Actins

Actin molecules possess four possible binding sites two binding sites for adjacent molecules to form strands (intrastrand binding) and two sites for binding of actin molecules in neighbouring strands (inter strand binding). 

Actin is the main constituent of the thin filament. It makes up ca. 22% of the total protein of the contractile apparatus. It is substantially less soluble than myosin, probably because it is 

The monomer G-actin (globular actin) consists of 375 amino acids, has a molecular weight of 42,000 and is able to bind ATP and a doubly charged cation. G-actin exists only at low ionic strengths. The addition of singly and doubly charged cations starts the polymerization to F-actin (fibrillar actin) with the cleavage of ATP to ADP, which remains in the bound state. 

F-actin in the thin filaments (1 1000 nm, d 8 nm) is in the form of a double-stranded helix in which the G-actin beads are stabilized by two 

Connective tissue proteins Proteins from organelles 10.5  

Mitochondrial proteins (including cytochrome c and insoluble enzymes) 5.0 

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Figure Bl.17.6. A protein complex (myosin SI decorated filamentous actin) embedded in a vitrified ice layer. Shown is a defociis series at (a) 580 mn, (b) 1130 mn, (c) 1700 mn and (d) 2600 mn underfocus. The pictures result from averagmg about 100 individual images from one electron micrograph the decorated filament length shown is 76.8 nm.

Figure Bl.17.12. Time-resolved visualization of the dissociation of myosin SI from filamentous actin (see also figure Bl.17.6). Shown are selected filament images before and after the release of a nucleotide analogue (AMPPNP) by photolysis (a) before flashing, (b) 20 ms, (c) 30 ms, (d) 80 ms and (e) 2 s after flashing. Note the change in obvious order (as shown by the diffraction insert in (a)) and the total dissociation of the complex in (e). The scale bar represents 35.4 mn. Picture with the courtesy of Academic Press.

Payment i, Hoiden H M, Whittacker M, Yohn C B, Lorenz M, Hoimes K C and Miiiigan R A 1993 Structure of the actin-myosin compiex and its impiications for muscie contraction Science 261 58-65... 

Schroder R R, Jahn W, Manstein D, Hoimes K C and Spudich J A 1993 Three-dimensionai atomic modei of F-actin decorated with Dictyostelium myosin SI Nature 364 171-4... 

Flenderson E, Flaydon P G and Sakaguchi D S 1992 Actin filament dynamics in living glial cells imaged by atomic force microscopy Science 257 1944... 

Ishijima A, Kojima H, Funatsu T, Tokunaga M, Higuchi H, Tanaka H and Yanagida T 1998 Simultaneous observation of individual ATPase and mechanical events by a single myosin molcule during interaction with actin Ce//92 161-71... 

Actin filaments are dynamic polymers whose assembly and disassembly in the cell cytoplasm drives shape changes (Small, 1989), cell locomotion (Theriot et al, 1992), and chemotactic migration (Theriot et al., 1992)Devreotes and... 

Zigmond, 1988). The ATP-hydrolysis that accompanies actin polymerization, ATP —> ADP + Pj, and the subsequent release of the cleaved phosphate (Pj) are believed to act as a clock (Pollard et ah, 1992 Allen et ah, 1996), altering in a time-dependent manner the mechanical properties of the filament and its propensity to depolymerize. Molecular dynamics simulations suggested a so-called back door mechanism for the hydrolysis reaction ATP ADP - - Pj in which ATP enters the actin from one side, ADP leaves from the same side, but Pj leaves from the opposite side, the back door (Wriggers and Schulten, 1997b). This hypothesis can explain the effect of the toxin phalloidin which blocks the exit of the putative back door pathway and, thereby, delays Pi release as observed experimentally (Dancker and Hess, 1990). 

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. 

Dancker and Hess, 1990] Dancker, P., and Hess, L. Phalloidin reduces the release of inorganic phosphate during actin polymerization. Biochira. Biophys. Acta. 1035 (1990) 197-200... 

Pollard et al., 1992] Pollard, T. D., Goldberg, I., and Schwarz, W. H. Nucleotide exchange, structure, and mechanical properties of filaments assembled from ATP-actin and ADP-actin. J. Biol. Chem. 267 (1992) 20339-20345... 

Theriot et al., 1992] Theriot, J. A., Mitchison, T. J., Tilney, L. G., and Portnoi, D. A. The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerization. Nature. 357 (1992) 257-260... 

Wriggers and Schulten, 1997b] Wriggers, W., and Schulten, K. Stability and dynamics of G-actin Back door water diffusion and behavior of a subdomain 3/4 loop. Biophys. J. 73 (1997b) 624-639... 

Fig. 1. The GP Ib-IX-V complex. The complex consists of seven transmembrane polypeptides denoted GP Iba (mol wt 145,000), GP IbP (mol wt 24,000), GPIX (mol wt 17,000) and GP V (mol wt 82,000), in a stoichiometry of 2 2 2 1. The hatched region represents the plasma membrane. The area above the hatched region represents the extracellular space that below represents the cytoplasm. The complex is a major attachment site between the plasma membrane and the cytoskeleton. Two molecules associated with the cytoplasmic domain are depicted a 14-3-3 dimer, which may mediate intracellular signaling, and actin-binding protein, which connects the complex to the cortical cytoskeleton and fixes its position and influences its function.

In the case of some types of polyesters, the dried treated sheet is very tacky, and must be interleaved with a plastic release film to prevent blocking in the roU prior to use. Certain polyesters and acryhcs can be converted to a gelled state by exposure to actinic radiation. 

Microfilaments and Microtubules. There are two important classes of fibers found in the cytoplasm of many plant and animal ceUs that are characterized by nematic-like organization. These are the microfilaments and microtubules which play a central role in the determination of ceU shape, either as the dynamic element in the contractile mechanism or as the basic cytoskeleton. Microfilaments are proteinaceous bundles having diameters of 6—10 nm that are chemically similar to actin and myosin muscle ceUs. Microtubules also are formed from globular elements, but consist of hoUow tubes that are about 30 nm in diameter, uniform, and highly rigid. Both of these assemblages are found beneath the ceU membrane in a linear organization that is similar to the nematic Hquid crystal stmcture. 

Contraction of muscle follows an increase of Ca " in the muscle cell as a result of nerve stimulation. This initiates processes which cause the proteins myosin and actin to be drawn together making the cell shorter and thicker. The return of the Ca " to its storage site, the sarcoplasmic reticulum, by an active pump mechanism allows the contracted muscle to relax (27). Calcium ion, also a factor in the release of acetylcholine on stimulation of nerve cells, influences the permeabiUty of cell membranes activates enzymes, such as adenosine triphosphatase (ATPase), Hpase, and some proteolytic enzymes and facihtates intestinal absorption of vitamin B 2 [68-19-9] (28). 

Thickness of the laminar layer is deterrnined both by the need to reproduce fine detail in the object and by the penetration depth of the actinic laser light into the monomer bath (21,76). There is thus a trade-off between precision of detail in the model and time required for stereohthography, ie, the number of layers that have to be written, and an optimum Light-absorbing initiator concentration in the monomer bath corresponding to the chosen layer thickness. Titanocene-based initiators, eg, bis-perfluorophenyltitanocene has been recommended for this apphcation (77). Mechanistic aspects of the photochemistry of titanocenes and mechanisms of photoinitiation have been reviewed (76). 

PyrogaUol has been cited for use in photosensitive compositions. It is used in the form of sulfonate esters of quinonediazides which hydrolyze when exposed to actinic light to Hberate the acid which, in turn, catalyzes further reaction of novolak resins (60). 

Proteins can be broadly classified into fibrous and globular. Many fibrous proteins serve a stmctural role (11). CC-Keratin has been described. Fibroin, the primary protein in silk, has -sheets packed one on top of another. CoUagen, found in connective tissue, has a triple-hehcal stmcture. Other fibrous proteins have a motile function. Skeletal muscle fibers are made up of thick filaments consisting of the protein myosin, and thin filaments consisting of actin, troponin, and tropomyosin. Muscle contraction is achieved when these filaments sHde past each other. Microtubules and flagellin are proteins responsible for the motion of ciUa and bacterial dageUa. 

For resistance to actinic degradation, the use of certain forms of titanium oxide is an alternative to chrome salts. Another approach has been the use of polymerized methylol melamine on cotton (145). In this case, the action of sunlight leads to gradual breakdown of the melamine polymer after several years. After this, actinic degradation of cotton proceeds as it does in unprotected cotton. 

Complexation of the initiator and/or modification with cocatalysts or activators affords greater polymerization activity (11). Many of the patented processes for commercially available polymers such as poly(MVE) employ BE etherate (12), although vinyl ethers can be polymerized with a variety of acidic compounds, even those unable to initiate other cationic polymerizations of less reactive monomers such as isobutene. Examples are protonic acids (13), Ziegler-Natta catalysts (14), and actinic radiation (15,16). 

Cross-linked PVP can also be obtained by cross-linking the preformed polymer chemically (with persulfates, hydrazine, or peroxides) or with actinic radiation (63). This approach requires a source of free radicals capable of hydrogen abstraction from one or another of the labile hydrogens attached alpha to the pyrrohdone carbonyl or lactam nitrogen. The subsequently formed PVP radical can combine with another such radical to form a cross-link or undergo side reactions such as scission or cyclization (64,65), thus ... 

In the presence of calcium, the primary contractile protein, myosin, is phosphorylated by the myosin light-chain kinase initiating the subsequent actin-activation of the myosin adenosine triphosphate activity and resulting in muscle contraction. Removal of calcium inactivates the kinase and allows the myosin light chain to dephosphorylate myosin which results in muscle relaxation. Therefore the general biochemical mechanism for the muscle contractile process is dependent on the avaUabUity of a sufficient intraceUular calcium concentration. 

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