Actin monomeric

Actin (Mr = 41,800) is widely distributed in eukaryotic cells, often being the most abundant protein, and commonly making up about 10 percent of the total cell protein. The protein is highly conserved with only 17 of the 375 amino acids different between slime mold actin and rabbit muscle actin. Monomeric actin is usually referred to as globular or G-actin while the polymer, filamentous actin, is termed F-actin. 

Phosphate release from actin. (a) Monomeric actin with ADP and Pi bound. The protein backbone (tube), ADP (grey spheres), and Ca -Pi (black spheres) are shown. The orientation of the spring indicates the pulling direction during P, unbinding. (b) Force exerted on the deprotonated (solid line) and protonated (dashed line) phosphate during the SMD simulations. 

Monomeric actin binds ATP very tightly with an association constant Ka of 1 O M in low ionic strength buffers in the presence of Ca ions. A polymerization cycle involves addition of the ATP-monomer to the polymer end, hydrolysis of ATP on the incorporated subunit, liberation of Pi in solution, and dissociation of the ADP-monomer. Exchange of ATP for bound ADP occurs on the monomer only, and precedes its involvement in another polymerization cycle. Therefore, monomer-polymer exchange reactions are linked to the expenditure of energy exactly one mol of ATP per mol of actin is incorporated into actin filaments. As a result, up to 40% of the ATP consumed in motile cells is used to maintain the dynamic state of actin. Thus, it is important to understand how the free energy of nucleotide hydrolysis is utilized in cytoskeleton assembly. 

Actin is a 42 kDa bent dumbbell-shaped globular monomer which is found in most eukaryotic cells. It is the primary protein of the thin (or actin) filaments. Also, by mass or molarity, actin is the largest constituent of the contractile apparatus, actually reaching millimolar concentrations. Actins from different sources seem to be more similar than myosins from the same sources. Actin binds ATP which is hydrolyzed to ADP, if the monomeric actin polymerizes. The backbone structure of the actin filament is a helix formed by two linear strands of polymerized actins like two strings of actin beads entwined. 

Enzymatic activities. The hydrolysis of ATP by actin-activated myosin is the characteristic enzymatic activity of muscle, smooth muscle included. All forms of smooth muscle myosin are slower than those of other muscles. The binding site for ATP and a reduced enzymatic activity are still present in monomeric myosin. The enzymatic activity of monomeric myosin is altered by a conformational change, (the 10S-6S transition) and the species of cations present in the reaction mixture. These differences relate to the possible mechanisms of regulation. 

Figure 8. (Continued). As described above, the packing of myosin molecules into the thick filament is such that a layer of heads is seen every 14.3 nm, and this reflection is thought to derive from this packing. Off the meridian the 42.9 nm myosin based layer line is shown. This arises from the helical pitch of the thick filament, due to the way in which the myosin molecules pack into the filament. The helical pitch is 42.9 nm. c) Meridional reflections from actin. Actin based layer lines can be seen at 35.5 nm, 5.9 nm and 5.1 nm (1st, 6th, and 7th layer lines)and they all arise from the various helical repeats along the thin filament. Only the 35.5 nm layer line is shown here.The 5.9 nm and 5.1 nm layer lines arise from the monomeric repeat. The 35.5 nm layer line arises from the long pitch helical repeat and is roughly equivalent to seven actin monomers. A meridional spot at 2.8 nm can also be seen, d) The equatorial reflections, 1,0 and 1,1 which arise from the spacings between crystal planes seen in cross section of muscle.

Other small monomeric GTPases (eg, ARF, Rab, Ras, and Rho) are important in various cellular processes such as vesicle formation and transport (ARF and Rab see below), cettain growth and differentiation processes (Ras), and formation of the actin cytoskele-ton. A process involving GTP and GDP is also crucial in the ttanspott of ptoteins across the membrane of the ER (see below). 

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. 

Two of the cytoskeletal components, the actin filaments and the microtubules have been studied with molecular rotors. The main component of the actin filaments is the actin protein, a 44 kD molecule found in two forms within the cell the monomeric globulin form (G-actin) and the filament form (F-actin). Actin binds with ATP to form the microfilaments that are responsible for cell shape and motility. The rate of polymerization from the monomeric form plays a vital role in cell movement and signaling. Actin filaments form the cortical mesh that is the basis of the cytoskeleton. The cytoskeleton has an active relationship with the plasma membrane. Functional proteins found in both structures... 

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. 

Figure 1. Nucleation and growth of actin filaments. Nucleation is shown here as a thermodynamically unfavored process, which in the presence of sufficient actin-ATP will undergo initial elongation to form small filament structures that subsequently elongate with rate constants that do not depend on filament length. Elongation proceeds until the monomeric actin (or G-actin) concentration equals the critical concentration for actin assembly.

Note that if a capping protein binds to monomeric actin, the capping protein will also be a monomer-sequestering agent. A good example of such behavior is profilin. See also ABM-1 ABM-2 Sequences inActin-Based Motors Actin-Based Bacterial Motility Actin Assembly Kinetics... 

True self-assembly is observed in the formation of many oligomeric proteins. Indeed, Friedman and Beychok reviewed efforts to define the subunit assembly and reconstitution pathways in multisubunit proteins, and all of the several dozen examples cited in their review represent true self-assembly. Polymeric species are also formed by true self-assembly, and the G-actin to F-actin transition is an excellent example. By contrast, there are strong indications that ribosomal RNA species play a central role in specifying the pathway to and the structure of ribosome particles. And it is interesting to note that the assembly of the tobacco mosaic virus (TMV) appears to be a two-step hybrid mechanism the coat protein subunits first combine to form 34-subunit disks by true self-assembly from monomeric and trimeric com-... 

Figure 7-10 (A) Model of the F-actin helix composed of eight monomeric subunits. The model was constructed from the known structure of the actin monomer with bound ADP using X-ray data from oriented gels of fibrous actin to deduce the helical arrangement of subunits. The main interactions appear to be along the two-start helix. See also Holmes ef a/.62 (B) Ribbon drawing of an actin monomer with the four domains labeled. Courtesy of Ivan Rayment.

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