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 ADE 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 ADE Immunoprecipitation of both isoforrns from extracts of cells prelabeled with [32p]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADE 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 ~,,18% to 150% of the amount of unphosphorylated ADE 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.

Abstract. To study the mechanisms underlying plasmalemmal expansion in the nerve growth cone, a cellfree assay was developed to quantify membrane addition, using ligand binding and sealed growth cone particles isolated by subcellular fractionation from fetal rat brain. Exposed versus total binding sites of t25I-wheat germ agglutinin were measured in the absence or presence of saponin, respectively, after incubation with various agents. Ca2+-ionophore A23187 in the presence of Ca 2+ increases the number of binding sites (Bm~,) but does not change their affinity (Kv), indicating that new receptors appear on the plasma membrane. Similarly, membrane depolarization by high K + or veratridine significantly induces, in a Ca2+-dependent manner, the externalization of lectin binding sites from an internal pool. Morphometric analysis of isolated growth cones indicates that A23187 and high K + treatment cause a significant reduction in a specific cytoplasmic membrane compartment, thus confirming the lectin labeling results and identifying the plasmalemmal precursor. The isolated growth cones take up 7-aminobutyric acid and serotonin, but show no evidence for Ca2+-dependent transmitter release so that transmitter exocytosis is dissociated from plasmalemmal expansion. The data demonstrate that plasmalemmal expansion in the growth cone is a regulated process and identify an internal pool of precursor membrane.

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