Myosin kinase

Upon entering the smooth muscle cell, Ca++ ions bind with calmodulin, an intracellular protein with a chemical structure similar to that of troponin. The resulting Ca++-calmodulin complex binds to and activates myosin kinase. This activated enzyme then phosphorylates myosin. Crossbridge cycling in smooth muscle may take place only when myosin has been phosphorylated. 

Walker, J. E., Saraste, M., Runswick, M. J. and Gay, N. J. (1982). Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold, EMBO J., 1, 945-951. 

Smooth muscle effects. The opposing effects on smooth muscle (A) of a-and p-adrenoceptor activation are due to differences in signal transduction (p. 66). This is exemplified by vascular smooth muscle (A). ai-Receptor stimulation leads to intracellular release of Ca + via activation of the inositol tris-phosphate (IP3) pathway. In concert with the protein calmodulin, Ca + can activate myosin kinase, leading to a rise in tonus via phosphorylation of the contractile protein myosin. cAMP inhibits activation of myosin kinase. Via the former effector pathway, stimulation of a-receptors results in vasoconstriction via the latter, P2-receptors mediate vasodilation, particularly in skeletal muscle - an effect that has little therapeutic use. 

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. 

The Ca2+ released from the endoplasmic reticulum by IP3 may perform a number of functions. The metabolically most important action of Ca2+, however, is to combine with a very ubiquitous protein called calmodulin. This protein has a molecular weight of about 17,500 and is in many ways similar to troponin. It has four Ca2+ binding sites and constitutes one of several subunits of several enzyme systems. Thus, as cellular [Ca2+] rises, the calmodulin subunit binds Ca2+. The result is that it changes its conformation to that of an a helix and thereby affects the catalytic activity of other constituent subunits. For instance, calmodulin is a component of myosin kinase, which phosphorylates one of the subunits of myosin. The structure of calmodulin is shown in Figure 16.22. 

Vasoconstriction occurs when calcium activates vascular myosin kinase, which in turn allows for phosphorylation of myosin and subsequent bridging with actin. Administration of calcium channel blockers will interfere with this process and produce vasodilation. 

An alternative mechanism by which cAMP may act to inhibit platelet function was proposed by Hathaway et al. (81). In this model (Figure 12), it is suggested that an increase in cAMP results in inhibition of myosin phosphorylation and consequent inhibition of platelet contractile activity (since unphos-phorylated myosin cannot interact with actin). Thus, it was proposed that cAMP causes the activation of a protein kinase which in turn phosphorylates myosin kinase. In the phosphory-lated form, myosin kinase is less capable of binding calmodulin and therefore is not as effective in phosphorylating myosin. 

Although this model would explain the ability of cAMP to interfere with platelet activation, the results to date were obtained using purified platelet myosin kinase. Consequently, more conclusive proof to support this mechanism of inhibition would require the demonstration that cAMP dependent phosphorylation of myosin kinase occurs under more physiological conditions, e.g. in intact platelets or platelet membrane fragments. 

Paul R], Doerman G, Zeugner C, Riiegg JC (1983) The dependence of unloaded shortening velocity on Ca, calmodulin, and duration of contraction in chemically skinned smooth muscle. Circ Res 53 342-351 Pearson RB, Misconi LY, Kemp BE (1986) Smooth muscle myosin kinase requires residues on the COOH-terminal site of the phosphorylation site. J Biol Chem 261 25-27... 

Strauss JD, de LaneroUe P, Paul RJ (1992) Effects of myosin kinase inhibiting peptide on contractility and LC20 phosphorylation in skinned smooth muscle. Am J Physiol 262 C1437-C1445... 

Conti MA, Adelstein RS (1981) The relationship between calmodulin binding and phosphorylation of smooth muscle myosin kinase by the catalytic subunit of 3 .5 cAMP-dependent protein kinase. J Biol Chem 256 3178-3181 Cornwell TL, Pryzwansky KB, Wyatt TA, Lincoln TM (1991) Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells. Mol Pharmacol 40 923-931... 

We have evaluated the effect of ionic strength on the kinetic parameters of lactose hydrolysis with (3-galactosidase from Aspergillus oryzae in Mcllvaine citrate-phosphate buffer at concentrations from 50 to 400 mM and found an increase in K but almost no effect on V (kcat). Studies on the effect of ionic strength on enzyme kinetics are not usual, so that works with acetylcholinesterase (Nolte et al. 1980), myosin kinase (Blumenthal and Stull 1982) and cytochrome C (Hazzard et al. 1991 Harris et al. 1994) are worth mentioning. Combined effect of pH and ionic strength on enzyme kinetics has been recently analyzed (Alberty 2006). 

Amoeba GPCR G-protein activation, PIP3 regulation of rho family G proteins, cGMP regulation of myosin kinases, ERK activation 5... 

Calmodulin, an intracellular calcium-combining protein, is involved in many bodily processes such as secretion, activation of myosin kinase, and cyclic nucleotide metabolism. A similar protein, troponin-r, regulates conformational changes in skeletal muscle. The control of skeletal muscle contraction depends entirely on intracellular calcium. Hence those drugs such as nifedepine (Section 14.2) which block calcium channels, have no effect. On the other hand, smooth and cardiac muscles are much influenced by external calcium levels. 

Figure 4.8. Schematic representation of the control of smooth muscle contraction [2 ]. Thick arrows indicate the tension accumulation and thin arrows its release, respectively. Mg-ATP-ase activity is maximal in the actin-myosin-Pa complex (CM - calmodulin, MLCK - light myosin kinase).

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