Calmodulin-dependent enzymes

The regulatory light chains from vertebrate forms of myosin-II undergo reversible phosphorylation by a calmodulin dependent enzyme called myosin light chain... 

The Ca transport ATPase of the surface membrane is a Ca -calmodulin-dependent enzyme of approximately 138-kDa mass that is structurally distinct from the sarcoplasmic reticulum Ca -ATPase, but shares with it some similarities in the mechanism of Ca translocation [2,3,34]. In both enzymes the Ca -dependent phosphorylation of an aspartyl-carboxyl-group by ATP leads to the formation of an acyl phosphate intermediate that provides the coupling between ATP hydrolysis and Ca translocation. 

An example for the reversible association of activator proteins with an enzyme is the Ca -calmodulin dependent enzymes. Calmodulin is a Ca -binding protein which can activate target enzymes, e.g. phosphorylase kinase (see 6.7.1 and 7.4) in its Ca -boimd form. Another example for activating proteins is the cyclins (see chapter 14). The cyc-lins are activators of protein kinases that regulate the cell cycle. 

Synthesis of NO Arginine, 02, and NADPH are substrates for cytosolic NO synthase (Figure 13.9). Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), heme, and tetrahydro- biopterin are coenzymes for the enzyme, and NO and citrulline are products of the reaction. Three NO synthases have been identified. Two are constitutive (synthesized at a constant rate regardless of physiologic demand), Ca2+-calmodulin-dependent enzymes. They are found primarily in endothelium (eNOS), and neural tissue... 

Deactivation of calmodulin-dependent enzymes may involve calmodulin-binding proteins such as calcineurin,229 although calcineurin has been shown to possess phosphatase activity.230... 

In addition to the psychoactive drugs, various agents with local anesthetic-like properties bind weakly in a Ca +-dependent manner (77). Another agent found to bind in the presence of Ca + and inhibit calmodulin-dependent enzymes is the sulfonamide,... 

The enzyme nitric oxide synthase (NOS), that produces NO and citrullin from arginine, occurs as several isoenzymes (Knowles et al., 1989). Type I NOS is a constitutive, calcium and calmodulin-dependent enzyme, present in neurons and, possibly, in glia. Type II NOS is calcium-independent and can be induced in macrophages and glial cells by exposure to bacterial lipopolysaccharide (Galea et al., 1992 Murphy et al., 1993). Type III NOS is the endothelial iso-enzyme. NOS-I, II and III are produced by diflferent genes (Bredt et al., 1991 Lamas et al., 1992 Xie et al., 1992 Lowenstein et al., 1992 Lyons et al. 1992 Ogura et al., 1993). NOS displays NADPH-dependent diaphorase... 

Another similarity to the catecholamine system is the fact that the initial enzyme, tryptophan hydroxylase, is subject to protein phosphorylation, which appears to increase its affinity for tetrahydrobiopterin. Of interest is the finding that, in this case, the protein kinase appears to be a specific calcium-calmodulin-dependent enzyme. While it is not known whether this regulation is physiologically significant, it has long been recognized that conditions that lead to increased activity of serotonin neurons and increased Ca " fluxes also lead to increased synthetic rates for serotonin. 

Thus, the individual pharmacological effects exerted by each calcium channel blocker may depend upon the extent to which the drug affects other intracellular systems as well as its potency at the calcium channel. Many of the effects described here would tend to increase the vasodilator action of the drugs, such as inhibition of calmodulin-dependent enzymes. However, these other effects are subsidiary to blockade of the calcium channel, as they occur mainly at concentrations higher than those required to block the channels therefore, at low concentrations the actions of the calcium channel blocking drugs are relatively specific. 

Jefferson, A. B., and Schulman, H. (1988). Sphingosine inhibits calmodulin-dependent enzymes. J Biol Chem 263, 15241-15244. 

The identification and development of specific inhibitors of MLCK could provide valuable experimental tools for exploring physiological functions of MLCK and myosin phosphorylation in smooth muscle and nonmuscle cells. Although inhibitors that act by binding calmodulin have been used experimentally, their usefulness is limited because they inhibit other calmodulin-dependent enzymes in tissues and cells (Asano and Stull, 1985 Nakanishi et al., 1992). Therefore, attempts have been made to develop novel reagents that inhibit MLCK activity directly. ML-9 [l-(5-chloronaphthalenesulfonyl)-lH-hexahydro-l, 4-diazepine] inhibits purified smooth muscle MLCK competitively with respect to ATP (Saitoh et al., 1987). Predictably, it inhibits RLC phosphorylation in ac-tomyosin, skinned fibers, and smooth muscle strips (Ishikawa et al., 1988). However, ML-9 also inhibits other protein kinases, so its specificity under different experimental conditions needs to be established. 

To further elucidate the activation mechanism, several inhibitors of MLCK have been applied. MLCK activity was inhibited by antagonising the binding of calmodulin to MLCK by calmodulin antagonists such as W-7 (Hidaka et al. 1978, Kanamori et al. 1981) or trifluoperazine (reviewed in Asano and Stull 1985). The disadvantage is that these inhibitors also inhibit other calmodulin-dependent enzymes in cells. ML-9 (Ishikawa et al. 

Candhi, C.R. Keenan, R.W. Calmodulin-dependent enzymic phosphorylation of dolichol in Tetrahymena pyriformis. Biochem. J., 216, 317-323 (1983)... 

Deactivation of calmodulin-dependent enzymes may involve calmodulin-binding proteins such... 

Vascular endothelial NOS (eNOS) is a calcium-calmodulin-dependent enzyme, with a monomeric molecular weight of 133 kDa that is bound to the cell membrane with a myristoylate bridge linked to the N-terminal glycine of the enzyme (Nathan 1992 Sessa et al. 1992). It is now well appreciated that eNOS is important for cardiovascular homeostasis, vessel remodeling, and angiogenesis (Nadaud et al. 2000 Chiou 2001). 

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