Monomeric G-actin

Monomeric G-actin (43 kDa G, globular) makes up 25% of muscle protein by weight. At physiologic ionic strength and in the presence of Mg, G-actin polymerizes noncovalently to form an insoluble double helical filament called F-actin (Figure 49-3). The F-actin fiber is 6-7 nm thick and has a pitch or repeating structure every 35.5 nm. 

Several experimental approaches can be employed to determine the pool sizes of polymerised and non-polymerised actin. Firstly, the enzyme DNAse I is inhibited by monomeric (G) actin, but not by polymerised (F) actin. Secondly, polymerised actin can be directly visualised by use of fluorescent derivatives of phalloidin, a cyclic peptide isolated from the toadstool Amanita phalloides that selectively binds to polymerised actin with high affinity. 

Actin comprises about 5-8% of the total protein of the neutrophil, and in resting cells about 50-70% of the actin pool exists as a monomer. This proportion of monomeric G-actin is far in excess of what would be predicted from the critical concentration for actin assembly in vitro. Thus, in vivo actin polymerisation and depolymerisation is regulated by the activities of a number of binding proteins, cations and other regulatory molecules, which are in turn regulated by the activation status of the cell. 

The relative F-actin changes in neutrophils are quantified by exposing the cells to a chemoattractant stimulus, and the reactions are stopped by formalin fixation of the cells. The cells are permeabilized by the addition of lysophosphatidyl choline to the samples, and fluorescently conjugated phallotoxin is added, which specifically labels F-actin, but not G-actin. After staining, the cells are washed to remove unbound phallotoxin, resuspended in phosphate-buffered saline (PBS), and analyzed for fluorescence intensity with a flow cytometer. All samples are compared with control, unstimulated (resting) neutrophils. The mean channel fluorescence (MCF) intensity of stimulated samples is divided by the MCF of resting cells and expressed as a relative F-actin ratio. Since phallotoxins bind well to both large and small F-actin polymers, but do not bind to monomeric G-actin, the fluorescence intensity of each cell is directly proportional to its F-actin content. 

ADP-ribosylation of actin depends on the native structure of the protein substrate. In the presence of EDTA, which chelates and removes the actin-bound magnesium ion, resulting in denaturation of actin, ADP-ribosylation is completely blocked (Just ef al., 1990). C. botulinum C2 toxin or C. perfringens iota toxin ADP-ribosylate monomeric G-actin, but not polymerized F-actin (Aktories et al., 1986b Schering et al., 1988). This is due to the fact that the acceptor amino acid arginine-177, which is located in domain III of actin, is at or near... 

Although the toxin s effects on the microfilament system are easily visualized by fluorescence microscopy, it may be important to determine the changes in the ratio of cellular F- and G-actin quantitatively. Because G-actin dissolves in Triton X-100, whereas F-actin (or at least a major fraction of F-actin) does not, fractionation by detergent solubility can be used to study changes in the G- and F-actin content of cells. A different approach is the determination of G-actin content by the DNAse inhibition assay according to Blikstad (Blikstad et al., 1978). The action of DNAse in cleaving DNA is inhibited by monomeric G-actin but not by F-actin. Thus, the extent of inhibition of DNAse is proportional to the concentration of G-actin in cell lysates. 

A FIGURE 19-3 Structures of monomeric G-actin and F-actin filament, (a) Model of a p-actin monomer from a nonmuscle cell shows It to be a platellke molecule (measuring... 

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