tissues where it is present from approximately 18% to 150% of the amount of unphosphorylated ADF. pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylation in vivo is catalyzed by a more specific kinase. We conclude that the ability to regulate ADF activity is important to muscle development since myocytes have both pre- and posttranslational mechanisms for regulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity.
Preview · Article · Sep 1993 · The Journal of Cell Biology
[Show abstract][Hide abstract] 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 E0.1%= 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.
No preview · Article · Feb 1991 · Methods in Enzymology