Thin filament proteins smooth muscle

The location of CD in the smooth muscle cell has been determined by immunofluorescence microscopy. A number of studies have shown the presence of two populations of thin filaments in smooth muscles one population is mainly p-actin and associated with the cytoskeleton, filamin, and calponin, whereas the remainder (about 60% of the total in chicken gizzard) is mainly a-actin and is associated with contractile proteins, myosin, TM, and CD (Furst et al., 1986 North et al., 1994a,b Lehman etal, 1987). 

Caldesmon is a major actin binding protein associated with thin filament of smooth muscle (Sobue etal., 1981) and nonmuscle cells (Owada and Kakiuchi,... 

The second aspect of the force-RLC phosphorylation relation that suggests additional regulation is illustrated by alterations in the slope of the relation (Fig. 1, lower right). Alterations in isometric force at fixed values of RLC phosphorylation are probably the strongest evidence for the in vivo operation of thin filament regulation. Smooth muscle contains two thin filament proteins, caldesmon and calponin, that inhibit actin-activated MgATPase activity of phosphorylated myosin. This inhibitory activity is reversed by the binding of Ca +ZCaM or by phosphorylation, and thus CD and CP may modulate the RLC phosphorylation... 

The thin filaments of smooth muscle cells are extremely stable during most fixation protocols and are the most abundant fQament in Smooth muscle. Actin is the primary protein constituent and forms the thin filament backbone. In addition, at least three other proteins, tropomyosin, caldesmon, and calponin, bind to actin and are localized to the thin filaments (Figure 5). 

Nieznanski K, Sobieszek A (1997) Telokin (kinase-related protein) modulates the oligomeric state of smooth-muscle myosin light-chain kinase and its interaction with myosin filaments. Biochem J 322 65-71 Nishida W, Abe M, Takahashi K, Hiwada K (1990) Do thin filaments of smooth muscle contain calponin A new method for the preparation. FEBS Lett 268 165-168 Nishikawa M, de LaneroUe P, Lincoln TM, Adelstein RS (1984) Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase. J Biol Chem 259 8429-8436... 

Apart from the phosphorylation theory, other regulatory mechanisms have also been suggested for smooth muscle contraction. A thin-filament protein that has been proposed as a regulatory component is caldesmon [102], Purified caldesmon is a potent inhibitor of actin-tropomyosin interaction with myosin. The mechanisms by which calcium removes this inhibition are controversial. Furthermore, phosphorylation of caldesmon by a caldesmon kinase in vitro has also been implicated in this... 

The key step in LC isolation is the preparation of pure smooth muscle myosin (Hasegawa et al., 1988). This is usually executed by homogenizing the muscle mince with salt solutions (60-100 mM ionic strength) to remove the cytoplasmic proteins and to a certain extent the thin filament proteins. A crude actomyosin is then extracted form the well-washed muscle residue with salt solutions containing ATP and the actin component is removed by ultracentrifugation. The LCs are... 

The principal molecular constituent of thin filaments is actin. Actin has been highly conserved during the course of evolution and is present in all eukaryotes, including single-celled organisms such as yeasts. Actin was first extracted and purified from skeletal muscle, where it forms the thin filaments of sarcomeres. It also is the main contractile protein of smooth muscle. Refined techniques for the detection of small amounts of actin (e.g., immunofluorescence microscopy, gel electrophoresis, and EM cytochemistry) subsequently confirmed the presence of actin in a great variety of nonmuscle cells. Muscle and nonmuscle actins are encoded by different genes and are isoforms. 

Contractile proteins which form the myofibrils are of two types myosin ( thick filaments each approximately 12 nm in diameter and 1.5 (im long) and actin ( thin filaments 6nm diameter and 1 (Am in length). These two proteins are found not only in muscle cells but widely throughout tissues being part of the cytoskeleton of all cell types. Filamentous actin (F-actin) is a polymer composed of two entwined chains each composed of globular actin (G-actin) monomers. Skeletal muscle F-actin has associated with it two accessory proteins, tropomyosin and troponin complex which are not found in smooth muscle, and which act to regulate the contraction cycle (Figure 7.1). 

The major allergen of molluscan shellfish is tropomyosin, a muscle protein. The term major allergen is used to define proteins that elicit IgE binding in the sera of half or more of patienfs wifh allergies to the specific source (Metcalfe et ah, 1996). Tropomyosin is a ubiquitous muscle protein in all animals. Tropomyosin is a 34- to 36-kDa protein that is highly water soluble and heat stable as evidenced by the fact that tropomyosin can be isolated from fhe water used to boil shrimp (Daul et ah, 1994). Tropomyosin can actually be found in bofh muscle and many nonmuscle cells in animals. In muscle cells, tropomyosin is associated with the thin filaments in muscle and plays a role in the contractile activity of muscle cells. In nonmuscle cells, tropomyosin is found in microfilaments but its fimction is less well imderstood. Tropomyosins are present in all eukaryotic cells. Different isoforms of tropomyosin are found in different types of muscle cells (skeletal, cardiac, smooth), brain, fibroblasts, and other nonmuscle cells. While these tropomyosins are highly homologous, small differences do exist in their... 

Actin and the thin filaments. There are at least six forms of actin in adult mammalian tissues a-cardiac, a-skeletal muscle, a- and y-smooth muscle, P- and y-cytoplasmic.87 89 All of them are closely homologous, e.g., the 42-kDa a-skeletal muscle actin differs in only 4 of 375 residues from the a-cardiac form and only in 6 residues from the y-smooth form. In almost all organisms actins contain one residue of N5-methylhistidine at position 73.87/88/90 Actin is an unusual protein in that... 

A family of actin-binding proteins that exist in various isoforms. As with other protein isoforms or isoenzymes, the expression of the isoforms is tissue-specific. The interaction of calponin with actin inhibits the actomyosin Mg-ATRase activity. See Winder, S. and Walsh, M., Inhibition of the actinomyosin MgATRase by chicken gizzard calponin. Prog. Clin. Biol. Res. 327, 141-148, 1990 Winder, S.J., Sutherland, C., and Walsh, M.R., Biochemical and functional characterization of smooth muscle calponin, Adv. Exp. Med. Biol. 304, 37-51, 1991 Winder, S.J. and Walsh, M.R., Calponin thin filament-linked regulation of smooth muscle contraction. Cell Signal. 5,677-686,1993 el-Mezgueldi, M., Calponin, Int. J. Biochem. 

Caldesmon (CD) is a ubiquitous protein in smooth muscle cells. The smooth muscle isoform has a sequence-derived molecular mass of 89-93 kDa, but migrates on SDS gels at 120-150 kDa. Within the smooth muscle cell, CD is localized within the contractile apparatus (Furst et al., 1986) and when the contractile filaments are isolated it is found to be tightly bound to the thin filaments (Marston and Lehman, 1985). It is suggested that its in vivo function involves regulation of thin filament activity and possibly a role in the assembly and stabilization of thick and thin filaments. 

Calmodulin is a prime candidate for the role of the Ca2+-sensitizing protein since it is the most abundant calcium binding protein in smooth muscles, with a total concentration in the same range as CD. Under particular reaction conditions (37°C/120 mM KCl), Ca " -regulated synthetic thin filaments may be reconstituted using only actin, TM, CD, and CM. In contrast, native thin filaments are Ca + sensitive over a wide range of conditions and in conditions optimal for Ca + sensitivity in native thin filaments, the synthetic thin filaments are usually completely Ca + insensitive (Smith et al., 1987 Pritchard and Marston, 1989 Marston et al., 1988a). 

Despite these complications, it is generally agreed that the thin filaments of the smooth muscle cell contain (on average) actin complexed with at least three proteins tropomyosin (TM), caldesmon (CD), and cal-ponin (CP). In the present chapter we aim to review what is currently known about CP and ideas about its... 

To account for activation of arterial smooth muscle independently of LC20 phosphorylation, attention has been focused on the possible roles of the thin filament-associated regulatory proteins, caldesmon and calponin. Both proteins have been localized in the actomyosin domain of the smooth muscle cell and both have been shown to inhibit actin-activated myosin ATPase by interacting with F-actin, tropomyosin, and/or myosin (Clark et al., 1986 Takahashi et al.,... 

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