Myosin-actin interaction, troponin

An enhancement of ATPase action comes through the phosphorylation of myosin light chains (MW 18,000). The phosphorylation is achieved because the high cellular [Ca2+] activates myosin kinase, an enzyme that contains calmodulin, a Ca2+-binding subunit. Phosphorylation of myosin is absolutely required for smooth muscle contraction, though not for the contraction of skeletal or cardiac muscle, because smooth muscle has no troponin. Thus, whereas contraction and relaxation in skeletal and cardiac muscle are achieved principally via the action of Ca2+ on troponin, in smooth muscle they must depend solely on the Ca2+-dependent phosphorylation of myosin. In skeletal and cardiac muscle, once the stimulus to the sarcolemma is removed, [Ca2+] in sarcoplasm drops rapidly back to 10 7 or 10 8 M via various Ca2+ pump mechanisms present in the sarcoplasmic reticulum, and tropomyosin can once again interfere with the myosin-actin interaction. 

When muscle is relaxed, troponin does not bind Ca because sarcoplasmic [Ca2+] is very low. This permits the tropomyosin to return to the relaxed state in the groove of the thin filaments and interfere with myosin-actin interaction. Since the thin filaments slide across (over) the thick filaments during contraction, the I zone, which represents the thin filaments, will decrease greatly in size. 

All except tropomyosin form larger molecules. Tubulin, under the influence of GTP, aggregates into microtubules G-actin polymerizes to F-actin myosin forms thick filaments by tail-to-tail interaction and /3-actin polymerizes to microfilaments. /3-Actin is similar but not identical to G-actin. Tropomyosin is a fibrous protein associated with the F-actin polymer. It controls myosin-actin interaction under the influence of troponin. 

After death, the ratio of ADP to ATP increases rapidly. In the ADP form, myosin motor domains bind tightly to actin. Myosin-actin interactions are possible because the drop in ATP concentration also allows the calcium concentration to rise, clearing the blockage of actin by tropomyosin through the action of the troponin complex. 

The inhibitory action of troponin I on actomyosin-tropomyosin represents the depression of contractile interaction of myosin-actin by troponin-tropomyosin in the absence of Ca. In the absence of tropomyosin, the inhibitory action of troponin I is weak, whereas about 80% of the ATPase of actomyosin is inhibited by troponin I in the presence of... 

Tropomyosin and the troponin complex regulate this sliding in response to nerve impulses. Under resting conditions, tropomyosin blocks the intimate interaction between myosin and actin. A nerve impulse leads to an in crease in calcium ion concentration within the muscle cell. A component ol the troponin complex senses the increase in Ca" and, in response, relieves the inhibition of myosin—actin interactions by tropomyosin. 

Relaxation occurs when sarcoplasmic Ca falls below 10 mol/L owing to its resequestration into the sarcoplasmic reticulum by Ca ATPase. TpC.dCa thus loses its Ca. Consequently, troponin, via interaction with tropomyosin, inhibits further myosin head and F-actin interaction, and in the presence of ATP the myosin head detaches from the F-actin. 

The interaction between actin and myosin must be regulated so that contraction occurs only in response to appropriate signals from the nervous system. The regulation is mediated by a complex of two proteins, tropomyosin and troponin. Tropomyosin binds to the thin filament, blocking the attachment sites for the myosin head groups. Troponin is a Ca2+-binding protein. 

Criddle, A. H., Geeves, M. A., andjeffries, T. (1985). The use of actin labeled with N-(l-pyrenyl)iodoacetamide to study the interaction of actin with myosin subfragments and troponin/tropomyosin. Biochem. J. 232, 343-349. 

Study of the molecular biology of calcium regulation of muscle contraction was initiated by the discovery of a new protein factor sensitizing actomyosin to calcium ions (Ebashi, 1963 Ebashi and Ebashi, 1964). This protein factor was called native tropomyosin, because of its similarity in amino acid composition to tropomyosin, which had been discovered earlier (Bailey, 1946, 1948). It was soon found that this factor is a complex of tropomyosin and a new globular protein, termed troponin (Ebashi and Kodama, 1965 Ebashi et al., 1968). Thus four proteins, i.e., myosin, actin, troponin, and tropomyosin, are involved in calcium-regulated physiological muscle contraction (Ebashi et al., 1968, 1969 Ebashi and Endo, 1968). The contractile interaction between myosin and actin is depressed by troponin and tropomyosin in the absence of calcium ions. When calcium ion acts on troponin, this depression is removed and the contractile interaction is then activated (Figs. 1 and 2). 

Fig. 2. Ca-regulatory mechanism of troponin and tropomyosin. Abscissa indicates the free calcium ion concentration in arbitrary units. Ordinates indicate (a) the extent of contractile interaction between myosin and actin in the presence of troponin and/or tropomyosin (Ebashi et al, 1969) and (b) the extent of contractile interaction of myosin-actin-tropomyosin in the presence of troponin components (Ohtsuki, 1980). TM, Tropomyosin TN, troponin TN -C, troponin C TN-I, troponin I TN-T, troponin T.

The discussion above stresses the importance of the CN4 region (residues 96-116) of troponin I in the inhibition of contractile interaction of myosin-actin in the presence of tropomyosin. The amino acid sequence of the CN4 region is shown in Fig. 3. Talbot and Hodges (1981) synthesized 12 peptide analogs of the CN4 sequence and examined their inhibitory action on actomyosin ATPase activity. The absence of residues 115 and 116 did not affect the activity, whereas the absence of residue 114 significantly decreased the inhibitory action. As to the N-terminal portion of the peptide, residues 96-103 were not essential, but the absence of Lys-105 decreased the inhibitory activity. A peptide containing the region of residues Lys-105-Val-l 14 showed about half of the inhibitory action of troponin I. The authors concluded that Lys-105 and the bulky side chain at Val-114 are essential for the inhibitory action of troponin I (Fig. 3). 

As mentioned in Section II,A, Ca regulation by troponin-tropomyosin consists of the depression of contractile interaction between myosin and actin by troponin-tropomyosin in the absence of Ca " and the release of the inhibition by Ca + (Ebashi et al., 1968,1969) (Fig. 2). In the regulatory processes of the troponin complex, the essential mechanism is considered to consist of the Ca -dependent interaction between troponin C and troponin I and of the inhibitory interaction of troponin I with actin-tropomyosin the two interactions are complementary to each other (Potter and Gergely, 1974 Ebashi, 1974a Perry, 1979). 

Schematic diagram of the organization of skeletal muscle thin filament, showing the position of tropo-myosin and the troponin complex on the actin filament. The binding of Ca " to TnC, the calcium-binding subunit of the troponin complex, removes Tnl, the inhibitory subunit, from actin and thus permits an interaction with a specialized protein, myosin, on neighboring thick muscle filaments (not shown). An ATP-driven conformation change in the myosin head group makes the thick and thin filaments move relative to one another, so that muscle contraction occurs.

The rapid decrease in the level of ATP following death has two consequences. First, the cytosolic level of calcium rises rapidly because the Ca i-ATPase pumps in the plasma membrane and sarcoplasmic reticulum membrane no longer operate. High Ca, through troponin and tropomyosin, enables myosin to interact with actin. Second, a large proportion of SI heads -will be associated with actin. Recall that ATP is required to dissociate the actomyosin complex. In the absence of ATP, skeletal muscle is locked in the contracted (rigor) state. 

Binds to actin and troponin-T. Inhibits the actin—myosin interaction unless calcium is bound to troponin-C Slow skeletal Cardiac Fast skeletal... 

Troponin C Troponin I Troponin T Minor M protein 18 21 31 165 2 M line Ca binding Inhibits actin-myosin interaction Binds to tropomyosin Binds to myosin... 

In striated muscle, there are two other proteins that are minor in terms of their mass but important in terms of their function. Tropomyosin is a fibrous molecule that consists of two chains, alpha and beta, that attach to F-actin in the groove between its filaments (Figure 49-3). Tropomyosin is present in all muscular and muscle-fike structures. The troponin complex is unique to striated muscle and consists of three polypeptides. Troponin T (TpT) binds to tropomyosin as well as to the other two troponin components. Troponin I (Tpl) inhibits the F-actin-myosin interaction and also binds to the other components of troponin. Troponin C (TpC) is a calcium-binding polypeptide that is structurally and functionally analogous to calmodulin, an important calcium-binding protein widely distributed in nature. Four molecules of calcium ion are bound per molecule of troponin C or calmodulin, and both molecules have a molecular mass of 17 kDa. 

Inhibitor of F-actin-myosin interaction (inhibitor of F-actin-dependent activation of ATPase) Troponin system (Tpl) Unphosphorylated myosin light chain... 

Troponins T or I Proteins found predominantly in cardiac muscle that regulate calcium-mediated interaction of actin and myosin troponins I and T are released into the blood from myocytes at the time of myocardial cell necrosis after infarction. These biochemical markers become elevated and are used in the diagnosis of myocardial infarction. 

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